Administrative Appointments

  • Co-Organizer, American Society of Microbiology Meeting on Viral Vectors (2002 - Present)
  • Executive Committee, Faculty Senate - Stanford (2002 - Present)
  • Chief Scientific Advisor, Benitec, LLC (2003 - 2005)
  • Vice President, ASGT (2003 - 2004)
  • Chair of Organizing Committee, Gordon Conference on Viral Vectors for Gene Therapy (2003 - 2004)
  • President Elect, American Society of Gene Therapy (2004 - 2005)
  • President, American Society of Gene Therapy (2005 - 2006)
  • Associate Chair for Basic Research, Department of Pediatrics (2012 - Present)

Honors & Awards

  • Board of Directors, Oligotherapeutics Society (2011-current)
  • 2017 Distinguished Alumni and Commencement Speaker- Lyman Briggs College, Michigan State University (2017)
  • 2017 Distinguished Alumni Award, Case Western Reserve University (2017)
  • Young Investigator Award, Western Society for Clinical Investigation (1996)
  • Arosenius Swedish Honorary Lectureship, - (1997)
  • Elected Member, American Society for Clinical Investigation (1997)
  • Pediatric Researcher of the Year, E. Mead Johnson Award (2000)
  • Researcher of the Year, National Hemophilia Foundation (2000)
  • Elected Member, AAP (2010)
  • Outstanding Achievement/Investigator Award, American Society for Cell and Gene Therapy (2013)
  • Sam Rosenthal Prize for Excellence in Pediatrics, Rosenthal Foundation (2011-2013)

Professional Education

  • B.S., Michigan State University, Physical Sciences (1980)
  • Ph.D., Case Western Reserve University, Developmental Genetics (1986)
  • M.D., Case Western Reserve University (1987)

Research & Scholarship

Current Research and Scholarly Interests

The goal of the Program in Human Gene Therapy is to develop gene transfer technologies and use them for hepatic gene therapy for the treatment of genetic and acquired diseases. The general approach is to develop new vector systems and delivery methods, test them in the appropriate animal models, uncover the mechanisms involved in vector transduction, and use the most promising approaches in clinical trials. Specifically, we work on a variety of viral and non-viral vector systems. Our major disease models are hemophilia, hepatitis C and B viral infections, and diabetes. The second major focus includes the role that small RNAs play in mammalian gene regulation.


2018-19 Courses

Stanford Advisees

Graduate and Fellowship Programs


All Publications

  • miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression. Nature communications Valdmanis, P. N., Kim, H. K., Chu, K., Zhang, F., Xu, J., Munding, E. M., Shen, J., Kay, M. A. 2018; 9 (1): 5321


    miR-122 is a highly expressed liver microRNA that is activated perinatally and aids in regulating cholesterol metabolism and promoting terminal differentiation of hepatocytes. Disrupting expression of miR-122 can re-activate embryo-expressed adult-silenced genes, ultimately leading to the development of hepatocellular carcinoma (HCC). Here we interrogate the liver transcriptome at various time points after genomic excision of miR-122 to determine the cellular consequences leading to oncogenesis. Loss of miR-122 leads to specific and progressive increases in expression of imprinted clusters of microRNAs and mRNA transcripts at the Igf2 and Dlk1-Dio3 loci that could be curbed by re-introduction of exogenous miR-122. mRNA targets of other abundant hepatic microRNAs are functionally repressed leading to widespread hepatic transcriptional de-regulation. Together, this reveals a transcriptomic framework for the hepatic response to loss of miR-122 and the outcome on other microRNAs and their cognate gene targets.

    View details for DOI 10.1038/s41467-018-07786-7

    View details for PubMedID 30552326

  • Transcriptional and Position Effect Contributions to rAAV-Mediated Homologous Recombination Spector, L. P., Tiffany, M., Kay, M. A. CELL PRESS. 2018: 368
  • Generide (TM), a Novel AAV Strategy to Treat Pediatric Patients with Methylmalonic Acidemia Liao, J., Rais, Y., Hayon, Y., Barzel, A., Lisowski, L., Kay, M. A., Chiang, K., Chau, N. CELL PRESS. 2018: 363
  • Promoterless Targeting without Nucleases of Hyperactive Factor IX Corrects the Bleeding Diathesis in Hemophilia B Mice Hayon, Y., Reines, N., Kilovaty, I., Rais, Y., Chau, N., Chiang, K., Lisowski, L., Kay, M. A., Barzel, A. CELL PRESS. 2018: 89–90
  • Alteration of AAV Capsid Lumenal Residues to Expand Vector Genome Packaging Capacity Tiffany, M., Pekrun, K., Zhang, F., Kay, M. A. CELL PRESS. 2018: 191
  • Disruption of the Heparin-Binding Site and Insertion of the PHB.P Peptide in AAV-DJ Improve Transduction of the Central Nervous System Song, R., Kay, M. A. CELL PRESS. 2018: 40–41
  • Improved Genome Editing through Inhibition of the FANCM Pathway de Alencastro, G., Pekrun, K., Zhang, F., Pillay, S., Majzoub, K., Carette, J., Kay, M. A. CELL PRESS. 2018: 433–34
  • Targeted Integration of MUT into the Albumin Locus Using a Promoterless AAV Vector (Generide (TM)) Confers a Hepatocellular Growth Advantage in Mice with Methylmalonic Acidemia Chandler, R. J., Chau, N., Chiang, K., Kay, M. A., Barzel, A., Venditti, C. P. CELL PRESS. 2018: 446–47
  • Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity MOLECULAR THERAPY Paulk, N. K., Pekrun, K., Zhu, E., Nygaard, S., Li, B., Xu, J., Chu, K., Leborgne, C., Dane, A. P., Haft, A., Zhang, Y., Zhang, F., Morton, C., Valentine, M. B., Davidoff, A. M., Nathwani, A. C., Mingozzi, F., Grompe, M., Alexander, I. E., Lisowski, L., Kay, M. A. 2018; 26 (1): 289–303


    Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

    View details for DOI 10.1016/j.ymthe.2017.09.021

    View details for Web of Science ID 000423013500028

    View details for PubMedID 29055620

    View details for PubMedCentralID PMC5763027

  • Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle. Molecular therapy. Methods & clinical development Paulk, N. K., Pekrun, K., Charville, G. W., Maguire-Nguyen, K., Wosczyna, M. N., Xu, J., Zhang, Y., Lisowski, L., Yoo, B., Vilches-Moure, J. G., Lee, G. K., Shrager, J. B., Rando, T. A., Kay, M. A. 2018; 10: 144–55


    Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.

    View details for DOI 10.1016/j.omtm.2018.06.001

    View details for PubMedID 30101152

    View details for PubMedCentralID PMC6077147

  • Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid alpha-glucosidase SCIENCE TRANSLATIONAL MEDICINE Puzzo, F., Colella, P., Biferi, M. G., Bali, D., Paulk, N. K., Vidal, P., Collaud, F., Simon-Sola, M., Charles, S., Hardet, R., Leborgne, C., Meliani, A., Cohen-Tannoudji, M., Astord, S., Gjata, B., Sellier, P., van Wittenberghe, L., Vignaud, A., Boisgerault, F., Barkats, M., Laforet, P., Kay, M. A., Koeberl, D. D., Ronzitti, G., Mingozzi, F. 2017; 9 (418)


    Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.

    View details for DOI 10.1126/scitranslmed.aam6375

    View details for Web of Science ID 000416522500001

    View details for PubMedID 29187643

    View details for PubMedCentralID PMC5826611

  • Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of alpha-1 Antitrypsin Deficiency MOLECULAR THERAPY Borel, F., Tang, Q., Gernoux, G., Greer, C., Wang, Z., Barzel, A., Kay, M. A., Shultz, L. D., Greiner, D. L., Flotte, T. R., Brehm, M. A., Mueller, C. 2017; 25 (11): 2477–89


    Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

    View details for DOI 10.1016/j.ymthe.2017.09.020

    View details for Web of Science ID 000414723500008

    View details for PubMedID 29032169

    View details for PubMedCentralID PMC5675605

  • Human and Baculovirus-Insect Manufacturing Platforms Generate Chemically and Functionally Distinct AAV Vectors with Sexually Dimorphic Liver Transduction. Paulk, N. K., Rumachik, N., Malaker, S., Adams, C., Leib, R., Bertozzi, C. R., Kay, M. WILEY. 2017: 373A
  • Promoterless gene targeting without nucleases rescues lethality of a Crigler-Najjar syndrome mouse model EMBO MOLECULAR MEDICINE Porro, F., Bortolussi, G., Barzel, A., De Caneva, A., Iaconcig, A., Vodret, S., Zentilin, L., Kay, M. A., Muro, A. F. 2017; 9 (10): 1346–55


    Crigler-Najjar syndrome type I (CNSI) is a rare monogenic disease characterized by severe neonatal unconjugated hyperbilirubinemia with a lifelong risk of neurological damage and death. Liver transplantation is the only curative option, which has several limitations and risks. We applied an in vivo gene targeting approach based on the insertion, without the use of nucleases, of a promoterless therapeutic cDNA into the albumin locus of a mouse model reproducing all major features of CNSI Neonatal transduction with the donor vector resulted in the complete rescue from neonatal lethality, with a therapeutic reduction in plasma bilirubin lasting for at least 12 months, the latest time point analyzed. Mutant mice, which expressed about 5-6% of WT Ugt1a1 levels, showed normal liver histology and motor-coordination abilities, suggesting no functional liver or brain abnormalities. These results proved that the promoterless gene therapy is applicable for CNSI, providing therapeutic levels of an intracellular ER membrane-bound enzyme responsible for a lethal liver metabolic disease.

    View details for DOI 10.15252/emmm.201707601

    View details for Web of Science ID 000412117800005

    View details for PubMedID 28751579

    View details for PubMedCentralID PMC5623861

  • Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals MOLECULAR THERAPY Lu, J., Zhang, F., Fire, A. Z., Kay, M. A. 2017; 25 (5): 1187-1198


    Conventional plasmid vectors are incapable of achieving sustained levels of transgene expression in vivo even in quiescent mammalian tissues because the transgene expression cassette is silenced. Transcriptional silencing results from the presence of the bacterial plasmid backbone or virtually any DNA sequence of >1 kb in length placed outside of the expression cassette. Here, we show that transcriptional silencing can be substantially forestalled by increasing the An/Tn sequence composition in the plasmid bacterial backbone. Increasing numbers of An/Tn sequences increased sustained transcription of both backbone sequences and adjacent expression cassettes. In order to recapitulate these expression profiles in compact and portable plasmid DNA backbones, we engineered the standard kanamycin or ampicillin antibiotic resistance genes, optimizing the number of An/Tn sequence without altering the encoded amino acids. The resulting vector backbones yield sustained transgene expression from mouse liver, providing generic DNA vectors capable of sustained transgene expression without additional genes or mammalian regulatory elements.

    View details for DOI 10.1016/j.ymthe.2017.03.003

    View details for Web of Science ID 000401082600016

    View details for PubMedID 28365028

  • Future of rAAV gene therapy: Platform for RNAi, Gene Editing and Beyond. Human gene therapy Valdmanis, P., Kay, M. A. 2017


    The use of recombinant adeno-associated viruses (rAAVs) ushered in a new millennium of gene transfer for therapeutic treatment of a number of conditions, including congenital blindness, hemophilia, and spinal muscular atrophy. rAAV vectors have remarkable staying power from a therapeutic standpoint, withstanding several ebbs and flows. As new technologies such as clustered regularly interspaced short palindromic repeat genome editing emerge, it is now the delivery tool-the AAV vector-that is the stalwart. The long-standing safety of this vector in a multitude of clinical settings makes rAAV a selling point in the advancement of approaches for gene replacement, gene knockdown, gene editing, and genome modification/engineering. The research community is building on these advances to develop more tailored delivery approaches and to tweak the genome in new and unique ways. Intertwining these approaches with newly engineered rAAV vectors is greatly expanding the available tools to manipulate gene expression with a therapeutic intent.

    View details for DOI 10.1089/hum.2016.171

    View details for PubMedID 28073291

    View details for PubMedCentralID PMC5399734

  • Regulated complex assembly safeguards the fidelity of Sleeping Beauty transposition. Nucleic acids research Wang, Y., Pryputniewicz-Dobrinska, D., Nagy, E. É., Kaufman, C. D., Singh, M., Yant, S., Wang, J., Dalda, A., Kay, M. A., Ivics, Z., Izsvák, Z. 2017; 45 (1): 311-326


    The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotoxic recombination activities, and the mobilization of internally deleted copies in the IR/DR subgroup, including Sleeping Beauty (SB). In contrast, internally deleted sequences (MITEs) are preferred substrates of mariner transposition, and this process is associated with the emergence of Hsmar1-derived miRNA genes in the human genome. Translating IR/DR regulation to in vitro evolution yielded an SB transposon version with optimized substrate recognition (pT4). The ends of SB transposons excised by a K248A excision(+)/integration(-) transposase variant are processed by hairpin resolution, representing a link between phylogenetically, and mechanistically different recombination reactions, such as V(D)J recombination and transposition. Such variants generated by random mutation might stabilize transposon-host interactions or prepare the transposon for a horizontal transfer.

    View details for DOI 10.1093/nar/gkw1164

    View details for PubMedID 27913727

    View details for PubMedCentralID PMC5224488

  • A 5 ' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo HUMAN GENE THERAPY Lu, J., Williams, J. A., Luke, J., Zhang, F., Chu, K., Kay, M. A. 2017; 28 (1): 125-134


    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo.

    View details for DOI 10.1089/hum.2016.140

    View details for Web of Science ID 000392817800006

    View details for PubMedID 27903072

    View details for PubMedCentralID PMC5278795

  • Multiplexed in vivo homology-directed repair and tumor barcoding enables parallel quantification of Kras variant oncogenicity. Nature communications Winters, I. P., Chiou, S. H., Paulk, N. K., McFarland, C. D., Lalgudi, P. V., Ma, R. K., Lisowski, L., Connolly, A. J., Petrov, D. A., Kay, M. A., Winslow, M. M. 2017; 8 (1): 2053


    Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded KrasHDRalleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.

    View details for DOI 10.1038/s41467-017-01519-y

    View details for PubMedID 29233960

    View details for PubMedCentralID PMC5727199

  • A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature Kim, H. K., Fuchs, G., Wang, S., Wei, W., Zhang, Y., Park, H., Roy-Chaudhuri, B., Li, P., Xu, J., Chu, K., Zhang, F., Chua, M. S., So, S., Zhang, Q. C., Sarnow, P., Kay, M. A. 2017; 552 (7683): 57–62


    Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

    View details for DOI 10.1038/nature25005

    View details for PubMedID 29186115

  • Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation. Experimental neurology Chak, K., Roy-Chaudhuri, B., Kim, H. K., Kemp, K. C., Porter, B. E., Kay, M. A. 2016; 286: 137-146


    MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine-induced status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3'UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-β receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene.

    View details for DOI 10.1016/j.expneurol.2016.10.003

    View details for PubMedID 27725160

    View details for PubMedCentralID PMC5331941

  • A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nature biotechnology Chu, J., Oh, Y., Sens, A., Ataie, N., Dana, H., Macklin, J. J., Laviv, T., Welf, E. S., Dean, K. M., Zhang, F., Kim, B. B., Tang, C. T., Hu, M., Baird, M. A., Davidson, M. W., Kay, M. A., Fiolka, R., Yasuda, R., Kim, D. S., Ng, H., Lin, M. Z. 2016; 34 (7): 760-767


    Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

    View details for DOI 10.1038/nbt.3550

    View details for PubMedID 27240196

    View details for PubMedCentralID PMC4942401

  • A universal system to select gene-modified hepatocytes in vivo SCIENCE TRANSLATIONAL MEDICINE Nygaard, S., Barzel, A., Haft, A., Major, A., Finegold, M., Kay, M. A., Grompe, M. 2016; 8 (342)


    Many genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often subtherapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles could be overcome if hepatocytes that have undergone genetic modification were to be selectively amplified or expanded. We describe a universally applicable system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background. In this system, the therapeutic transgene is coexpressed with a short hairpin RNA (shRNA) that confers modified hepatocytes with resistance to drug-induced toxicity. An shRNA against the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase protected hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate, a small-molecule inhibitor of fumarylacetoacetate hydrolase. To select for specific gene targeting events, the protective shRNA was embedded in a microRNA and inserted into a recombinant adeno-associated viral vector designed to integrate site-specifically into the highly active albumin locus. After selection of the gene-targeted cells, transgene expression increased 10- to 1000-fold, reaching supraphysiological levels of human factor 9 protein (50,000 ng/ml) in mice. This drug resistance system can be used to achieve therapeutically relevant transgene levels in hepatocytes in any setting.

    View details for DOI 10.1126/scitranslmed.aad8166

    View details for Web of Science ID 000377443800003

    View details for PubMedID 27280686

  • RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice NATURE MEDICINE Valdmanis, P. N., Gu, S., Chu, K., Jin, L., Zhang, F., Munding, E. M., Zhang, Y., Huang, Y., Kutay, H., Ghoshal, K., Lisowski, L., Kay, M. A. 2016; 22 (5): 557-562


    Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity-including hepatocyte turnover, loss of gene repression and lethality-in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122-knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.

    View details for DOI 10.1038/nm.4079

    View details for Web of Science ID 000375514000019

    View details for PubMedID 27064447

    View details for PubMedCentralID PMC4860119

  • RNA interference. Drugging RNAi. Science Haussecker, D., Kay, M. A. 2015; 347 (6226): 1069-1070

    View details for DOI 10.1126/science.1252967

    View details for PubMedID 25745148

  • Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs HUMAN GENE THERAPY CLINICAL DEVELOPMENT Nichols, T. C., Whitford, M. H., Arruda, V. R., Stedman, H. H., Kay, M. A., High, K. A. 2015; 26 (1): 5-14


    Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches.

    View details for DOI 10.1089/humc.2014.153

    View details for Web of Science ID 000350951100001

    View details for PubMedID 25675273

  • Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature BARZEL, A., Paulk, N. K., Shi, Y., Huang, Y., Chu, K., Zhang, F., Valdmanis, P. N., Spector, L. P., Porteus, M. H., Gaensler, K. M., Kay, M. A. 2015; 517 (7534): 360-364


    Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ∼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.

    View details for DOI 10.1038/nature13864

    View details for PubMedID 25363772

    View details for PubMedCentralID PMC4297598

  • Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature BARZEL, A., Paulk, N. K., Shi, Y., Huang, Y., Chu, K., Zhang, F., Valdmanis, P. N., Spector, L. P., Porteus, M. H., Gaensler, K. M., Kay, M. A. 2015; 517 (7534): 360-364

    View details for DOI 10.1038/nature13864

    View details for PubMedID 25363772

  • Upregulation of the microRNA cluster at the Dlkl-Dio3 locus in lung adenocarcinoma ONCOGENE Valdmanis, P. N., Roy-Chaudhuri, B., Kim, H. K., Sayles, L. C., Zheng, Y., Chuang, C., Caswell, D. R., Chu, K., Zhang, Y., Winslow, M. M., Sweet-Cordero, E. A., Kay, M. A. 2015; 34 (1): 94-103


    Mice in which lung epithelial cells can be induced to express an oncogenic Kras(G12D) develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of Kras(G12D) tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of ∼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured Kras(G12D) tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.Oncogene advance online publication, 9 December 2013; doi:10.1038/onc.2013.523.

    View details for DOI 10.1038/onc.2013.523

    View details for Web of Science ID 000349740100009

  • Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Scientific reports Diecke, S., Lu, J., Lee, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., Ebert, A. D., Churko, J. M., Sharma, A., Kay, M. A., Wu, J. C. 2015; 5: 8081-?


    The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.

    View details for DOI 10.1038/srep08081

    View details for PubMedID 25628230

  • Novel codon-optimized mini-intronic plasmid for efficient, inexpensive, and xeno-free induction of pluripotency. Scientific reports Diecke, S., Lu, J., Lee, J., Termglinchan, V., Kooreman, N. G., Burridge, P. W., Ebert, A. D., Churko, J. M., Sharma, A., Kay, M. A., Wu, J. C. 2015; 5: 8081-?


    The development of human induced pluripotent stem cell (iPSC) technology has revolutionized the regenerative medicine field. This technology provides a powerful tool for disease modeling and drug screening approaches. To circumvent the risk of random integration into the host genome caused by retroviruses, non-integrating reprogramming methods have been developed. However, these techniques are relatively inefficient or expensive. The mini-intronic plasmid (MIP) is an alternative, robust transgene expression vector for reprogramming. Here we developed a single plasmid reprogramming system which carries codon-optimized (Co) sequences of the canonical reprogramming factors (Oct4, Klf4, Sox2, and c-Myc) and short hairpin RNA against p53 ("4-in-1 CoMiP"). We have derived human and mouse iPSC lines from fibroblasts by performing a single transfection. Either independently or together with an additional vector encoding for LIN28, NANOG, and GFP, we were also able to reprogram blood-derived peripheral blood mononuclear cells (PBMCs) into iPSCs. Taken together, the CoMiP system offers a new highly efficient, integration-free, easy to use, and inexpensive methodology for reprogramming. Furthermore, the CoMIP construct is color-labeled, free of any antibiotic selection cassettes, and independent of the requirement for expression of the Epstein-Barr Virus nuclear antigen (EBNA), making it particularly beneficial for future applications in regenerative medicine.

    View details for DOI 10.1038/srep08081

    View details for PubMedID 25628230

  • Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa SCIENCE TRANSLATIONAL MEDICINE Sebastiano, V., Zhen, H. H., Derafshi, B. H., Bashkirova, E., Melo, S. P., Wang, P., Leung, T. L., Siprashvili, Z., Tichy, A., Li, J., Ameen, M., Hawkins, J., Lee, S., Li, L., Schwertschkow, A., Bauer, G., Lisowski, L., Kay, M. A., Kim, S. K., Lane, A. T., Wernig, M., Oro, A. E. 2014; 6 (264)


    Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

    View details for DOI 10.1126/scitranslmed.3009540

    View details for Web of Science ID 000345595200003

    View details for PubMedCentralID PMC4428910

  • Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa. Science translational medicine Sebastiano, V., Zhen, H. H., Haddad, B., Bashkirova, E., Melo, S. P., Wang, P., Leung, T. L., Siprashvili, Z., Tichy, A., Li, J., Ameen, M., Hawkins, J., Lee, S., Li, L., Schwertschkow, A., Bauer, G., Lisowski, L., Kay, M. A., Kim, S. K., Lane, A. T., Wernig, M., Oro, A. E. 2014; 6 (264): 264ra163-?


    Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack functional type VII collagen owing to mutations in the gene COL7A1 and suffer severe blistering and chronic wounds that ultimately lead to infection and development of lethal squamous cell carcinoma. The discovery of induced pluripotent stem cells (iPSCs) and the ability to edit the genome bring the possibility to provide definitive genetic therapy through corrected autologous tissues. We generated patient-derived COL7A1-corrected epithelial keratinocyte sheets for autologous grafting. We demonstrate the utility of sequential reprogramming and adenovirus-associated viral genome editing to generate corrected iPSC banks. iPSC-derived keratinocytes were produced with minimal heterogeneity, and these cells secreted wild-type type VII collagen, resulting in stratified epidermis in vitro in organotypic cultures and in vivo in mice. Sequencing of corrected cell lines before tissue formation revealed heterogeneity of cancer-predisposing mutations, allowing us to select COL7A1-corrected banks with minimal mutational burden for downstream epidermis production. Our results provide a clinical platform to use iPSCs in the treatment of debilitating genodermatoses, such as RDEB.

    View details for DOI 10.1126/scitranslmed.3009540

    View details for PubMedID 25429056

    View details for PubMedCentralID PMC4428910

  • Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B NEW ENGLAND JOURNAL OF MEDICINE Nathwani, A. C., Reiss, U. M., Tuddenham, E. G., Rosales, C., Chowdary, P., Mcintosh, J., Della Peruta, M., Lheriteau, E., Patel, N., Raj, D., Riddell, A., Pie, J., Rangarajan, S., Bevan, D., Recht, M., Shen, Y., HALKA, K. G., Basner-Tschakarjan, E., Mingozzi, F., High, K. A., ALLAY, J., Kay, M. A., Ng, C. Y., Zhou, J., Cancio, M., Morton, C. L., Gray, J. T., Srivastava, D., NIENHUIS, A. W., Davidoff, A. M. 2014; 371 (21): 1994-2004
  • Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic acids research Gu, S., Zhang, Y., Jin, L., Huang, Y., Zhang, F., Bassik, M. C., Kampmann, M., Kay, M. A. 2014; 42 (19): 12169-12176


    The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3' regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency. These studies provide new rules that should significantly improve on siRNA/shRNA design.

    View details for DOI 10.1093/nar/gku854

    View details for PubMedID 25270879

    View details for PubMedCentralID PMC4231738

  • Regulation of microRNA-mediated gene silencing by microRNA precursors. Nature structural & molecular biology Roy-Chaudhuri, B., Valdmanis, P. N., Zhang, Y., Wang, Q., Luo, Q., Kay, M. A. 2014; 21 (9): 825-832


    Processing of microRNAs (miRNAs) from their precursors to their biologically active mature forms is regulated during development and cancer. We show that mouse pri- or pre-miR-151 can bind to and compete with mature miR-151-5p and miR-151-3p for binding sites contained within the complementary regions of the E2f6 mRNA 3' untranslated region (UTR). E2f6 mRNA levels were directly regulated by pri- or pre-miR-151. Conversely, miR-151-mediated repression of ARHGDIA mRNA was dependent on the level of mature miR-151 because only the mature miRNA binds the 3' UTR. Thus, processing of miR-151 can have different effects on separate mRNA targets within a cell. A bioinformatics pipeline revealed additional candidate regions where precursor miRNAs can compete with their mature miRNA counterparts. We validated this experimentally for miR-124 and the SNAI2 3' UTR. Hence, miRNA precursors can serve as post-transcriptional regulators of miRNA activity and are not mere biogenesis intermediates.

    View details for DOI 10.1038/nsmb.2862

    View details for PubMedID 25086740

  • Regulation of microRNA-mediated gene silencing by microRNA precursors. Nature structural & molecular biology Roy-Chaudhuri, B., Valdmanis, P. N., Zhang, Y., Wang, Q., Luo, Q., Kay, M. A. 2014; 21 (9): 825-832


    Processing of microRNAs (miRNAs) from their precursors to their biologically active mature forms is regulated during development and cancer. We show that mouse pri- or pre-miR-151 can bind to and compete with mature miR-151-5p and miR-151-3p for binding sites contained within the complementary regions of the E2f6 mRNA 3' untranslated region (UTR). E2f6 mRNA levels were directly regulated by pri- or pre-miR-151. Conversely, miR-151-mediated repression of ARHGDIA mRNA was dependent on the level of mature miR-151 because only the mature miRNA binds the 3' UTR. Thus, processing of miR-151 can have different effects on separate mRNA targets within a cell. A bioinformatics pipeline revealed additional candidate regions where precursor miRNAs can compete with their mature miRNA counterparts. We validated this experimentally for miR-124 and the SNAI2 3' UTR. Hence, miRNA precursors can serve as post-transcriptional regulators of miRNA activity and are not mere biogenesis intermediates.

    View details for DOI 10.1038/nsmb.2862

    View details for PubMedID 25086740

  • Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Recapitulates Long QT Phenotype for Drug Testing. Journal of the American College of Cardiology Wang, Y., Liang, P., Lan, F., Wu, H., Lisowski, L., Gu, M., Hu, S., Kay, M. A., Urnov, F. D., Shinnawi, R., Gold, J. D., Gepstein, L., Wu, J. C. 2014; 64 (5): 451-459


    Human induced pluripotent stem cells (iPSCs) play an important role in disease modeling and drug testing. However, the current methods are time-consuming and lack an isogenic control.This study sought to establish an efficient technology to generate human PSC-based disease models with isogenic control.The ion channel genes KCNQ1 and KCNH2 with dominant negative mutations causing long QT syndrome types 1 and 2, respectively, were stably integrated into a safe harbor AAVS1 locus using zinc finger nuclease technology.Patch-clamp recording revealed that the edited iPSC-derived cardiomyocytes (iPSC-CMs) displayed characteristic long QT syndrome phenotype and significant prolongation of the action potential duration compared with the unedited control cells. Finally, addition of nifedipine (L-type calcium channel blocker) or pinacidil (KATP-channel opener) shortened the action potential duration of iPSC-CMs, confirming the validity of isogenic iPSC lines for drug testing in the future.Our study demonstrates that iPSC-CM-based disease models can be rapidly generated by overexpression of dominant negative gene mutants.

    View details for DOI 10.1016/j.jacc.2014.04.057

    View details for PubMedID 25082577

  • Organ Size Control Is Dominant over Rb Family Inactivation to Restrict Proliferation In Vivo CELL REPORTS Ehmer, U., Zmoos, A., Auerbach, R. K., Vaka, D., Butte, A. J., Kay, M. A., Sage, J. 2014; 8 (2): 370-380
  • Organ Size Control Is Dominant over Rb Family Inactivation to Restrict Proliferation In Vivo. Cell reports Ehmer, U., Zmoos, A., Auerbach, R. K., Vaka, D., Butte, A. J., Kay, M. A., Sage, J. 2014; 8 (2): 371-381


    In mammals, a cell's decision to divide is thought to be under the control of the Rb/E2F pathway. We previously found that inactivation of the Rb family of cell cycle inhibitors (Rb, p107, and p130) in quiescent liver progenitors leads to uncontrolled division and cancer initiation. Here, we show that, in contrast, deletion of the entire Rb gene family in mature hepatocytes is not sufficient for their long-term proliferation. The cell cycle block in Rb family mutant hepatocytes is independent of the Arf/p53/p21 checkpoint but can be abrogated upon decreasing liver size. At the molecular level, we identify YAP, a transcriptional regulator involved in organ size control, as a factor required for the sustained expression of cell cycle genes in hepatocytes. These experiments identify a higher level of regulation of the cell cycle in vivo in which signals regulating organ size are dominant regulators of the core cell cycle machinery.

    View details for DOI 10.1016/j.celrep.2014.06.025

    View details for PubMedID 25017070

  • Characterization of Vector-Based Delivery of Neurogenin-3 in Murine Diabetes HUMAN GENE THERAPY Phillips, N., Kay, M. A. 2014; 25 (7): 651-661


    Treatment of type 1 diabetes with gene transfer-induced cellular reprogramming requires a pancreatic transcription factor such as Neurogenin-3 (Ngn3) and as of yet unknown component of the adenoviral particle. Despite intensive study, there are many unsolved processes related to the mechanisms and physiological parameters related to diabetes correction using this approach. While we confirm that systemic delivery of adenovirus (Ad)-Ngn3 provides long-lasting correction of streptozotocin (STZ)-induced hyperglycemia and restoration of growth curves, we found that insulin levels and glucose tolerance tests are not fully restored. By altering the innate and antigen-specific immune responses, we establish that the former likely plays some role in the reprogramming process. Interestingly, Ad-hNgn3 therapy in diabetic animals appeared to protect them from secondary STZ challenge. The resistance to secondary STZ response was more pronounced at later time points, indicating that a period of cell maturation and/or expansion may be required in order to promote lasting correction. More importantly, these results suggest that the long-term reprogrammed cells are not fully reprogrammed into β-cells, which in the case of autoimmune diabetes may be advantageous in a long-term treatment strategy. Finally, we show that the prophylactic administration of Ad-hNgn3 before diabetic induction protected mice from developing hyperglycemia, demonstrating the potential for reducing or eliminating disease progression should treatment be initiated early or before onset of symptoms.

    View details for DOI 10.1089/hum.2013.206

    View details for Web of Science ID 000339658800011

    View details for PubMedID 24635696

  • Somatic Correction of Junctional Epidermolysis Bullosa by a Highly Recombinogenic AAV Variant. Molecular therapy : the journal of the American Society of Gene Therapy Melo, S. P., Lisowski, L., Bashkirova, E., Zhen, H. H., Chu, K., Keene, D. R., Marinkovich, M. P., Kay, M. A., Oro, A. E. 2014; 22 (4): 725-733


    Definitive correction of disease causing mutations in somatic cells by homologous recombination (HR) is an attractive therapeutic approach for the treatment of genetic diseases. However, HR-based somatic gene therapy is limited by the low efficiency of gene targeting in mammalian cells and replicative senescence of primary cells ex vivo, forcing investigators to explore alternative strategies such as retro- and lentiviral gene transfer, or genome editing in induced pluripotent stem cells. Here, we report correction of mutations at the LAMA3 locus in primary keratinocytes derived from a patient affected by recessive inherited Herlitz junctional epidermolysis bullosa (H-JEB) disorder using recombinant adenoassociated virus (rAAV)-mediated HR. We identified a highly recombinogenic AAV serotype, AAV-DJ, that mediates efficient gene targeting in keratinocytes at clinically relevant frequencies with a low rate of random integration. Targeted H-JEB patient cells were selected based on restoration of adhesion phenotype, which eliminated the need for foreign sequences in repaired cells, enhancing the clinical use and safety profile of our approach. Corrected pools of primary cells assembled functional laminin-332 heterotrimer and fully reversed the blistering phenotype both in vitro and in skin grafts. The efficient targeting of the LAMA3 locus by AAV-DJ using phenotypic selection, together with the observed low frequency of off-target events, makes AAV-DJ based somatic cell targeting a promising strategy for ex vivo therapy for this severe and often lethal epithelial disorder.

    View details for DOI 10.1038/mt.2013.290

    View details for PubMedID 24390279

    View details for PubMedCentralID PMC3982486

  • Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference MOLECULAR THERAPY Borel, F., Kay, M. A., Mueller, C. 2014; 22 (4): 692-701


    RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed.

    View details for DOI 10.1038/mt.2013.285

    View details for Web of Science ID 000334061100003

    View details for PubMedID 24352214

  • Selection and evaluation of clinically relevant AAV variants in a xenograft liver model NATURE Lisowski, L., Dane, A. P., Chu, K., Zhang, Y., Cunningham, S. C., Wilson, E. M., Nygaard, S., Grompe, M., Alexander, I. E., Kay, M. A. 2014; 506 (7488): 382-?


    Recombinant adeno-associated viral (rAAV) vectors have shown early promise in clinical trials. The therapeutic transgene cassette can be packaged in different AAV capsid pseudotypes, each having a unique transduction profile. At present, rAAV capsid serotype selection for a specific clinical trial is based on effectiveness in animal models. However, preclinical animal studies are not always predictive of human outcome. Here, in an attempt to further our understanding of these discrepancies, we used a chimaeric human-murine liver model to compare directly the relative efficiency of rAAV transduction in human versus mouse hepatocytes in vivo. As predicted from preclinical and clinical studies, rAAV2 vectors functionally transduced mouse and human hepatocytes at equivalent but relatively low levels. However, rAAV8 vectors, which are very effective in many animal models, transduced human hepatocytes rather poorly-approximately 20 times less efficiently than mouse hepatocytes. In light of the limitations of the rAAV vectors currently used in clinical studies, we used the same murine chimaeric liver model to perform serial selection using a human-specific replication-competent viral library composed of DNA-shuffled AAV capsids. One chimaeric capsid composed of five different parental AAV capsids was found to transduce human primary hepatocytes at high efficiency in vitro and in vivo, and provided species-selected transduction in primary liver, cultured cells and a hepatocellular carcinoma xenograft model. This vector is an ideal clinical candidate and a reagent for gene modification of human xenotransplants in mouse models of human diseases. More importantly, our results suggest that humanized murine models may represent a more precise approach for both selecting and evaluating clinically relevant rAAV serotypes for gene therapeutic applications.

    View details for DOI 10.1038/nature12875

    View details for Web of Science ID 000331477800044

    View details for PubMedID 24390344

    View details for PubMedCentralID PMC3939040

  • The expanding repertoire of circular RNAs. Molecular therapy : the journal of the American Society of Gene Therapy Valdmanis, P. N., Kay, M. A. 2013; 21 (6): 1112-1114

    View details for DOI 10.1038/mt.2013.101

    View details for PubMedID 23728253

  • Upregulation of a Cluster of microRNAs on Mouse Chromosome 12qF1 in a Kras Mutant Mouse Model of Lung Adenocarcinoma 16th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Valdmanis, P. N., Roy-Chaudhuri, B., Kim, H. K., Sayles, L. C., Zheng, Y., Chuang, C., Caswell, D., Winslow, M., Sweet-Cordero, E. A., Kay, M. A. NATURE PUBLISHING GROUP. 2013: S168–S168
  • Benchtop DNA Synthesizer: Oligo-Templated Polymerization (OTP) 16th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Barzel, A., Kay, M. A. NATURE PUBLISHING GROUP. 2013: S132–S133
  • A Mini-intronic Plasmid (MIP): A Novel Robust Transgene Expression Vector In Vivo and In Vitro. Molecular therapy : the journal of the American Society of Gene Therapy Lu, J., Zhang, F., Kay, M. A. 2013; 21 (5): 954-963


    The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.

    View details for DOI 10.1038/mt.2013.33

    View details for PubMedID 23459514

  • The anti-genomic (negative) strand of Hepatitis C Virus is not targetable by shRNA. Nucleic acids research Lisowski, L., Elazar, M., Chu, K., Glenn, J. S., Kay, M. A. 2013; 41 (6): 3688-3698


    Hepatitis C Virus (HCV) and other plus-strand RNA viruses typically require the generation of a small number of negative genomes (20-100× lower than the positive genomes) for replication, making the less-abundant antigenome an attractive target for RNA interference(RNAi)-based therapy. Because of the complementarity of duplex short hairpin RNA/small interfering RNA (shRNA/siRNAs) with both genomic and anti-genomic viral RNA strands, and the potential of both shRNA strands to become part of the targeting complexes, preclinical RNAi studies cannot distinguish which viral strand is actually targeted in infected cells. Here, we addressed the question whether the negative HCV genome was bioaccessible to RNAi. We first screened for the most active shRNA molecules against the most conserved regions in the HCV genome, which were then used to generate asymmetric anti-HCV shRNAs that produce biologically active RNAi specifically directed against the genomic or antigenomic HCV sequences. Using this simple but powerful and effective method to screen for shRNA strand selectivity, we demonstrate that the antigenomic strand of HCV is not a viable RNAi target during HCV replication. These findings provide new insights into HCV biology and have important implications for the design of more effective and safer antiviral RNAi strategies seeking to target HCV and other viruses with similar replicative strategies.

    View details for DOI 10.1093/nar/gkt068

    View details for PubMedID 23396439

    View details for PubMedCentralID PMC3616702

  • Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Molecular therapy : the journal of the American Society of Gene Therapy Gracey Maniar, L. E., Maniar, J. M., Chen, Z., Lu, J., Fire, A. Z., Kay, M. A. 2013; 21 (1): 131-138


    Current efforts in nonviral gene therapy are plagued by a pervasive difficulty in sustaining therapeutic levels of delivered transgenes. Minicircles (plasmid derivatives with the same expression cassette but lacking a bacterial backbone) show sustained expression and hold promise for therapeutic use where persistent transgene expression is required. To characterize the widely-observed silencing process affecting expression of foreign DNA in mammals, we used a system in which mouse liver presented with either plasmid or minicircle consistently silences plasmid but not minicircle expression. We found that preferential silencing of plasmid DNA occurs at a nuclear stage that precedes transport of mRNA to the cytoplasm, evident from a consistent >25-fold minicircle/plasmid transcript difference observed in both nuclear and total RNA. Among possible mechanisms of nuclear silencing, our data favor chromatin-linked transcriptional blockage rather than targeted degradation, aberrant processing, or compromised mRNA transport. In particular, we observe dramatic enrichment of H3K27 trimethylation on plasmid sequences. Also, it appears that Pol II can engage the modified plasmid chromatin, potentially in a manner that is not productive in the synthesis of high levels of new transcript. We outline a scenario in which sustained differences at the chromatin level cooperate to determine the activity of foreign DNA.

    View details for DOI 10.1038/mt.2012.244

    View details for PubMedID 23183534

  • Minicircle DNA Vectors Achieve Sustained Expression Reflected by Active Chromatin and Transcriptional Level MOLECULAR THERAPY Maniar, L. E., Maniar, J. M., Chen, Z., Lu, J., Fire, A. Z., Kay, M. A. 2013; 21 (1): 131-138
  • Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging CIRCULATION RESEARCH Wang, Y., Zhang, W. Y., Hu, S., Lan, F., Lee, A. S., Huber, B., Lisowski, L., Liang, P., Huang, M., de Almeida, P. E., Won, J. H., Sun, N., Robbins, R. C., Kay, M. A., Urnov, F. D., Wu, J. C. 2012; 111 (12): 1494-?


    Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression.To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging.We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine.Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.

    View details for DOI 10.1161/CIRCRESAHA.112.274969

    View details for Web of Science ID 000311994700042

    View details for PubMedID 22967807

    View details for PubMedCentralID PMC3518748

  • Stable Factor IX Activity Following AAV-Mediated Gene Transfer in Patients with Severe Hemophilia B 54th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Davidoff, A., Tuddenham, E. G., Rangarajan, S., Rosales, C., McIntosh, J., Chowdary, P., Riddell, A., Glader, B., Rustagi, P., Ng, C., Kay, M., Zhou, J., Spence, Y., Morton, C., Allay, J., Sleep, S., Basner-Tschakarjan, E., Mingozzi, F., High, K. A., Gray, J. T., Reiss, U. M., Nienhuis, A. W., Nathwani, A. C. AMER SOC HEMATOLOGY. 2012
  • The Loop Position of shRNAs and Pre-miRNAs Is Critical for the Accuracy of Dicer Processing In Vivo CELL Gu, S., Jin, L., Zhang, Y., Huang, Y., Zhang, F., Valdmanis, P. N., Kay, M. A. 2012; 151 (4): 900-911


    Short hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that, in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3' counting rules. These promiscuous noncanonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a "loop-counting rule," we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

    View details for DOI 10.1016/j.cell.2012.09.042

    View details for Web of Science ID 000310921200019

    View details for PubMedID 23141545

    View details for PubMedCentralID PMC3499986

  • The Extragenic Spacer Length Between the 5 ' and 3 ' Ends of the Transgene Expression Cassette Affects Transgene Silencing From Plasmid-based Vectors MOLECULAR THERAPY Lu, J., Zhang, F., Xu, S., Fire, A. Z., Kay, M. A. 2012; 20 (11): 2111-2119


    In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5' and 3' ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3'-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.

    View details for DOI 10.1038/mt.2012.65

    View details for Web of Science ID 000310764500014

    View details for PubMedID 22565847

  • rAAV-Mediated Tumorigenesis: Still Unresolved After an AAV Assault MOLECULAR THERAPY Valdmanis, P. N., Lisowski, L., Kay, M. A. 2012; 20 (11): 2014-2017

    View details for DOI 10.1038/mt.2012.220

    View details for Web of Science ID 000310764500004

    View details for PubMedID 23131853

  • Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression MOLECULAR THERAPY Lisowski, L., Lau, A., Wang, Z., Zhang, Y., Zhang, F., Grompe, M., Kay, M. A. 2012; 20 (10): 1912-1923


    Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8-13 times more frequently than control vectors, providing an estimate that 23-39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells.

    View details for DOI 10.1038/mt.2012.164

    View details for Web of Science ID 000309519000014

    View details for PubMedID 22990671

    View details for PubMedCentralID PMC3464642

  • Optimization of liver gene transfer for hemophilia B Collaborative Congress of the European-Society-of-Gene-and-Cell-Therapy/French-Society-of-Cell-and-Gene-Therapy Anguela, X. M., Chen, Y., Davidson, R. J., Zhou, S., Nichols, T. C., Kay, M. A., Mingozzi, F., High, K. A. MARY ANN LIEBERT INC. 2012: A53–A53
  • AAV Vectors Containing rDNA Homology Display Increased Chromosomal Integration and Transgene Persistence MOLECULAR THERAPY Wang, Z., Lisowski, L., Finegold, M. J., Nakai, H., Kay, M. A., Grompe, M. 2012; 20 (10): 1902-1911


    Although recombinant adeno-associated viral (rAAV) vectors are promising tools for gene therapy of genetic disorders, they remain mostly episomal and hence are lost during cell replication. For this reason, rAAV vectors capable of chromosomal integration would be desirable. Ribosomal DNA (rDNA) repeat sequences are overrepresented during random integration of rAAV. We therefore sought to enhance AAV integration frequency by including 28S rDNA homology arms into our vector design. A vector containing ~1 kb of homology on each side of a cDNA expression cassette for human fumarylacetoacetate hydrolase (FAH) was constructed. rAAV of serotypes 2 and 8 were injected into Fah-deficient mice, a model for human tyrosinemia type 1. Integrated FAH transgenes are positively selected in this model and rDNA-containing AAV vectors had a ~30× higher integration frequency than controls. Integration by homologous recombination (HR) into the 28S rDNA locus was seen in multiple tissues. Furthermore, rDNA-containing AAV vectors for human factor IX (hFIX) demonstrated increased transgene persistence after liver regeneration. We conclude that rDNA containing AAV vectors may be superior to conventional vector design for the treatment of genetic diseases, especially those associated with increased hepatocyte replication.

    View details for DOI 10.1038/mt.2012.157

    View details for Web of Science ID 000309519000013

    View details for PubMedID 22990673

  • Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts NATURE Shiba, Y., Fernandes, S., Zhu, W., Filice, D., Muskheli, V., Kim, J., Palpant, N. J., Gantz, J., Moyes, K. W., Reinecke, H., Van Biber, B., Dardas, T., Mignone, J. L., Izawa, A., Hanna, R., Viswanathan, M., Gold, J. D., Kotlikoff, M. I., Sarvazyan, N., Kay, M. W., Murry, C. E., Laflamme, M. A. 2012; 489 (7415): 322-?


    Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

    View details for DOI 10.1038/nature11317

    View details for Web of Science ID 000308635900048

    View details for PubMedID 22864415

  • Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain CURRENT BIOLOGY Gu, S., Jin, L., Huang, Y., Zhang, F., Kay, M. A. 2012; 22 (16): 1536-1542


    Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood. The Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3' end of small RNAs. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells. We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs, PAZ-disrupted Agos bound duplex small interfering RNAs, but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics.

    View details for DOI 10.1016/j.cub.2012.06.040

    View details for Web of Science ID 000307795000029

    View details for PubMedID 22795694

    View details for PubMedCentralID PMC3604743

  • Targeting 2A-Fusions to Endogenous Genes 15th Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT) Barzel, A., Voit, R., Porteus, M., Kay, M. A. NATURE PUBLISHING GROUP. 2012: S234–S234
  • Expression determinants of mammalian argonaute proteins in mediating gene silencing NUCLEIC ACIDS RESEARCH Valdmanis, P. N., Gu, S., Schuermann, N., Sethupathy, P., Grimm, D., Kay, M. A. 2012; 40 (8): 3704-3713


    RNA interference occurs by two main processes: mRNA site-specific cleavage and non-cleavage-based mRNA degradation or translational repression. Site-specific cleavage is carried out by argonaute-2 (Ago2), while all four mammalian argonaute proteins (Ago1-Ago4) can carry out non-cleavage-mediated inhibition, suggesting that Ago1, Ago3 and Ago4 may have similar but potentially redundant functions. It has been observed that in mammalian tissues, expression of Ago3 and Ago4 is dramatically lower compared with Ago1; however, an optimization of the Ago3 and Ago4 coding sequences to include only the most common codon at each amino acid position was able to augment the expression of Ago3 and Ago4 to levels comparable to that of Ago1 and Ago2. Thus, we examined whether particular sequence features exist in the coding region of Ago3 and Ago4 that may prevent a high level of expression. Swapping specific sub-regions of wild-type and optimized Ago sequence identified the portion of the coding region (nucleotides 1-1163 for Ago-3 and 1-1494 for Ago-4) that is most influential for expression. This finding has implications for the evolutionary conservation of Ago proteins in the mammalian lineage and the biological role that potentially redundant Ago proteins may have.

    View details for DOI 10.1093/nar/gkr1274

    View details for Web of Science ID 000303333500040

    View details for PubMedID 22210886

  • Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B NEW ENGLAND JOURNAL OF MEDICINE Nathwani, A. C., Tuddenham, E. G., Rangarajan, S., Rosales, C., McIntosh, J., Linch, D. C., Chowdary, P., Riddell, A., Pie, A. J., Harrington, C., O'Beirne, J., Smith, K., Pasi, J., Glader, B., Rustagi, P., Ng, C. Y., Kay, M. A., Zhou, J., Spence, Y., Morton, C. L., Allay, J., Coleman, J., Sleep, S., Cunningham, J. M., Srivastava, D., Basner-Tschakarjan, E., Mingozzi, F., High, K. A., Gray, J. T., Reiss, U. M., Nienhuis, A. W., Davidoff, A. M. 2011; 365 (25): 2357-2365


    Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months.AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values.Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; number, NCT00979238.).

    View details for DOI 10.1056/NEJMoa1108046

    View details for Web of Science ID 000298320200002

    View details for PubMedID 22149959

  • Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration JOURNAL OF CLINICAL INVESTIGATION Malato, Y., Naqvi, S., Schuermann, N., Ng, R., Wang, B., Zape, J., Kay, M. A., Grimm, D., Willenbring, H. 2011; 121 (12): 4850-4860


    Recent evidence has contradicted the prevailing view that homeostasis and regeneration of the adult liver are mediated by self duplication of lineage-restricted hepatocytes and biliary epithelial cells. These new data suggest that liver progenitor cells do not function solely as a backup system in chronic liver injury; rather, they also produce hepatocytes after acute injury and are in fact the main source of new hepatocytes during normal hepatocyte turnover. In addition, other evidence suggests that hepatocytes are capable of lineage conversion, acting as precursors of biliary epithelial cells during biliary injury. To test these concepts, we generated a hepatocyte fate-tracing model based on timed and specific Cre recombinase expression and marker gene activation in all hepatocytes of adult Rosa26 reporter mice with an adenoassociated viral vector. We found that newly formed hepatocytes derived from preexisting hepatocytes in the normal liver and that liver progenitor cells contributed minimally to acute hepatocyte regeneration. Further, we found no evidence that biliary injury induced conversion of hepatocytes into biliary epithelial cells. These results therefore restore the previously prevailing paradigms of liver homeostasis and regeneration. In addition, our new vector system will be a valuable tool for timed, efficient, and specific loop out of floxed sequences in hepatocytes.

    View details for DOI 10.1172/JCI59261

    View details for Web of Science ID 000297599500032

    View details for PubMedID 22105172

  • Adeno-Associated Viral Vector Mediated Gene Transfer for Hemophilia B 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Nathwani, A. C., Tuddenham, E. G., Rangarajan, S., Rosales, C., McIntosh, J. H., Linch, D. C., Chowdary, P., Griffioen, A., Riddell, A., Pie, J., Harrington, C., O'Beirne, J., Smith, K., Pasi, J., Glader, B., Rustagi, P., Ng, C., Kay, M., Zhou, J., Spence, Y., Morton, C., Allay, J., Coleman, J., Sleep, S., Cunningham, J. M., Basner-Tschakarjan, E., Mingozzi, F., High, K. A., Gray, J. T., Reiss, U. M., Nienhuis, A. W., Davidoff, A. AMER SOC HEMATOLOGY. 2011: 4–5
  • THE MECHANISM OF miRNA AND siRNA ARGONAUTE LOADING IN MAMMALS 7th Annual Meeting of the Oligonucleotide-Therapeutics-Society Kay, M. A., Gu, S., Jin, L. MARY ANN LIEBERT INC. 2011: A50–A51
  • Double Knockdown of Prolyl Hydroxylase and Factor-Inhibiting Hypoxia-Inducible Factor With Nonviral Minicircle Gene Therapy Enhances Stem Cell Mobilization and Angiogenesis After Myocardial Infarction Annual Meeting of the American-Heart-Association Huang, M., Nguyen, P., Jia, F., Hu, S., Gong, Y., de Almeida, P. E., Wang, L., Nag, D., Kay, M. A., Giaccia, A. J., Robbins, R. C., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2011: S46–S54


    Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure.PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively.We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.

    View details for DOI 10.1161/CIRCULATIONAHA.110.014019

    View details for Web of Science ID 000294782800006

    View details for PubMedID 21911818

    View details for PubMedCentralID PMC3181087

  • Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gu, S., Jin, L., Zhang, F., Huang, Y., Grimm, D., Rossi, J. J., Kay, M. A. 2011; 108 (22): 9208-9213


    MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage- and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.

    View details for DOI 10.1073/pnas.1018023108

    View details for Web of Science ID 000291106200060

    View details for PubMedID 21576459

    View details for PubMedCentralID PMC3107324

  • State-of-the-art gene-based therapies: the road ahead NATURE REVIEWS GENETICS Kay, M. A. 2011; 12 (5): 316-328


    Improvements in the gene transfer vectors used in therapeutic trials have led to substantial clinical successes in patients with serious genetic conditions, such as immunodeficiency syndromes, blindness and some cancer types. Several barriers need to be overcome before this type of therapy becomes a widely accepted treatment for a broad group of medical diseases. However, recent progress in the field is finally realizing some of the promises made more than 20 years ago, providing optimism for additional successes in the near future.

    View details for DOI 10.1038/nrg2971

    View details for Web of Science ID 000289637600010

    View details for PubMedID 21468099

  • Minicircle DNA-based Gene Therapy Coupled With Immune Modulation Permits Long-term Expression of alpha-L-Iduronidase in Mice With Mucopolysaccharidosis Type I MOLECULAR THERAPY Osborn, M. J., McElmurry, R. T., Lees, C. J., DeFeo, A. P., Chen, Z., Kay, M. A., Naldini, L., Freeman, G., Tolar, J., Blazar, B. R. 2011; 19 (3): 450-460


    Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease characterized by mutations to the α-L-iduronidase (IDUA) gene resulting in inactivation of the IDUA enzyme. The loss of IDUA protein results in the progressive accumulation of glycosaminoglycans within the lysosomes resulting in severe, multi-organ system pathology. Gene replacement strategies have relied on the use of viral or nonviral gene delivery systems. Drawbacks to these include laborious production procedures, poor efficacy due to plasmid-borne gene silencing, and the risk of insertional mutagenesis. This report demonstrates the efficacy of a nonintegrating, minicircle (MC) DNA vector that is resistant to epigenetic gene silencing in vivo. To achieve sustained expression of the immunogenic IDUA protein we investigated the use of a tissue-specific promoter in conjunction with microRNA target sequences. The inclusion of microRNA target sequences resulted in a slight improvement in long-term expression compared to their absence. However, immune modulation by costimulatory blockade was required and permitted for IDUA expression in MPS I mice that resulted in the biochemical correction of pathology in all of the organs analyzed. MC gene delivery combined with costimulatory pathway blockade maximizes safety, efficacy, and sustained gene expression and is a new approach in the treatment of lysosomal storage disease.

    View details for DOI 10.1038/mt.2010.249

    View details for Web of Science ID 000287911600004

    View details for PubMedID 21081900

  • Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors NATURE PROTOCOLS Narsinh, K. H., Jia, F., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2011; 6 (1): 78-88


    Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

    View details for DOI 10.1038/nprot.2010.173

    View details for Web of Science ID 000285965000008

    View details for PubMedID 21212777

    View details for PubMedCentralID PMC3657506

  • A robust system for production of minicircle DNA vectors NATURE BIOTECHNOLOGY Kay, M. A., He, C., Chen, Z. 2010; 28 (12): 1287-U96


    Minicircle DNA vectors allow sustained transgene expression in quiescent cells and tissues. To improve minicircle production, we genetically modified Escherichia coli to construct a producer strain that stably expresses a set of inducible minicircle-assembly enzymes, ΦC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use minicircles in place of plasmids in mammalian transgene expression studies.

    View details for DOI 10.1038/nbt.1708

    View details for Web of Science ID 000285088400022

    View details for PubMedID 21102455

  • Early Clinical Trial Results Following Administration of a Low Dose of a Novel Self Complementary Adeno-Associated Viral Vector Encoding Human Factor IX In Two Subjects with Severe Hemophilia B 52nd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Nathwani, A., Tuddenham, E., Rosales, C., McIntosh, J., Riddell, A., Rustagi, P., Glader, B., Kay, M., Allay, J., Coleman, J., Sleep, S., High, K. A., Mingozzi, F., Gray, J. T., Reiss, U. M., Nienhuis, A. W., Davidoff, A. AMER SOC HEMATOLOGY. 2010: 114–14
  • Hyperactive Sleeping Beauty Transposase Enables Persistent Phenotypic Correction in Mice and a Canine Model for Hemophilia B MOLECULAR THERAPY Hausl, M. A., Zhang, W., Muether, N., Rauschhuber, C., Franck, H. G., Merricks, E. P., Nichols, T. C., Kay, M. A., Ehrhardt, A. 2010; 18 (11): 1896-1906


    Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.

    View details for DOI 10.1038/mt.2010.169

    View details for Web of Science ID 000283712400003

    View details for PubMedID 20717103

    View details for PubMedCentralID PMC2990515

  • Early clinical trial results following administration of a low dose of a novel self complementary adenohyphen;associated viral vector encoding human factor ix in two subjects with severe Haemophilia B 18th Annual Congress of the European-Society-of-Gene-and-Cell-Therapy Nathwani, A. C., Tuddenham, E. G., Rosales, C., Mcintosh, J., Riddell, A., Rustagi, P., Galder, B., Kay, M., ALLAY, J., Coleman, J., Sleep, S., High, K. A., Mingozzi, F., Gray, J., Reiss, U., NIENHUIS, A. W., Davidoff, A. M. MARY ANN LIEBERT INC. 2010: 1362–62
  • Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver JOURNAL OF CLINICAL INVESTIGATION Grimm, D., Wang, L., Lee, J. S., Schuermann, N., Gu, S., Boerner, K., Storm, T. A., Kay, M. A. 2010; 120 (9): 3106-3119


    shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.

    View details for DOI 10.1172/JCI43565

    View details for Web of Science ID 000281458800017

    View details for PubMedID 20697157

    View details for PubMedCentralID PMC2929739

  • FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl-CoA synthetase AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM Falcon, A., Doege, H., Fluitt, A., Tsang, B., Watson, N., Kay, M. A., Stahl, A. 2010; 299 (3): E384-E393


    Fatty acid transport protein (FATP)2, a member of the FATP family of fatty acid uptake mediators, has independently been identified as a hepatic peroxisomal very long-chain acyl-CoA synthetase (VLACS). Here we address whether FATP2 is 1) a peroxisomal enzyme, 2) a plasma membrane-associated long-chain fatty acid (LCFA) transporter, or 3) a multifunctional protein. We found that, in mouse livers, only a minor fraction of FATP2 localizes to peroxisomes, where it contributes to approximately half of the peroxisomal VLACS activity. However, total hepatic (V)LACS activity was not significantly affected by loss of FATP2, while LCFA uptake was reduced by 40%, indicating a more prominent role in hepatic LCFA uptake. This suggests FATP2 as a potential target for a therapeutic intervention of hepatosteatosis. Adeno-associated virus 8-based short hairpin RNA expression vectors were used to achieve liver-specific FATP2 knockdown, which significantly reduced hepatosteatosis in the face of continued high-fat feeding, concomitant with improvements in liver physiology, fasting glucose, and insulin levels. Based on our findings, we propose a model in which FATP2 is a multifunctional protein that shows subcellular localization-dependent activity and is a major contributor to peroxisomal (V)LACS activity and hepatic fatty acid uptake, suggesting FATP2 as a potential novel target for the treatment of nonalcoholic fatty liver disease.

    View details for DOI 10.1152/ajpendo.00226.2010

    View details for Web of Science ID 000281260500006

    View details for PubMedID 20530735

  • An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo EPIGENETICS & CHROMATIN Gracey, L. E., Chen, Z., Maniar, J. M., Valouev, A., Sidow, A., Kay, M. A., Fire, A. Z. 2010; 3


    The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA.We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active). This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent.These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

    View details for DOI 10.1186/1756-8935-3-13

    View details for Web of Science ID 000283775900001

    View details for PubMedID 20594331

  • Prevention of spontaneous bleeding in dogs with haemophilia A and haemophilia B 10th Novo Nordisk Symposium on Haemostasis Management Nichols, T. C., Raymer, R. A., Franck, H. W., Merricks, E. P., Bellinger, D. A., Defriess, N., Margaritis, P., Arruda, V. R., Kay, M. A., High, K. A. WILEY-BLACKWELL. 2010: 19–23


    Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

    View details for Web of Science ID 000276947800005

    View details for PubMedID 20586797

    View details for PubMedCentralID PMC3101869

  • Human tRNA-derived small RNAs in the global regulation of RNA silencing RNA-A PUBLICATION OF THE RNA SOCIETY Haussecker, D., Huang, Y., Lau, A., Parameswaran, P., Fire, A. Z., Kay, M. A. 2010; 16 (4): 673-695


    Competition between mammalian RNAi-related gene silencing pathways is well documented. It is therefore important to identify all classes of small RNAs to determine their relationship with RNAi and how they affect each other functionally. Here, we identify two types of 5'-phosphate, 3'-hydroxylated human tRNA-derived small RNAs (tsRNAs). tsRNAs differ from microRNAs in being essentially restricted to the cytoplasm and in associating with Argonaute proteins, but not MOV10. The first type belongs to a previously predicted Dicer-dependent class of small RNAs that we find can modestly down-regulate target genes in trans. The 5' end of type II tsRNA was generated by RNaseZ cleavage downstream from a tRNA gene, while the 3' end resulted from transcription termination by RNA polymerase III. Consistent with their preferential association with the nonslicing Argonautes 3 and 4, canonical gene silencing activity was not observed for type II tsRNAs. The addition, however, of an oligonucleotide that was sense to the reporter gene, but antisense to an overexpressed version of the type II tsRNA, triggered robust, >80% gene silencing. This correlated with the redirection of the thus reconstituted fully duplexed double-stranded RNA into Argonaute 2, whereas Argonautes 3 and 4 were skewed toward less structured small RNAs, particularly single-strand RNAs. We observed that the modulation of tsRNA levels had minor effects on the abundance of microRNAs, but more pronounced changes in the silencing activities of both microRNAs and siRNAs. These findings support that tsRNAs are involved in the global control of small RNA silencing through differential Argonaute association, suggesting that small RNA-mediated gene regulation may be even more finely regulated than previously realized.

    View details for DOI 10.1261/rna.2000810

    View details for Web of Science ID 000275951000004

    View details for PubMedID 20181738

    View details for PubMedCentralID PMC2844617

  • Adeno-Associated Virus Gene Repair Corrects a Mouse Model of Hereditary Tyrosinemia In Vivo HEPATOLOGY Paulk, N. K., Wursthorn, K., Wang, Z., Finegold, M. J., Kay, M. A., Grompe, M. 2010; 51 (4): 1200-1208


    Adeno-associated virus (AAV) vectors are ideal for performing gene repair due to their ability to target multiple different genomic loci, low immunogenicity, capability to achieve targeted and stable expression through integration, and low mutagenic and oncogenic potential. However, many handicaps to gene repair therapy remain. Most notable is the low frequency of correction in vivo. To date, this frequency is too low to be of therapeutic value for any disease. To address this, a point-mutation-based mouse model of the metabolic disease hereditary tyrosinemia type I was used to test whether targeted AAV integration by homologous recombination could achieve high-level stable gene repair in vivo. Both neonatal and adult mice were treated with AAV serotypes 2 and 8 carrying a wild-type genomic sequence for repairing the mutated Fah (fumarylacetoacetate hydrolase) gene. Hepatic gene repair was quantified by immunohistochemistry and supported with reverse transcription polymerase chain reaction and serology for functional correction parameters. Successful gene repair was observed with both serotypes but was more efficient with AAV8. Correction frequencies of up to 10(-3) were achieved and highly reproducible within typical dose ranges. In this model, repaired hepatocytes have a selective growth advantage and are thus able to proliferate to efficiently repopulate mutant livers and cure the underlying metabolic disease. Conclusion: AAV-mediated gene repair is feasible in vivo and can functionally correct an appropriate selection-based metabolic liver disease in both adults and neonates.

    View details for DOI 10.1002/hep.23481

    View details for Web of Science ID 000276538100016

    View details for PubMedID 20162619

  • A nonviral minicircle vector for deriving human iPS cells NATURE METHODS Jia, F., Wilson, K. D., Sun, N., Gupta, D. M., Huang, M., Li, Z., Panetta, N. J., Chen, Z. Y., Robbins, R. C., Kay, M. A., Longaker, M. T., Wu, J. C. 2010; 7 (3): 197-U46


    Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem cells (iPSCs). We report the use of 'minicircle' DNA, a vector type that is free of bacterial DNA and capable of high expression in cells, for this purpose. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells.

    View details for DOI 10.1038/NMETH.1426

    View details for Web of Science ID 000275058200018

    View details for PubMedID 20139967

    View details for PubMedCentralID PMC2892897

  • miR-122 Continues to Blaze the Trail for MicroRNA Therapeutics MOLECULAR THERAPY Haussecker, D., Kay, M. A. 2010; 18 (2): 240-242

    View details for DOI 10.1038/mt.2009.313

    View details for Web of Science ID 000274447300003

    View details for PubMedID 20125164

  • Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems PLOS PATHOGENS Parameswaran, P., Sklan, E., Wilkins, C., Burgon, T., Samuel, M. A., Lu, R., Ansel, K. M., Heissmeyer, V., Einav, S., Jackson, W., Doukas, T., Paranjape, S., Polacek, C., dos Santos, F. B., Jalili, R., Babrzadeh, F., Gharizadeh, B., Grimm, D., Kay, M., Koike, S., Sarnow, P., Ronaghi, M., Ding, S., Harris, E., Chow, M., Diamond, M. S., Kirkegaard, K., Glenn, J. S., Fire, A. Z. 2010; 6 (2)


    We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from "vanishingly rare" (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs "miRNAs"). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3' overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.

    View details for DOI 10.1371/journal.ppat.1000764

    View details for Web of Science ID 000275295900016

    View details for PubMedID 20169186

    View details for PubMedCentralID PMC2820531

  • Low-level shRNA Cytotoxicity Can Contribute to MYC-induced Hepatocellular Carcinoma in Adult Mice MOLECULAR THERAPY Beer, S., Bellovin, D. I., Lee, J. S., Komatsubara, K., Wang, L. S., Koh, H., Boerner, K., Storm, T. A., Davis, C. R., Kay, M. A., Felsher, D. W., Grimm, D. 2010; 18 (1): 161-170


    Short hairpin RNAs (shRNAs) have emerged as a novel therapeutic modality, but there is increasing concern over nonspecific effects in vivo. Here, we used viral vectors to express shRNAs against endogenous p53 in livers of conditional MYC-transgenic mice. As expected, the shRNAs silenced hepatic p53 and accelerated liver tumorigenesis when MYC was concurrently expressed. Surprisingly, various irrelevant control shRNAs similarly induced a rapid onset of tumorigenesis, comparable to carbon tetrachloride (CCl4), a potent carcinogen. We found that even marginal shRNA doses can already trigger histologically detectable hepatoxicity and increased hepatocyte apoptosis. Moreover, we noted that shRNA expression globally dysregulated hepatic microRNA (miRNA) expression, and that shRNA levels and activity further increased in the presence of MYC. In MYC-expressing transgenic mice, the marginal shRNA-induced liver injury sufficed to further stimulate hepatocellular division that was in turn associated with markedly increased expression of the mitotic cyclin B1. Hence, even at low doses, shRNAs can cause low-level hepatoxicity that can facilitate the ability of the MYC oncogene to induce liver tumorigenesis. Our data warrant caution regarding the possible carcinogenic potential of shRNAs when used as clinical agent, particularly in circumstances where tissues are genetically predisposed to cellular transformation and proliferation.

    View details for DOI 10.1038/mt.2009.222

    View details for Web of Science ID 000274447200023

    View details for PubMedID 19844192

    View details for PubMedCentralID PMC2839214

  • How do miRNAs mediate translational repression? Silence Gu, S., Kay, M. A. 2010; 1 (1): 11-?


    Micro(mi)RNAs regulate gene expression by what are believed to be related but separate mechanistic processes. The relative contribution that each process plays, their mechanistic overlap, and the degree by which they regulate complex genetic networks is still being unraveled. One process by which miRNAs inhibit gene expression occurs through translational repression. In recent years, there has been a plethora of studies published, which have resulted in various molecular models of how miRNAs impair translation. At first evaluation, it appears that these models are quite different and incompatible with one another. In this paper, we focus on possible explanations for the various interpretations of these data sets, and provide a model that we believe is consistent with many of the observations published to date.

    View details for DOI 10.1186/1758-907X-1-11

    View details for PubMedID 20459656

  • Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication RNA-A PUBLICATION OF THE RNA SOCIETY Cao, D., Haussecker, D., Huang, Y., Kay, M. A. 2009; 15 (11): 1971-1979


    Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated with FLAG-HDAg-S, many of which had predicted roles in RNA metabolism. The biological relevance of this screen was strongly supported by the identification of nine out of the 12 subunits of the RNA polymerase II complex thought to mediate HDV replication. To further investigate the significance of these factors for HDV replication, we selected 65 proteins to look for factors that would also affect the accumulation of HDV RNA following siRNA knockdown. Fifteen and three factors were found to regulate HDV RNA accumulation negatively and positively, respectively, upon RNAi knockdown. Our results provide a valuable resource for future research to advance our mechanistic understanding of HDV replication and RNA-directed transcription in mammalian cells.

    View details for DOI 10.1261/rna.1782209

    View details for Web of Science ID 000270851000004

    View details for PubMedID 19776158

  • Novel Minicircle Vector for Gene Therapy in Murine Myocardial Infarction 81st Annual Scientific Session of the American-Heart-Association Huang, M., Chen, Z., Hu, S., Jia, F., Li, Z., Hoyt, G., Robbins, R. C., Kay, M. A., Wu, J. C. LIPPINCOTT WILLIAMS & WILKINS. 2009: S230–S237


    Conventional plasmids for gene therapy produce low-level and short-term gene expression. In this study, we develop a novel nonviral vector that robustly and persistently expresses the hypoxia-inducible factor-1 alpha (HIF-1alpha) therapeutic gene in the heart, leading to functional benefits after myocardial infarction.We first created minicircles (MC) carrying double-fusion reporter gene consisting of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) for noninvasive measurement of transfection efficiency. Mouse C2C12 myoblasts and normal FVB/N mice were used for in vitro and in vivo confirmation, respectively. Bioluminescence imaging showed stable MC gene expression in the heart for >12 weeks and the activity level was 5.6+/-1.2-fold stronger than regular plasmid at day 4 (P<0.01). Next, we created MC carrying HIF-1alpha (MC-HIF-1alpha) therapeutic gene for treatment of myocardial infarction. Adult FVB/N mice underwent left anterior descending ligation and were injected intramyocardially with: (1) MC-HIF-1alpha; (2) regular plasmid carrying HIF-1alpha (PL-HIF-1alpha) as positive control; and (3) PBS as negative control (n=10/group). Echocardiographic study showed a significantly greater improvement of left ventricular ejection fraction in the MC group (51.3%+/-3.6%) compared to regular plasmid group (42.3%+/-4.1%) and saline group (30.5%+/-2.8%) at week 4 (P<0.05 for both). Histology demonstrated increased neoangiogenesis in both treatment groups. Finally, Western blot showed MC express >50% higher HIF-1alpha level than regular plasmid.Taken together, this is the first study to our knowledge to demonstrate that MC can significantly improve transfection efficiency, duration of transgene expression, and cardiac contractility. Given the serious drawbacks associated with most viral vectors, we believe this novel nonviral vector can be of great value for cardiac gene therapy protocols.

    View details for DOI 10.1161/CIRCULATIONAHA.108.841155

    View details for Web of Science ID 000269773000033

    View details for PubMedID 19752373

    View details for PubMedCentralID PMC3163107

  • FACING THE BLEEDING OBVIOUS: AAV-MEDIATED GENE THERAPY FOR HAEMOPHILIA B 6th Meeting of the Australasian-Gene-Therapy-Society Rasko, J. E., High, K., Tigges, M., Manno, C., Sabatino, D., Dake, M., McDonnell, J. W., Razavi, M., Arruda, V., Herzog, R., Rustagi, P., Sommer, J., Ragni, M., Konkle, B., Lessard, R., Luk, A., Glader, B., Pierce, G., Couto, L., Jiang, H., Kay, M. JOHN WILEY & SONS LTD. 2009: 843–43
  • Biological basis for restriction of microRNA targets to the 3 ' untranslated region in mammalian mRNAs NATURE STRUCTURAL & MOLECULAR BIOLOGY Gu, S., Jin, L., Zhang, F., Sarnow, P., Kay, M. A. 2009; 16 (2): 144-150


    MicroRNAs (miRNAs) interact with target sites located in the 3' untranslated regions (3' UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3' UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

    View details for DOI 10.1038/nsmb.1552

    View details for Web of Science ID 000263286600010

    View details for PubMedID 19182800

    View details for PubMedCentralID PMC2713750

  • Gene Transfer to Mouse Heart and Skeletal Muscles Using a Minicircle Expressing Human Vascular Endothelial Growth Factor JOURNAL OF CARDIOVASCULAR PHARMACOLOGY Stenler, S., Andersson, A., Simonson, O. E., Lundin, K. E., Chen, Z., Kay, M. A., Smith, C. I., Sylven, C., Blomberg, P. 2009; 53 (1): 18-23


    : Gene transfer to heart muscle is a promising modality to treat ischemic heart disease. However, current vectors are inefficient and need to be improved. A novel vector system that shows great promise is the minicircle (MC) vector being smaller than conventional plasmid vectors and devoid of bacterial sequences.: To study gene transfer of MC DNA, expressing the human vascular endothelial growth factor (hVEGF), to mouse heart and skeletal muscles and to compare it with one of the efficient plasmids used in cardiovascular trials, the phVEGF165 containing the same expression cassette as the MC.: The MC and the phVEGF165 plasmid show similar expression patterns both in vitro and in mouse heart and skeletal muscle studies in vivo on molar basis (equal expression in heart 24 hours, 0.9 fold lower expression from MC in heart and 1.9 fold higher in skeletal muscle at 7 days), whereas on weight basis the MC construct was more efficient in skeletal muscle (5.6 fold higher expression, P < 0.05), and at least as efficient in heart (1.6 fold higher expression).: The gene expression is similar in the 2 vector systems, so the smaller size and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene therapy.

    View details for Web of Science ID 000263316100003

    View details for PubMedID 19129741

  • A rapid protocol for construction and production of high-capacity adenoviral vectors NATURE PROTOCOLS Jager, L., Hausl, M. A., Rauschhuber, C., Wolf, N. M., Kay, M. A., Ehrhardt, A. 2009; 4 (4): 547-564


    High-capacity adenoviral vectors (HC-AdVs) lacking all viral coding sequences were shown to result in long-term transgene expression and phenotypic correction in small and large animal models. It has been established that HC-AdVs show significantly reduced toxicity profiles compared with early-generation adenoviral vectors. Furthermore, with capsid-modified HC-AdV becoming available, we are just starting to understand the full potential of this vector system. However, for many researchers, the wide-scale use of HC-AdV is hampered by labor-intensive and complex production procedures. Herein, we provide a feasible and detailed protocol for efficient generation of HC-AdV. We introduce an efficient cloning strategy for the generation of recombinant HC-AdV vector genomes. Infection and amplification of the HC-AdV are performed in a spinner culture system. For purification, we routinely apply cesium chloride gradients. Finally, we describe various methods for establishing vector titers. Generation of high-titer HC-AdV can be achieved in 3 weeks.

    View details for DOI 10.1038/nprot.2009.4

    View details for Web of Science ID 000265782100011

    View details for PubMedID 19373227

  • Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic MOLECULAR THERAPY Giering, J. C., Grimm, D., Storm, T. A., Kay, M. A. 2008; 16 (9): 1630-1636


    It has been observed that overexpression of some short-hairpin RNAs (shRNAs) can induce acute cytotoxicity. This has raised concerns about the safety of using RNA interference (RNAi) technology as a potential therapeutic tool. We have sought to address this challenge of expression control by developing a mono-cistronic vector for the tissue-specific expression of an shRNA from a liver-derived polymerase (pol) II promoter. This new construct efficiently induces target silencing in hepatoma cells in vitro and in mouse livers in vivo. In order to demonstrate the therapeutic potential and improved safety of this approach, we selected an shRNA targeting the envelope surface antigen (sAg) of hepatitis B virus (HBV), which is among the most toxic when expressed from the commonly used U6 promoter. Packaging it as a double-stranded DNA into an adeno-associated virus (AAV) pseudotype 8 and delivering it at a high particle dose (1 x 10(12)) to HBV transgenic mice resulted in the stable reduction of serum sAg to 85% of starting levels, without any concomitant sign of liver damage. With this improved tolerability, the liver-specific pol II shRNA expression persisted for more than one year after the injection. We conclude that this pol II shRNA expression system combined with a potent delivery vector represents an effective alternative to either U6-based strategies or systems that achieve tissue specificity through the use of additional elements.

    View details for DOI 10.1038/mt.2008.144

    View details for Web of Science ID 000258782400018

    View details for PubMedID 18665161

  • Silencing of hepatic fatty acid transporter protein 5 in vivo reverses diet-induced non-alcoholic fatty liver disease and improves Hyperglycemia JOURNAL OF BIOLOGICAL CHEMISTRY Doege, H., Grimm, D., Falcon, A., Tsang, B., Storm, T. A., Xu, H., Ortegon, A. M., Kazantzis, M., Kay, M. A., Stahl, A. 2008; 283 (32): 22186-22192


    Non-alcoholic fatty liver disease is a serious health problem linked to obesity and type 2 diabetes. To investigate the biological outcome and therapeutic potential of hepatic fatty acid uptake inhibition, we utilized an adeno-associated virus-mediated RNA interference technique to knock down the expression of hepatic fatty acid transport protein 5 in vivo prior to or after establishing non-alcoholic fatty liver disease in mice. Using this approach, we demonstrate here the ability to achieve specific, non-toxic, and persistent knockdown of fatty acid transport protein 5 in mouse livers from a single adeno-associated virus injection, resulting in a marked reduction of hepatic dietary fatty acid uptake, reduced caloric uptake, and concomitant protection from diet-induced non-alcoholic fatty liver disease. Importantly, knockdown of fatty acid transport protein 5 was also able to reverse already established non-alcoholic fatty liver disease, resulting in significantly improved whole-body glucose homeostasis. Thus, continued activity of hepatic fatty acid transport protein 5 is required to sustain caloric uptake and fatty acid flux into the liver during high fat feeding and may present a novel avenue for the treatment of non-alcoholic fatty liver disease.

    View details for DOI 10.1074/jbc.M803510200

    View details for Web of Science ID 000258114700038

    View details for PubMedID 18524776

  • Radioprotection in vitro and in vivo by minicircle plasmid carrying the human manganese superoxide dismutase transgene HUMAN GENE THERAPY Zhang, X., Epperly, M. W., Kay, M. A., Chen, Z., Dixon, T., Franicola, D., Greenberger, B. A., Komanduri, P., Greenberger, J. S. 2008; 19 (8): 820-826


    Manganese superoxide dismutase plasmid liposomes (MnSOD-PL) confer organ-specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 microg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 +/- 0.5 U/mg compared with 2.7 +/- 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 +/- 0.2 and n = 4.6 +/- 0.2, respectively, compared with 1.5 +/- 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

    View details for DOI 10.1089/hum.2007.141

    View details for Web of Science ID 000259034300006

    View details for PubMedID 18699723

  • Capped small RNAs and MOV10 in human hepatitis delta virus replication NATURE STRUCTURAL & MOLECULAR BIOLOGY Haussecker, D., Cao, D., Huang, Y., Parameswaran, P., Fire, A. Z., Kay, M. A. 2008; 15 (7): 714-721


    The evolutionary origin of human hepatitis delta virus (HDV) replication by RNA-directed transcription is unclear. Here we identify two species of 5'-capped, approximately 18-25-nucleotide small RNAs. One was of antigenomic polarity, corresponding to the 5' end of hepatitis delta antigen (HDAg) mRNA, and interacted with HDAg and RNA polymerase II (Pol II), whereas the other mapped to a structurally analogous region on the genomic RNA hairpin. An HDAg-interaction screen indicated that HDAg interacts with MOV10, the human homolog of the Arabidopsis thaliana RNA amplification factor gene SDE3 and Drosophila melanogaster RISC-maturation factor gene Armitage (armi). MOV10 knockdown inhibited HDV replication, but not HDAg mRNA translation, further supporting a role for MOV10 in RNA-directed transcription. Together, our studies define RNA hairpins as critical elements for the initiation of HDV-related, RNA-directed transcription. The identification of capped small RNAs and the involvement of MOV10 in HDV replication further suggest a conserved mechanism related to RNA-directed transcription in lower eukaryotes.

    View details for DOI 10.1038/nsmb.1440

    View details for Web of Science ID 000257412500014

    View details for PubMedID 18552826

  • In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses JOURNAL OF VIROLOGY Grimm, D., Lee, J. S., Wang, L., Desai, T., Akache, B., Storm, T. A., Kay, M. A. 2008; 82 (12): 5887-5911


    Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.

    View details for DOI 10.1128/JVI.00254-08

    View details for Web of Science ID 000256453600021

    View details for PubMedID 18400866

    View details for PubMedCentralID PMC2395137

  • The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver MOLECULAR THERAPY McCaffrey, A. P., Fawcett, P., Nakai, H., McCaffrey, R. L., Ehrhardt, A., Pham, T. T., Pandey, K., Xu, H., Feuss, S., Storm, T. A., Kay, M. A. 2008; 16 (5): 931-941


    Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction.

    View details for DOI 10.1038/mt.2008.37

    View details for Web of Science ID 000255516600022

    View details for PubMedID 18388926

  • Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation MOLECULAR THERAPY Chen, Z., Riu, E., He, C., Xu, H., Kay, M. A. 2008; 16 (3): 548-556


    Minicircle DNA vectors devoid of plasmid bacterial backbone, (BB) DNAs, are transcriptionally persistent, whereas their parent plasmid counterparts are silenced in the liver. In this study we establish that circular plasmid BB provided in trans did not silence a transgene expression cassette in vivo, further confirming our previous conclusions that the covalent attachment of the plasmid BB to the expression cassette is required for DNA silencing. Given the high concentration of CpG dinucleotides in the plasmid BB, we investigated the role of DNA methylation on transgene silencing in vivo. The presence or absence of methylation in CpG motifs in routine plasmid BBs had no significant effect on transcriptional silencing. Furthermore, the removal of the CpG motifs from the BB did not ameliorate transcriptional silencing. Transgene silencing was partially inhibited when two tandem copies of the chicken cHS4 insulator at each end of a routine plasmid vector were used. These results are consistent with the idea that the transcriptional repression observed with plasmid DNA vectors in the liver is caused by formation of repressive heterochromatin on the plasmid DNA backbone, which then spreads and inactivates the transgene in cis, and that CpG content or methylation has little or no influence in the process.

    View details for DOI 10.1038/

    View details for Web of Science ID 000253548100017

    View details for PubMedID 18253155

  • Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells HEPATOLOGY Streetz, K. L., Doyonnas, R., Grimm, D., Jenkins, D. D., Fuess, S., Perryman, S., Lin, J., Trautwein, C., Shizuru, J., Blau, H., Sylvester, K. G., Kay, M. A. 2008; 47 (2): 706-718


    The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT.The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.

    View details for DOI 10.1002/hep.22012

    View details for Web of Science ID 000252939500040

    View details for PubMedID 18220289

  • Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? JOURNAL OF CLINICAL INVESTIGATION Grimm, D., Kay, M. A. 2007; 117 (12): 3633-3641


    With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising biotherapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near.

    View details for DOI 10.1172/JCI34129

    View details for Web of Science ID 000251396600007

    View details for PubMedID 18060021

  • Cis-acting gene regulatory activities in the terminal regions of Sleeping Beauty DNA transposon-based vectors HUMAN GENE THERAPY Moldt, B., Yant, S. R., Andersen, P. R., Kay, M. A., Mikkelsen, J. G. 2007; 18 (12): 1193-1204


    Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of nonviral integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles. Because of concerns related to mutagenesis and/or activation of cellular genes by integrating vectors, much attention has been paid to integration site preferences and the ability of vectors to influence expression of neighboring genes. Here, we test the hypothesis that terminal repeats of transposons carry cis-acting regulatory sequences. In transient gene expression studies, we demonstrate that the inverted repeats of SB direct gene expression in HeLa cells to levels that are 3-fold higher than in promoter-deficient controls. Inverted repeats pointing toward the transposon center consistently facilitate the highest levels of activity in a number of cell lines. We show that transposon sequences flanking the inverted repeats of SB are required for positive effects on gene expression and, moreover, that these regions contain both stimulatory and inhibitory cis-acting elements. In the context of an integrated SB vector the regulatory activities of the transposon termini are sufficient to drive expression of selectable marker genes carried by the transposon, indicating that opposing transcriptional activities originating from the transposon termini may influence expression of its genetic cargo. Finally, detection of regulatory properties of the terminal repeats of the active Tc3 element from Caenorhabditis elegans leads to the suggestion that transcriptional activities of the inverted repeats are conserved among Tc1/mariner transposons in nature. Our data suggest that SB-based gene vectors may carry ancient properties of self-regulation with potential relevance for SB-directed therapeutic gene transfer.

    View details for DOI 10.1089/hum.2007.099

    View details for Web of Science ID 000251699300003

    View details for PubMedID 17988194

  • Postintegrative gene silencing within the Sleeping Beauty transposition system MOLECULAR AND CELLULAR BIOLOGY Garrison, B. S., Yant, S. R., Mikkelsen, J. G., Kay, M. A. 2007; 27 (24): 8824-8833


    The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates approximately 20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2'-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.

    View details for DOI 10.1128/MCB.00498-07

    View details for Web of Science ID 000251527300035

    View details for PubMedID 17938204

  • Characterization of the relationship of AAV capsid domain swapping to liver transduction efficiency MOLECULAR THERAPY Shen, X., Storm, T., Kay, M. A. 2007; 15 (11): 1955-1962


    Recombinant adeno-associated virus (AAV) vectors show promise for use in gene therapy. For liver-targeted gene transfer in animals, AAV vectors pseudotyped with the AAV serotype 8 (AAV8) capsid have definite advantages over the widely used but less efficient serotype AAV2, even though the capsid amino acid sequences are 82% conserved. To demonstrate the mechanism behind the higher liver transduction efficiency associated with AAV8 capsids, we adopted a domain-swapping strategy that would generate 27 chimeric capsid genes containing exchanged domains between AAV2 and AAV8. The resulting chimeric capsids were then used to package AAV genomes with a liver-specific human coagulation factor IX (hFIX) expression cassette. By comparing the transduction efficiencies between vectors pseudotyped with chimeric, AAV2 and AAV8 capsids, we found that the more efficient liver transduction achieved by AAV8 was closely related to the components of its interstrand Loop IV domain, particularly the subloops 1 and 4. These subloops are exposed on opposite sides of a threefold proximal peak on the virion surface, which may function as a critical structural determinant for AAV transduction. Because a single specific peptide component could not explain all the observed differences in the transduction parameters, we suggest that important subloop regions require interaction with other portions of the capsid for their functioning.

    View details for DOI 10.1038/

    View details for Web of Science ID 000250382400015

    View details for PubMedID 17726459

  • DNA palindromes with a modest arm length of greater than or similar to s20 base pairs are a significant target for recombinant adeno-associated virus vector integration in the liver, muscles, and heart in mice JOURNAL OF VIROLOGY Inagaki, K., Lewis, S. M., Wu, X., Ma, C., Munroe, D. J., Fuess, S., Storm, T. A., Kay, M. A., Nakai, H. 2007; 81 (20): 11290-11303


    Our previous study has shown that recombinant adeno-associated virus (rAAV) vector integrates preferentially in genes, near transcription start sites and CpG islands in mouse liver (H. Nakai, X. Wu, S. Fuess, T. A. Storm, D. Munroe, E. Montini, S. M. Burgess, M. Grompe, and M. A. Kay, J. Virol. 79:3606-3614, 2005). However, the previous method relied on in vivo selection of rAAV integrants and could be employed for the liver but not for other tissues. Here, we describe a novel method for high-throughput rAAV integration site analysis that does not rely on marker gene expression, selection, or cell division, and therefore it can identify rAAV integration sites in nondividing cells without cell manipulations. Using this new method, we identified and characterized a total of 997 rAAV integration sites in mouse liver, skeletal muscle, and heart, transduced with rAAV2 or rAAV8 vector. The results support our previous observations, but notably they have revealed that DNA palindromes with an arm length of greater, similar 20 bp (total length, greater, similar 40 bp) are a significant target for rAAV integration. Up to approximately 30% of total integration events occurred in the vicinity of DNA palindromes with an arm length of greater, similar 20 bp. Considering that DNA palindromes may constitute fragile genomic sites, our results support the notion that rAAV integrates at chromosomal sites susceptible to breakage or preexisting breakage sites. The use of rAAV to label fragile genomic sites may provide an important new tool for probing the intrinsic source of ongoing genomic instability in various tissues in animals, studying DNA palindrome metabolism in vivo, and understanding their possible contributions to carcinogenesis and aging.

    View details for DOI 10.1128/JVI.00963-07

    View details for Web of Science ID 000250019400044

    View details for PubMedID 17686840

  • Rapid and stable knockdown of an endogenous gene in retinal pigment epithelium HUMAN GENE THERAPY Paskowitz, D. M., Greenberg, K. P., Yasumura, D., Grimm, D., Yang, H., Duncan, J. L., Kay, M. A., LaVail, M. M., Flannery, J. G., Vollrath, D. 2007; 18 (10): 871-880


    The selective silencing of target genes in specific cell types by RNA interference (RNAi) represents a powerful approach both to gene therapy of dominantly active mutant alleles, and to the investigation of normal gene function in animal models in vivo. We established a simple and versatile in vitro method for screening the efficacy of DNA-based short hairpin RNAs (shRNAs), and identified a highly effective shRNA targeting basic fibroblast growth factor (bFGF), a gene thought to play important roles in endogenous neuroprotective responses in the rat retina. We used two viral vectors, based on lentivirus and adeno-associated virus (AAV), to deliver shRNAs and silence bFGF in retinal pigment epithelial cells in vivo. The AAV experiments made use of a "stabilized double-stranded" version of these vectors with rapid onset of gene expression. In the rat retinal pigment epithelium, shRNAs delivered by either vector reduced bFGF immunoreactivity to undetectable levels in transduced cells, whereas a nonfunctional control construct incorporating a two-base pair mutation had no measurable effect on bFGF expression. Silencing commenced within a few days after injection of virus and remained stable throughout the period of observation, as long as 60 days. Viral delivery of RNAi constructs offers a powerful and versatile approach for both gene therapy and the analysis of fundamental questions in retinal biology.

    View details for DOI 10.1089/hum.2007.065

    View details for Web of Science ID 000250339500001

    View details for PubMedID 17892416

  • Liver directed AAV-mediated homologous recombination is largely independent of serotype 58th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases Wursthorn, K., Paulk, N., Storm, T., Finegold, M. J., Kay, M., Grompe, M. WILEY-BLACKWELL. 2007: 887A–887A
  • AAV vectors and turnorigenicity NATURE BIOTECHNOLOGY Kay, M. A. 2007; 25 (10): 1111-1113

    View details for DOI 10.1038/nbt1007-1111

    View details for Web of Science ID 000250226600020

    View details for PubMedID 17921994

  • The role of DNA-PKcs and artemis in opening viral DNA hairpin termini in various tissues in mice JOURNAL OF VIROLOGY Inagaki, K., Ma, C., Storm, T. A., Kay, M. A., Nakai, H. 2007; 81 (20): 11304-11321


    A subset of cellular DNA hairpins at double-strand breaks is processed by DNA-dependent protein kinase catalytic subunit (DNA-PKcs)- and Artemis-associated endonuclease. DNA hairpin termini of adeno-associated virus (AAV) are processed by DNA repair machinery; however, how and what cellular factors are involved in the process remain elusive. Here, we show that DNA-PKcs and Artemis open AAV inverted terminal repeat (ITR) hairpin loops in a tissue-dependent manner. We investigated recombinant AAV (rAAV) genome metabolism in various tissues of DNA-PKcs- or Artemis-proficient or -deficient mice. In the absence of either factor, ITR hairpin opening was impaired, resulting in accumulation of double-stranded linear rAAV genomes capped with covalently closed hairpins at termini. The 5' end of 3-base hairpin loops of the ITR was the primary target for DNA-PKcs- and Artemis-mediated cleavage. In the muscle, heart, and kidney, DNA-PKcs- and Artemis-dependent hairpin opening constituted a significant pathway, while in the liver, undefined alternative pathways effectively processed hairpins. In addition, our study revealed a Holliday junction resolvase-like activity in the liver that cleaved T-shaped ITR hairpin shoulders by making nicks at diametrically opposed sites. Thus, our approach furthers our understanding of not only rAAV biology but also fundamental DNA repair systems in various tissues of living animals.

    View details for DOI 10.1128/JVI.01225-07

    View details for Web of Science ID 000250019400045

    View details for PubMedID 17686847

  • Distinct pathways of genomic progression to benign and malignant tumors of the liver PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tward, A. D., Jones, K. D., Yant, S., Cheung, S. T., Fan, S. T., Chen, X., Kay, M. A., Wang, R., Bishop, J. M. 2007; 104 (37): 14771-14776


    We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

    View details for DOI 10.1073/pnas.0706578104

    View details for Web of Science ID 000249513000041

    View details for PubMedID 17785413

    View details for PubMedCentralID PMC1964540

  • Robust expansion of human hepatocytes in Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice NATURE BIOTECHNOLOGY Azuma, H., Paulk, N., Ranade, A., Dorrell, C., Al-Dhalimy, M., Ellis, E., Strom, S., Kay, M. A., Finegold, M., Grompe, M. 2007; 25 (8): 903-910


    Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinase-expressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.

    View details for DOI 10.1038/nbt1326

    View details for Web of Science ID 000248725800029

    View details for PubMedID 17664939

  • Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo MOLECULAR THERAPY Riu, E., Chen, Z., Xu, H., He, C., Kay, M. A. 2007; 15 (7): 1348-1355


    One of the major obstacles to success in non-viral gene therapy is transcriptional silencing of the DNA vector. The mechanisms underlying gene silencing/repression in mammalian cells are complex and remain unclear. Because changes in chromatin structure and, in particular, histone modifications are involved in transcriptional regulation of endogenous genes, we hypothesized that changes in the pattern of histone modifications were related to the observed transcriptional silencing of exogenous DNA vectors. We used antibodies against specific modified histones to perform chromatin immunoprecipitation (ChIP) analyses on liver lysates from mice transfected with two types of plasmids: (i) DNA minicircles (MCs) devoid of bacterial plasmid backbone DNA, which showed marked persistence of transgene expression, and (ii) their parental plasmids, which were silenced over time. Silencing of the transgene from the parental vectors was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the MC DNA was consistent with euchromatin. Our data indicates that (i) episomal vectors undergo chromatinization in vivo, and (ii) both persistence and silencing of transgene expression are associated with specific histone modifications.

    View details for DOI 10.1038/

    View details for Web of Science ID 000247646400022

    View details for PubMedID 17457320

  • Correction of DNA protein kinase deficiency by spliceosome-mediated RNA trans-splicing and Sleeping Beauty transposon delivery MOLECULAR THERAPY Zayed, H., Xia, L., Yerich, A., Yant, S. R., Kay, M. A., Puttaraju, M., McGarrity, G. J., Wiest, D. L., McIvor, R. S., Tolar, J., Blazar, B. R. 2007; 15 (7): 1273-1279


    Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology for the repair of defective pre-messenger RNA (pre-mRNA) molecules. It is especially useful in the treatment of genetic disorders involving large genes. Although viral vectors have been used for achieving long-lasting expression of trans-splicing molecules, the immunogenicity and suboptimal safety profiles associated with viral-based components could limit the widespread application of SMaRT in the repair of genetic defects. Here, we tested whether the non-viral Sleeping Beauty (SB) transposon system could mediate stable delivery of trans-splicing molecules designed to correct the genetic defect responsible for severe combined immune deficiency (SCID). This immunological disorder is caused by a point mutation within the 12.4 kilobase (kb) gene encoding the DNA protein kinase catalytic subunit (DNA-PKcs) and is associated with aberrant DNA repair, defective T- and B-cell production, and hypersensitivity to radiation-induced injury. Using a novel SB-based trans-splicing vector, we demonstrate stable mRNA correction, proper DNA-PKcs protein production, and conference of a radiation-resistant phenotype in a T-cell thymoma cell line and SCID multipotent adult progenitor cells (MAPCs). These results suggest that SB-based trans-splicing vectors should prove useful in facilitating the correction of endogenous mutated mRNA transcripts, including the DNA-PKcs defect present in SCID cells.

    View details for DOI 10.1038/

    View details for Web of Science ID 000247646400013

    View details for PubMedID 17457319

  • Dual system of RNA trans-splicing element and sleeping beauty transposon corrects dna protein kinase mutation in severe combined immune deficiency 12th Congress of the European-Hematology-Association Tolar, J., Zayed, H., Xia, L., Yerich, A., Yant, S. R., Kay, M. A., Puttaraju, M., Mansfield, S. G., Wiest, D., McIvor, R. S., Blazar, B. R. FERRATA STORTI FOUNDATION. 2007: 154–154
  • Combinatorial RNAi: A winning strategy for the race against evolving targets? MOLECULAR THERAPY Grimm, D., Kay, M. A. 2007; 15 (5): 878-888


    The ability to use double-stranded RNA to inhibit gene expression sequence-specifically (RNA interference, or RNAi) is currently revolutionizing science and medicine alike. Numerous pre-clinical studies are evaluating RNAi as a novel therapeutic modality in the battle against gain-of-function autosomal dominant diseases, cancer, and viral infections. One emerging concern is that RNAi mono-therapies might ultimately fail to control viruses that can escape silencing by mutation and/or RNAi suppression. Thus, sophisticated strategies are being developed that aim to avert viral resistance by combining RNAi effectors with each other or with further gene expression inhibitors. Several reports already validate this new concept of "combinatorial RNAi" (coRNAi) and illustrate its versatility by describing co-expression of RNAi triggers directed against single or multiple, viral or cellular, targets. Other studies document the successful delivery of these triggers with additional RNA- or protein-based silencers. Moreover, vectors have been engineered to blend RNAi-mediated gene inhibition with conventional gene replacement strategies. Collectively, these efforts open up exciting new therapeutic avenues but could also augment the inherent risks of RNAi technology, including immune responses, off-targeting, and oversaturation of endogenous pathways. Here, we critically review all coRNAi strategies and discuss the requirements for their transition into clinical application.

    View details for DOI 10.1038/

    View details for Web of Science ID 000245999500006

    View details for PubMedID 17311009

  • Site-directed transposon integration in human cells NUCLEIC ACIDS RESEARCH Yant, S. R., Huang, Y., Akache, B., Kay, M. A. 2007; 35 (7)


    The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.

    View details for DOI 10.1093/nar/gkm089

    View details for Web of Science ID 000246294700004

    View details for PubMedID 17344320

  • Adenovirus transduction is required for the correction of diabetes using Pdx-1 or neurogenin-3 in the liver MOLECULAR THERAPY Wang, A. Y., Ehrhardt, A., Xu, H., Kay, M. A. 2007; 15 (2): 255-263


    The regeneration of insulin-producing cells in vivo has emerged as a promising method for treating type I diabetes. Pancreatic duodenal homeobox-1 (Pdx-1), NeuroD, and Neurogenin-3 (Ngn3) are pancreatic transcription factors important for the development of insulin-producing cells in the liver. Other groups have demonstrated that adenoviral-mediated transgene expression of these transcription factors in the liver can reverse hyperglycemia in diabetic mice. We delivered Pdx-1 and Ngn3 to the livers of diabetic mice using adeno-associated virus (AAV) serotype 8, a vector that has been shown to result in non-toxic, persistent, high level expression of the transgene. We were unable to correct hyperglycemia in mice with streptozotocin-induced diabetes using AAV vectors expressing Pdx-1 and Ngn3. However, when we co-delivered these transcription factor expression cassettes in non-viral vectors with an irrelevant adenoviral vector, we were able to correct hyperglycemia in diabetic animals. Further studies demonstrated that an antigen-dependent immune response elicited by the adenoviral capsid together with the expression of a pancreatic transcription factor was required for restoration of serum insulin levels by the liver. Our results suggest that a host response to adenovirus in combination with expression of a pro-endocrine pancreas transcription factor is sufficient to induce insulin production in the livers of diabetic mice.

    View details for DOI 10.1038/

    View details for Web of Science ID 000244405200007

    View details for PubMedID 17235302

  • Sarcoma derived from cultured mesenchymal stem cells STEM CELLS Tolar, J., Nauta, A. J., Osborn, M. J., Mortari, A. P., McElmurry, R. T., Bell, S., Xia, L., Zhou, N., Riddle, M., Schroeder, T. M., Westendorf, J. J., McIvor, R. S., Hogendoorn, P. C., Szuhai, K., Oseth, L., Hirsch, B., Yant, S. R., Kay, M. A., Peister, A., Prockop, D. J., Fibbe, W. E., Blazar, B. R. 2007; 25 (2): 371-379


    To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole-body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain-specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon-related genetic abnormality, and p53 was not overexpressed. Infusion of MSC-derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

    View details for DOI 10.1634/stemcells.2005-0620

    View details for Web of Science ID 000244070600014

    View details for PubMedID 17038675

  • A two-hybrid screen identifies cathepsins B and L as uncoating factors for adeno-associated virus 2 and 8 MOLECULAR THERAPY Akache, B., Grimm, D., Shen, X., Fuess, S., Yant, S. R., Glazer, D. S., Park, J., Kay, M. A. 2007; 15 (2): 330-339


    Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.

    View details for DOI 10.1038/

    View details for Web of Science ID 000244405200016

    View details for PubMedID 17235311

  • RNAi and gene therapy: a mutual attraction. Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program Grimm, D., Kay, M. A. 2007: 473-481


    The phylogenetically conserved cellular phenomenon of RNA interference (RNAi)-the sequence-specific post-transcriptional silencing of gene expression mediated by small double-stranded RNAs-holds substantial promise for basic research and for drug development. Particularly attractive from a medical standpoint is the juxtaposition of new RNAi methodology with established gene transfer strategies, especially viral vectors for efficient and tissue-specific RNAi delivery to patients. Here, we summarize the latest experimental and clinical advances in RNAi-based gene therapy approaches. We briefly portray emerging nonviral strategies for siRNA transfer, before comparing the three viral vectors currently predominantly developed as shRNA delivery vehicles, adenovirus, lentivirus, and adeno-associated virus (AAV). Moreover, we describe the most clinically relevant genetic, acquired or infectious targets being pursued for therapeutic purposes. Specifically, we assess the use of vector-mediated RNAi for treatment of viral processes, solid cancers, lymphoproliferative disorders, and neurodegenerative and ocular diseases. In addition, we highlight further emerging applications, including stem cell therapies and animal transgenesis, as well as discuss some of the potential pitfalls and limitations inherent to the individual approaches. While we predict that eventual schemes will be shaped by our increasing understanding of the complexities of human RNAi biology, as well as by progressive refinements of viral shuttle designs, the potential scientific and medical benefits from a successful marriage of RNAi and gene therapy seem enormous.

    View details for PubMedID 18024667

  • Somatic integration from an adenoviral hybrid vector into a hot spot in mouse liver results in persistent transgene expression levels in vivo MOLECULAR THERAPY Ehrhardt, A., Yant, S. R., Giering, J. C., Xu, H., Engler, J. A., Kay, M. A. 2007; 15 (1): 146-156


    We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.

    View details for DOI 10.1038/

    View details for Web of Science ID 000244404700025

    View details for PubMedID 17164786

  • Molecular analysis of chromosomal rearrangements in mammalian cells after phi C31-mediated integration HUMAN GENE THERAPY Ehrhardt, A., Engler, J. A., Xu, H., Cherry, A. M., Kay, M. A. 2006; 17 (11): 1077-1094


    Reports on insertional mutagenesis due to integration of gene therapy vectors into the host genome have raised concerns about the genetic manipulation of somatic cells. Previously, it was demonstrated that integrase phiC31 derived from a Streptomyces phage mediates site-specific integration into the host genome of mammalian cells in vitro and in vivo by recombining the attB recognition site in an episomal plasmid and one or more pseudoattP sites in the host chromosomes. In the present study we investigated whether cryptic phiC31 recognition sites in the host genome may result in chromosomal rearrangements. Of 69 independent integration events analyzed in human cells, 6 (8.7%) integrated into human chromosome 19 (19q13.31) and 10 (14.49%) integrated into human chromosome 12 (12q22). Most importantly, of all integration sites analyzed, 15% were found to contain an integrated transgene that was flanked by DNA sequences originating from two different chromosomes. To confirm chromosomal translocations we performed a polymerase chain reaction analysis of chromosomal DNA flanking the transgene and also performed limited studies to determine the genotype of single-cell clones. Although the mechanism responsible for chromosomal translocations needs to be further characterized, we speculate that cryptic phiC31 attachment sites flanking the transgene and cryptic phiC31 attachment sites in the host genome recombine with each other.

    View details for Web of Science ID 000242211300003

    View details for PubMedID 17069535

  • The 37/67-kilodalton laminin receptor is a receptor for adeno-associated virus serotypes 8, 2, 3, and 9 JOURNAL OF VIROLOGY Akache, B., Grimm, D., Pandey, K., Yant, S. R., Xu, H., Kay, M. A. 2006; 80 (19): 9831-9836


    Adeno-associated virus serotype 8 (AAV8) is currently emerging as a powerful gene transfer vector, owing to its capability to efficiently transduce many different tissues in vivo. While this is believed to be in part due to its ability to uncoat more readily than other AAV serotypes such as AAV2, understanding all the processes behind AAV8 transduction is important for its application and optimal use in human gene therapy. Here, we provide the first report of a cellular receptor for AAV8, the 37/67-kDa laminin receptor (LamR). We document binding of LamR to AAV8 capsid proteins and intact virions in vitro and demonstrate its contribution to AAV8 transduction of cultured cells and mouse liver in vivo. We also show that LamR plays a role in transduction by three other closely related serotypes (AAV2, -3, and -9). Sequence and deletion analysis allowed us to map LamR binding to two protein subdomains predicted to be exposed on the AAV capsid exterior. Use of LamR, which is constitutively expressed in many clinically relevant tissues and is overexpressed in numerous cancers, provides a molecular explanation for AAV8's broad tissue tropism. Along with its robust transduction efficiency, our findings support the continued development of AAV8-based vectors for clinical applications in humans, especially for tumor gene therapy.

    View details for DOI 10.1128/JVI.00878-06

    View details for Web of Science ID 000240647200047

    View details for PubMedID 16973587

  • Correction of severe combined immune deficiency in multipotent adult progenitor cells by spliceosome-mediated RNA trans-splicing and Sleeping Beauty transposon delivery 35th Annual Meeting of the International-Society-for-Experimental-Hematology Zayed, H., Xia, L., Yerich, A., Yant, S. R., Kay, M. A., Puttaraju, M., Mansfield, G., Wiest, D. L., McIvor, R. S., Tolar, J., Blazar, B. R. ELSEVIER SCIENCE INC. 2006: 49–49
  • Robust systemic transduction with AAV9 vectors in mice: Efficient global cardiac gene transfer superior to that of AAV8 MOLECULAR THERAPY Inagaki, K., Fuess, S., Storm, T. A., Gibson, G. A., McTiernan, C. F., Kay, M. A., Nakai, H. 2006; 14 (1): 45-?


    It has been recently shown that recombinant adeno-associated virus serotype 8 (rAAV8) is a robust alternative serotype vector that overcomes many of the limitations of rAAV2 and transduces various tissues efficiently and globally through systemic vector administration. AAV9 is a serotype newly isolated from human tissues, but our knowledge of the biology of rAAV9 in vivo is currently limited. Here, we demonstrate by a series of comprehensive side-by-side experiments with rAAV8 and 9 vectors delivered via different routes or at various doses in mice that rAAV9 vectors share the robustness of rAAV8, i.e., (1) very high liver transduction efficiency irrespective of whether vectors are administered intravascularly or extravascularly and (2) substantial transduction in the heart, skeletal muscle, and pancreas by peripheral vein injection. Importantly, rAAV9 transduced myocardium 5- to 10-fold higher than rAAV8, resulting in over 80% cardiomyocyte transduction following tail vein injection of as low as 1.0 x 10(11) particles per mouse. Thus rAAV9, as well as rAAV8, is a robust vector for gene therapy applications and rAAV9 is superior to rAAV8 specifically for cardiac gene delivery by systemic vector administration.

    View details for DOI 10.1016/j.ymthe.2006.03.014

    View details for Web of Science ID 000238805400009

    View details for PubMedID 16713360

  • Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways NATURE Grimm, D., Streetz, K. L., Jopling, C. L., Storm, T. A., Pandey, K., Davis, C. R., Marion, P., Salazar, F., Kay, M. A. 2006; 441 (7092): 537-541


    RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

    View details for Web of Science ID 000237778900056

    View details for PubMedID 16724069

  • Host factors that impact the biodistribution and persistence of multipotent adult progenitor cells BLOOD Tolar, J., O'Shaughnessy, M. J., Panoskaltsis-Mortari, A., McElmurry, R. T., Bell, S., Riddle, M., McIvor, R. S., Yant, S. R., Kay, M. A., Krause, D., Verfaillie, C. M., Blazar, B. R. 2006; 107 (10): 4182-4188


    Multipotent adult progenitor cells (MAPCs) are marrow-derived pluripotent stem cells with a broad differentiation potential. We sought to identify factors that affect adoptively transferred MAPCs. In vitro, MAPCs expressed low levels of major histocompatibility complex (MHC) antigens, failed to stimulate CD4(+) and CD8(+) T-cell alloresponses, and were targets of NK cytolysis. To study in vivo biodistribution, we labeled MAPCs with luciferase for sequential quantification of bioluminescence and DsRed2 for immunohistochemical analysis. C57BL /6 MAPCs were infused intravenously into C57BL /6, Rag-2(-/-) (T- and B-cell-deficient), and Rag-2(-/-)/IL-2Rgamma(c)(-/-) (T-, B-, and NK-cell-deficient) mice. In C57BL /6 mice, MAPCs were transiently detected only in the chest compared with long-term persistence in T- and B-cell-deficient mice. NK depletion reduced MAPC elimination. Because the lungs were the major uptake site after intravenous injection, intra-arterial injections were tested and found to result in more widespread biodistribution. Widespread MAPC biodistribution and long-term persistence were seen in irradiated recipients given allogeneic marrow and MAPCs; such MAPCs expressed MHC class I antigens in tissues. Our data indicate that the biodistribution and persistence of reporter gene-labeled MAPCs are maximized after intra-arterial delivery or host irradiation and that T cells, B cells, and NK cells contribute to in vivo MAPC rejection.

    View details for DOI 10.1182/blood-2005-08-3289

    View details for Web of Science ID 000237584500059

    View details for PubMedID 16410448

    View details for PubMedCentralID PMC1895284

  • Therapeutic short hairpin RNA expression in the liver: viral targets and vectors GENE THERAPY Grimm, D., Kay, M. A. 2006; 13 (6): 563-575


    Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.

    View details for DOI 10.1038/

    View details for Web of Science ID 000235836000014

    View details for PubMedID 16453009

  • Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response NATURE MEDICINE Manno, C. S., Arruda, V. R., Pierce, G. F., Glader, B., Ragni, M., Rasko, J., Ozelo, M. C., Hoots, K., Blatt, P., Konkle, B., Dake, M., Kaye, R., Razavi, M., Zajko, A., Zehnder, J., Nakai, H., Chew, A., Leonard, D., Wright, J. F., Lessard, R. R., Sommer, J. M., TIGGES, M., Sabatino, D., Luk, A., Jiang, H. Y., Mingozzi, F., Couto, L., Ertl, H. C., High, K. A., Kay, M. A. 2006; 12 (3): 342-347


    We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

    View details for DOI 10.1038/nm1358

    View details for Web of Science ID 000235802900035

    View details for PubMedID 16474400

  • Analysis of vector genome integration sites in various tissues following systemic administration of AAV serotype 8 vector in mice 11th Annual Meeting of the Japan-Society-of-Gene-Therapy Inagaki, K., Wu, X., Fuess, S., Storm, T. A., Kay, M. A., Nakai, H. WILEY-BLACKWELL. 2006: 391–91
  • Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype JOURNAL OF VIROLOGY Grimm, D., Pandey, K., Nakai, H., Storm, T. A., Kay, M. A. 2006; 80 (1): 426-439


    We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.

    View details for DOI 10.1128/JVI.80.1.426-439.2006

    View details for Web of Science ID 000235091700041

    View details for PubMedID 16352567

  • In vivo selection of primary and bone-marrow-derived hepatocytes after allogeneic transplantation in mice 41st Annual Meeting of the European-Association-for-the-Study-of-the-Liver Streetz, K. L., Doyonnas, R., Jenkins, D., Perryman, S., Fuess, S., Lin, S., Shizuru, J., Blau, H., Trautwein, C., Sylvester, K., Kay, M. A. ELSEVIER SCIENCE BV. 2006: S33–S33
  • In vivo selection of transplanted allogeneic hepatocytes and bone-marrow derived hepatocytes after allogeneic bone-marrow transplantation in mice 56th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases Streetz, K., Doyonnas, R., Jenkins, D., Lin, S., Shizuru, J., Blau, H., Sylvester, K., Kay, M. WILEY-BLACKWELL. 2005: 370A–371A
  • Cell therapy for hepatocyte replacement through bone marrow derived myelomonocytic progenitors 91st Annual Clinical Congress of the American-College-of-Surgeons Sylvester, K. G., Jenkins, D., Streetz, K., Doyannis, R., Perryman, S., Kay, M., Blau, H. ELSEVIER SCIENCE INC. 2005: S47–S48
  • Lessons from a clinical trial of liver-directed AAV gene transfer in hemophilia B 4th Meeting of the Australasian-Gene-Therapy-Society Rasko, J., High, K., TIGGES, M., Manno, C., Sabatino, D., Dake, M., Razavi, M., Arruda, V., Herzog, R., Rustagi, P., Sommer, J., Ragni, M., Konkle, B., Lessard, R., Luk, A., Glader, B., Pierce, G., Couto, L., Jiang, H. Y., Kay, M. WILEY-BLACKWELL. 2005: 1117–18
  • Real-time in vivo imaging transgenesis by of stem cells following transposition MOLECULAR THERAPY Tolar, J., Osborn, M., Bell, S., McElmurry, R., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., Jiang, Y. H., McIvor, R. S., Contag, C. H., Yant, S. R., Kay, M. A., Verfaillie, C. M., Blazar, B. R. 2005; 12 (1): 42-48
  • Real-time in vivo imaging of stem cells following transgenesis by transposition. Molecular therapy : the journal of the American Society of Gene Therapy Tolar, J., Osborn, M., Bell, S., McElmurry, R., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., Jiang, Y., McIvor, R. S., Contag, C. H., Yant, S. R., Kay, M. A., Verfaillie, C. M., Blazar, B. R. 2005; 12 (1): 42-48


    Previous studies have identified Sleeping Beauty transposons as efficient vectors for nonviral gene delivery in mammalian cells. However, studies demonstrating the usefulness of transposons as gene delivery vehicles into adult stem cells are lacking. Multipotent adult progenitor cells (MAPC) are nonhematopoietic stem cells with the capacity to form most, if not all, cell types of the body and as such hold great therapeutic potential. The whole-body biodistribution and persistence of MAPC are unknown, and such data would help direct clinical applications. We have nucleofected murine MAPC with two plasmid-based Sleeping Beauty transposons encoding the red fluorescent protein (DsRed2) and firefly luciferase. Transgenic euploid MAPC clones maintained their characteristic multilineage differentiation potential in vitro. DsRed2 and luciferase expression allowed for MAPC detection in vivo and in tissue sections. To confirm that transgenesis occurred by transposition into the genome of MAPC, we mapped Sleeping Beauty transposon integration sites in two MAPC clones using splinkerette PCR. This novel dual-reporter imaging approach based on the transgenesis of MAPC with Sleeping Beauty transposons sheds light on the homing patterns of MAPC and paves the way for quantification of MAPC engraftment in real time in vivo.

    View details for PubMedID 15963919

  • A direct comparison of two nonviral gene therapy vectors for somatic integration: In vivo evaluation of the bacteriophage integrase phi c31 and the Sleeping Beauty transposase MOLECULAR THERAPY Ehrhardt, A., Xu, H., Huang, Z., Engler, J. A., Kay, M. A. 2005; 11 (5): 695-706


    In this study we performed a head-to-head comparison of the integrase phiC31 derived from a Streptomyces phage and the Sleeping Beauty (SB) transposase, a member of the TC1/mariner superfamily of transposable elements. Mouse liver was cotransfused with a vector containing our most robust human coagulation factor IX expression cassette and the appropriate recombinase recognition site and either a phiC31- or a SB transposase-expressing vector. To analyze transgene persistence and to prove somatic integration in vivo we induced cell cycling of mouse hepatocytes and found that the transgene expression levels dropped by only 16 to 21% and 56 to 66% in mice that received phiC31 and SB, respectively. Notably, no difference in the toxicity profile was detected in mice treated with either recombinase. Moreover we observed that with the integrase-mediated gene transfer, transgene expression levels were dependent on the remaining noncoding vector sequences, which also integrate into the host genome. Further analyses of a hot spot of integration after phiC31-mediated integration revealed small chromosomal deletions at the target site and that the recombination process was not dependent on the orientation in which the phiC31 recognition site attached to the pseudo-recognition sites in the host genome. Coupled together with ongoing improvements in both systems this study suggests that both nonviral vector systems will have important roles in achieving stable gene transfer in vivo.

    View details for DOI 10.1016/j.ymthe.2005.01.010

    View details for Web of Science ID 000228966100008

    View details for PubMedID 15851008

  • Increased maintenance and persistence of transgenes by excision of expression cassettes from plasmid sequences in vivo HUMAN GENE THERAPY Riu, E., Grimm, D., Huang, Z., Kay, M. A. 2005; 16 (5): 558-570


    Persistence of transgene expression is a major limitation for nonvirus-mediated gene therapy approaches. We have suggested that covalent linkage of bacterial DNA to the expression cassette plays a critical role in transcriptional silencing of transgenes in vivo. To gain insight into the role of the covalent linkage of plasmid DNA to the expression cassette and transcriptional repression, and whether this silencing effect could be alleviated by altering the molecular structure of vector DNAs in vivo, we generated a scheme for converting routine plasmids into a purified expression cassette, free of bacterial DNA after gene transfer in vivo. To do this, the human alpha-1-antitrypsin (hAAT) and human clotting factor IX (hfIX) reporter genes were flanked by two ISceI endonuclease recognition sites, and coinjected together with a plasmid encoding the I-SceI cDNA or a control plasmid into mouse liver. Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed low serum levels of hAAT or hFIX, which remained low throughout the length of the experiment. However, animals that expressed I-SceI had a 5- to 10-fold increase in serum hAAT or hFIX that persisted for at least 8 months (length of study). Expression of I-SceI resulted in cleavage and excision of the expression cassettes from the plasmid backbone, forming mostly circles devoid of bacterial DNA sequences, as established by a battery of different Southern blot and polymerase chain reaction analyses in both C57BL/6 and scid treated mice. In contrast, only the input parental circular plasmid DNA band was detected in mice injected with the reporter gene alone, or an I-SceI plasmid together with the hAAT reporter plasmid lacking the I-SceI sites. Similar results were obtained when the Flp recombinase system was used to make mini-plasmids in mouse liver in vivo. This study presents further independent evidence that removing the covalent linkage between plasmid and transgene sequences leads to a marked increase in and persistence of transgene expression. Unraveling the mechanisms by which the covalent linkage of bacterial DNA to the expression cassette is connected to gene silencing is fundamental to establishing the mechanism of transcriptional regulation in mammalian systems and will be important for the development of versatile nonviral vectors that can be used to achieve persistent gene expression in different cell types.

    View details for Web of Science ID 000229503300003

    View details for PubMedID 15916481

  • Allogeneic bone-marrow transplantation in mice provides tolerance and promotes massive in vivo selection of subsequently transplanted hepatocytes 40th Annual Meeting of the European-Association-for-the-Study-of-the-Liver Streetz, K. L., Doyonnas, R., Jenkins, D., Sylvester, K., Kay, M. A. ELSEVIER SCIENCE BV. 2005: 6–6
  • Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver JOURNAL OF VIROLOGY Nakai, H., Wu, X. L., Fuess, S., Storm, T. A., Munroe, D., Montini, E., Burgess, S. M., Grompe, M., Kay, M. A. 2005; 79 (6): 3606-3614


    Recombinant adeno-associated virus (rAAV) vector holds promise for gene therapy. Despite a low frequency of chromosomal integration of vector genomes, recent studies have raised concerns about the risk of rAAV integration because integration occurs preferentially in genes and accompanies chromosomal deletions, which may lead to loss-of-function insertional mutagenesis. Here, by analyzing 347 rAAV integrations in mice, we elucidate novel features of rAAV integration: the presence of hot spots for integration and a strong preference for integrating near gene regulatory sequences. The most prominent hot spot was a harmless chromosomal niche in the rRNA gene repeats, whereas nearly half of the integrations landed near transcription start sites or CpG islands, suggesting the possibility of activating flanking cellular disease genes by vector integration, similar to retroviral gain-of-function insertional mutagenesis. Possible cancer-related genes were hit by rAAV integration at a frequency of 3.5%. In addition, the information about chromosomal changes at 218 integration sites and 602 breakpoints of vector genomes have provided a clue to how vector terminal repeats and host chromosomal DNA are joined in the integration process. Thus, the present study provides new insights into the risk of rAAV-mediated insertional mutagenesis and the mechanisms of rAAV integration.

    View details for DOI 10.1128/JVI.79.6.3606-3614.2005

    View details for Web of Science ID 000227366900038

    View details for PubMedID 15731255

  • Modified infusion procedures affect recombinant adeno-associated virus vector type 2 transduction in the liver HUMAN GENE THERAPY Ohashi, K., Nakai, H., Couto, L. B., Kay, M. A. 2005; 16 (3): 299-306


    Recombinant adeno-associated virus (rAAV) vectors have therapeutic potential for the treatment of several types of liver diseases including hepato-deficiency disorders. Most of the preclinical and clinical applications involve the use of adeno-associated vector serotype 2 (AAV-2). However, when this vector is delivered at high doses into the portal vein or hepatic artery, a relatively small number of hepatocytes are stably transduced. We elected to determine if the route of vector administration and altering the vascular delivery route within the liver influenced the relative level of transduction. First, we delivered an AAV vector expressing the human factor IX gene from a liver-specific promoter into the hepatic artery, portal vein, or general circulation of rats. Transgene expression was equal with hepatic artery and portal vein infusion, which was higher than vector administered via peripheral venous infusion. Next, we determined how localized perfusion or changing the vector dwell time affected AAV transduction in vivo. To do this, we infused an AAV vector lacking a functional expression and quantified transduction by quantifying the number of double-stranded vector DNA genomes. By increasing vector dwell time in the liver to 5 min, vector transduction was enhanced approximately 4- to 5- fold. To establish if gene transduction could be restricted to a specific anatomic location in the liver, we delivered vector into specific liver lobes by clamping the venous inflow to the middle and left liver lobes (noninfused lobes) and infusing vector into the right two liver lobes through the hepatic artery followed by vector circulation between the two right lobes and general circulation for 5 min. With this selective infusion, 40 to 120 times higher vector genome was observed in the perfused lobes than the nonperfused lobes. All the procedures described in this study were performed without detectable liver injury or toxicity. In all, the present study clearly demonstrated that hepatic arterial infusion of rAAV is effective for liver-directed gene therapy and that other parameters related to blood flow can be adjusted to further optimize gene transfer.

    View details for Web of Science ID 000228276300003

    View details for PubMedID 15812225

  • High-resolution genome-wide mapping of transposon integration in mammals MOLECULAR AND CELLULAR BIOLOGY Yant, S. R., Wu, X. L., Huang, Y., Garrison, B., Burgess, S. M., Kay, M. A. 2005; 25 (6): 2085-2094


    The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon- and virus-based integrating vectors.

    View details for DOI 10.1128/MCB.25.6.2085-2094.2005

    View details for Web of Science ID 000227475300002

    View details for PubMedID 15743807

  • Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases HEPATOLOGY Ohashi, K., Waugh, J. M., Dake, M. D., Yokoyama, T., Kuge, H., Nakajima, Y., Yamanouchi, M., Naka, H., Yoshioka, A., Kay, M. A. 2005; 41 (1): 132-140


    Liver tissue engineering using hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation or liver-directed gene therapy to correct various types of hepatic insufficiency. Hepatocytes are not sustained when transplanted under the kidney capsule of syngeneic mice. However, when we transplanted hepatocytes with the extracellular matrix components extracted from Engelbreth-Holm-Swarm cells, hepatocytes survived for at least 140 days and formed small liver tissues. Liver engineering in hemophilia A mice reconstituted 5% to 10% of normal clotting activity, enough to reduce the bleeding time and have a therapeutic benefit. Conversely, the subcutaneous space did not support the persistent survival of hepatocytes with Engelbreth-Holm-Swarm gel matrix. We hypothesized that establishing a local vascular network at the transplantation site would reduce graft loss. To test this idea, we provided a potent angiogenic agent before hepatocyte transplantation into the subcutaneous space. With this procedure, persistent survival was achieved for the length of the experiment (120 days). To establish that these engineered liver tissues also retained their native regeneration potential in vivo, we induced two different modes of proliferative stimulus to the naive liver and confirmed that hepatocytes within the extrahepatic tissues regenerated with activity similar to that of naive liver. In conclusion, our studies indicate that liver tissues can be engineered and maintained at extrahepatic sites, retain their capacity for regeneration in vivo, and used to successfully treat genetic disorders.

    View details for DOI 10.1002/hep.20484

    View details for Web of Science ID 000226245000019

    View details for PubMedID 15619229

  • Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice 3rd Annual Meeting of the Japanese-Society-for-Regenerative-Medicine Ohashi, K., Kay, M. A., Yokoyama, T., Kuge, H., Kanehiro, H., Hisanaga, M., Ko, S., Nakajima, Y. COGNIZANT COMMUNICATION CORP. 2005: 621–27


    Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 +/- 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.

    View details for Web of Science ID 000234411000003

    View details for PubMedID 16405072

  • Unrestricted hepatocyte transduction with adeno-associated virus serotype 8 vectors in mice JOURNAL OF VIROLOGY Nakai, H., Fuess, S., Storm, T. A., Muramatsu, S., Nara, Y., Kay, M. A. 2005; 79 (1): 214-224


    Recombinant adeno-associated virus (rAAV) vectors can mediate long-term stable transduction in various target tissues. However, with rAAV serotype 2 (rAAV2) vectors, liver transduction is confined to only a small portion of hepatocytes even after administration of extremely high vector doses. In order to investigate whether rAAV vectors of other serotypes exhibit similar restricted liver transduction, we performed a dose-response study by injecting mice with beta-galactosidase-expressing rAAV1 and rAAV8 vectors via the portal vein. The rAAV1 vector showed a blunted dose-response similar to that of rAAV2 at high doses, while the rAAV8 vector dose-response remained unchanged at any dose and ultimately could transduce all the hepatocytes at a dose of 7.2 x 10(12) vector genomes/mouse without toxicity. This indicates that all hepatocytes have the ability to process incoming single-stranded vector genomes into duplex DNA. A single tail vein injection of the rAAV8 vector was as efficient as portal vein injection at any dose. In addition, intravascular administration of the rAAV8 vector at a high dose transduced all the skeletal muscles throughout the body, including the diaphragm, the entire cardiac muscle, and substantial numbers of cells in the pancreas, smooth muscles, and brain. Thus, rAAV8 is a robust vector for gene transfer to the liver and provides a promising research tool for delivering genes to various target organs. In addition, the rAAV8 vector may offer a potential therapeutic agent for various diseases affecting nonhepatic tissues, but great caution is required for vector spillover and tight control of tissue-specific gene expression.

    View details for DOI 10.1128/JVI.79.1.214-224.2005

    View details for Web of Science ID 000225904700021

    View details for PubMedID 15596817

  • Genomic progression in mouse models for liver tumors 70th Cold Spring Harbor Symposium on Quantitative Biology Tward, A. D., Jones, K. D., Yant, S., Kay, M. A., Wang, R., BISHOP, J. M. COLD SPRING HARBOR LABORATORY PRESS. 2005: 217–224


    The principal cause of human liver cancer is infection with hepatitis viruses B and C, but tumor progression is fueled by ensuing perturbations that confer gain of function on proto-oncogenes or loss of function on tumor suppressor genes. Frequent among these perturbations is overexpression of the proto-oncogene MET. We have modeled the pathogenesis of liver tumors by expressing conditional transgenes of MET in the hepatocytes of inbred mice. The response to the MET transgene varied with both the magnitude and timing of its expression but included hyperplasia of hepatic progenitor cells, as well as benign and malignant tumors that display both phenotypic and genotypic resemblances to human counterparts. The results reveal MET to be a crucial switch in the development of the liver; dramatize how different cellular compartments within a developmental lineage can give rise to distinctive tumor stem cells; delineate rules of tumor progression; provide evidence that the experimental tumors in mice are authentic models for human tumors; and support a role for MET in the genesis of human liver tumors. The models should be useful in elucidating the mechanisms of tumorigenesis and in the preclinical testing of new therapeutics.

    View details for Web of Science ID 000239915900025

    View details for PubMedID 16869757

  • Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo HUMAN GENE THERAPY Chen, Z. Y., He, C. Y., Kay, M. A. 2005; 16 (1): 126-131


    We have shown previously that minicircle DNA vectors free of plasmid bacterial DNA sequences are capable of persistent high level of transgene expression in vivo. The minicircle is generated in bacteria from a parental plasmid containing an inducible phage oC31 integrase gene and a therapeutic expression cassette flanked with attB and attP sites. The oC31-mediated intramolecular recombination between attB and attP results in the formation of two circular DNA molecules, one containing the eukaryotic expression cassette (minicircle), and the other the plasmid bacterial DNA backbone (BB). Previously, the minicircle was purified away from the plasmid BB by a restriction enzyme digestion step and ultracentrifugation in cesium chloride. We have now included the endonuclease I-SceI gene together with its recognition site in the minicircle-producing plasmid to allow the linearization and degradation of the plasmid BB in bacteria. The minicircle can then be isolated by routine plasmid purification procedures such as a one-step affinity column. With additional modifications to our previous strategy, we can prepare a minicircle encoding a 4-kb human factor IX expression cassette, up to 1.8 mg of minicircle with 97% purity was prepared from a 1 liter bacterial culture. The high yield, simple purification, and robust and persistent transgene expression make these vectors viable for gene therapy applications.

    View details for Web of Science ID 000227063400012

    View details for PubMedID 15703495

  • Adeno-associated virus vectors for short hairpin RNA expression RNA INTERFERENCE Grimm, D., Pandey, K., Kay, M. A. 2005; 392: 381-405


    Five recent publications have documented the successful development and use of gene transfer vectors based on adeno-associated virus (AAV) for expressing short hairpin RNA (shRNA). In cultured mammalian cells and in whole animals, infection with these vectors was shown to result in specific, efficient, and stable knockdown of various targeted endo- or exogenous genes. Here we review this exciting approach, to trigger RNA interference in vitro and in vivo by shRNA expressed from AAV vectors, and describe the state-of-the-art technology for vector particle generation. In particular, we present a set of novel AAV vector plasmids that were specifically designed for the easy and rapid cloning of shRNA expression cassettes into AAV. The plasmids contain alternative RNA polymerase III promoters (U6, H1, or 7SK) together with a respective terminator sequence, as well as stuffer DNA to guarantee an optimal vector size for efficient packaging into AAV capsids. To provide maximum versatility and user-friendliness, the constructs were also engineered to contain a set of unique restriction enzyme recognition sites, allowing the simple and straightforward replacement of the shRNA cassette or other vector components with customized sequences. Our novel vector plasmids complement existing AAV vector technology and should help further establish AAV as a most promising alternative to using adeno- or retro-?lentiviral vectors as shRNA delivery vehicles.

    View details for Web of Science ID 000228160100023

    View details for PubMedID 15644194

  • Human immune responses to AAV-2 capsid may limit duration of expression in liver-directed gene transfer in humans with hemophilia B. 46th Annual Meeting of the American-Society-of-Hematology High, K., TIGGES, M., Manno, C., Sabatino, D., Arruda, V., Herzog, R., Rustagi, P., Rasko, J., Sommer, J., Jaworski, K., Ragni, M., Glader, B., Lessard, R., Luk, A., Couto, L., Jiang, H. Y., Pierce, G., Kay, M. AMER SOC HEMATOLOGY. 2004: 121A–121A
  • Transgenesis of multipotent adult progenitor cells (MAPC) with sleeping beauty transposons to determine MAPC homing and persistence in real-time in vivo. 46th Annual Meeting of the American-Society-of-Hematology Tolar, J., Osborn, M., Bell, S., Xia, L., Riddle, M., Panoskaltsis-Mortari, A., McIvor, S., Stephen, Y. R., Kay, M. A., Contag, C. H., Verfaillie, C. M., Blazar, B. R. AMER SOC HEMATOLOGY. 2004: 577A–578A
  • Real-time in vivo biodistribution of multipotent adult progenitor cells (MAPC): Role of the immune system in MAPC resistance in non-transplanted and bone marrow transplanted mice. 46th Annual Meeting of the American-Society-of-Hematology Tolar, J., Bell, S., McElmurry, R., Xia, L. L., McIvor, R. S., Yam, S. R., Kay, M. A., Contag, C. H., Verfaillie, C. M., Blazar, B. R. AMER SOC HEMATOLOGY. 2004: 147A–148A
  • Efficient inhibition of in-stent restenosis by controlled stent-based inhibition of elastase: A pilot study JOURNAL OF VASCULAR AND INTERVENTIONAL RADIOLOGY Ganaha, F., Ohashi, K., Do, Y. S., Lee, J., Sugimoto, K., Minamiguchi, H., Elkins, C. J., Sameni, D., Modanlou, S., Kao, E. Y., Kay, M. A., Waugh, J. M., Dake, M. D. 2004; 15 (11): 1287-1293


    It is proposed that local elastase inhibition could suppress the extracellular matrix (ECM) degradation and subsequent smooth muscle cell migration and limit subsequent in-stent restenosis. This study evaluated the effect of stent-based controlled elastase inhibition on restenosis after stent implantation in a rabbit model.Biodegradable microspheres containing the potent elastase inhibitor alpha-1-antitrypsin (AAT) were prepared. Daily release of AAT from the microspheres was confirmed in vitro. The microspheres were loaded into stents with an abluminal polymer reservoir. Implantation of the stent with AAT microspheres and blank microspheres (control) was performed in the abdominal aortae of six rabbits in each group. After stent deployment, all stents were overdilated to 125% diameter. Stent-implanted arteries were harvested after 7 days (n = 3 each) or 28 days (n = 3 each). To assess the effect of local delivery of AAT, elastase activity and elastin content of the stent-implanted aortae were analyzed. As an endpoint, intima-to-media (I/M) ratio was determined in the 7-day and 28-day specimens.Significant inhibition of elastase was confirmed in treated vessels versus controls at 7 days after stent implantation (P < .05). This reduction in elastase activity was sufficient to afford early and late reduction of in-stent neointima. Plaque progression in the 28-day specimens decreased to 67% with elastase inhibition relative to controls (P < .05).Stent-based controlled release of elastase inhibitor may significantly reduce ECM degradation and might limit in-stent restenosis.

    View details for DOI 10.1097/01.RVI.0000141340.67588.4F

    View details for Web of Science ID 000227678800014

    View details for PubMedID 15525749

  • Extracellular matrix component cotransplantation prolongs survival of heterotopically transplanted human hepatocytes in mice 8th Congress of the Asian-Society-of-Transplantation Ohashi, K., Kay, M. A. ELSEVIER SCIENCE INC. 2004: 2469–70
  • Mutational analysis of the N-terminal DNA-binding domain of Sleeping Beauty transposase: Critical residues for DNA binding and hyperactivity in mammalian cells MOLECULAR AND CELLULAR BIOLOGY Yant, S. R., Park, J., Huang, Y., Mikkelsen, J. G., Kay, M. A. 2004; 24 (20): 9239-9247


    The N-terminal domain of the Sleeping Beauty (SB) transposase mediates transposon DNA binding, subunit multimerization, and nuclear translocation in vertebrate cells. For this report, we studied the relative contributions of 95 different residues within this multifunctional domain by large-scale mutational analysis. We found that each of four amino acids (leucine 25, arginine 36, isoleucine 42, and glycine 59) contributes to DNA binding in the context of the N-terminal 123 amino acids of SB transposase, as indicated by electrophoretic mobility shift analysis, and to functional activity of the full-length transposase, as determined by a quantitative HeLa cell-based transposition assay. Moreover, we show that amino acid substitutions within either the putative oligomerization domain (L11A, L18A, L25A, and L32A) or the nuclear localization signal (K104A and R105A) severely impair its ability to mediate DNA transposition in mammalian cells. In contrast, each of 10 single amino acid changes within the bipartite DNA-binding domain is shown to greatly enhance SB's transpositional activity in mammalian cells. These hyperactive mutations functioned synergistically when combined and are shown to significantly improve transposase affinity for transposon end sequences. Finally, we show that enhanced DNA-binding activity results in improved cleavage kinetics, increased SB element mobilization from host cell chromosomes, and dramatically improved gene transfer capabilities of SB in vivo in mice. These studies provide important insights into vertebrate transposon biology and indicate that Sleeping Beauty can be readily improved for enhanced genetic research applications in mammals.

    View details for DOI 10.1128/MCB.24.20.92309-9247.2004

    View details for Web of Science ID 000224239100038

    View details for PubMedID 15456893

  • Donor-derived, liver-specific protein expression after bone marrow transplantation TRANSPLANTATION Jenkins, D. D., Streetz, K., Tataria, M., Sahar, D., Kurobe, M., Longaker, M. T., Kay, M. A., Sylvester, K. G. 2004; 78 (4): 530-536


    Bone marrow transplantation (BMT) may represent a novel mechanism to deliver a functional gene to a deficient liver. Bone marrow-derived hepatocytes are rare and without a defined contribution to liver function. Consequently, the clinical significance of BMT to treat liver disease is unclear. We sought to quantify bone marrow-derived hepatocyte protein expression after BMT and determine whether the process is inducible with liver injury.Mice transgenic for human alpha-1 antitrypsin (hAAT) under a hepatocyte-specific promoter were used as bone marrow donors. Adenoviral transduction of modified urokinase plasminogen activator (Ad-muPA) was used to induce liver injury. Eight weeks after lethal irradiation and BMT, recipients were stratified into two groups: BMT alone (n = 5) and BMT + Ad-muPA (n= 10). Both groups of animals were bled before (t = 0) and at 2, 4, 8, and 16 weeks after Ad-muPA administration, and the serum samples were assessed for hAAT by enzyme-linked immunosorbent assay.Transgenic donor mice expressed 5 to 10 mg/mL of hAAT. Recipients of BMT alone expressed less than 80 ng/mL of hAAT over all time periods. Animals receiving BMT + Ad-muPA showed sustained and stable hAAT expression of approximately 200 ng/mL. Differences were statistically significant at each time point.Serum protein levels from liver-specific transgene expression are detectable and persist after BMT. Expression is low, but inducible with liver injury. We are currently developing strategies to augment donor-derived, liver-specific protein expression after BMT.

    View details for DOI 10.1097/01.TP.0000130180.42573.BI

    View details for Web of Science ID 000223486500008

    View details for PubMedID 15446311

  • Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo GENE THERAPY Chen, Z. Y., He, C. Y., Meuse, L., Kay, M. A. 2004; 11 (10): 856-864


    We previously demonstrated that sustainable enhanced levels of transgene products could be expressed from a bacterial DNA-free expression cassette either formed from a fragmented plasmid in mouse liver or delivered as a minicircle vector. This suggested that bacterial DNA sequences played a role in episomal transgene silencing. To further understand the silencing mechanism, we systematically altered the DNA components in both the expression cassette and the bacterial backbone, and compared the gene expression profiles from mice receiving different DNA forms. In nine vectors tested, animals that received the purified expression cassette alone always expressed persistently higher levels of transgene compared to 2fDNA groups. In contrast, animals that received linearized DNA by a single cut in the bacterial backbone had similar expression profiles to that of intact plasmid groups. All three linear DNAs formed large concatemers and small circles in mouse liver, while ccDNA remained intact. In all groups, the relative amount of vector DNA in liver remained similar. Together, these results further established that the DNA silencing effect was mediated by a covalent linkage of the expression cassette and the bacteria DNA elements.

    View details for DOI 10.1038/

    View details for Web of Science ID 000221242700007

    View details for PubMedID 15029228

  • Comparison of adenoviral and adeno-associated viral vectors for pancreatic gene delivery in vivo 7th Annual Meeting of the American-Society-of-Gene-Therapy Wang, A. Y., Peng, P. D., Ehrhardt, A., Storm, T. A., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S66–S66
  • Mechanistic insights into the persistence of non-viral mediated gene transfer in vivo 7th Annual Meeting of the American-Society-of-Gene-Therapy Riu, E., Huang, Z., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S398–S398
  • In vivo activity of nuclease-resistant siRNAs RNA-A PUBLICATION OF THE RNA SOCIETY Layzer, J. M., McCaffrey, A. P., Tanner, A. K., Huang, Z., Kay, M. A., Sullenger, B. A. 2004; 10 (5): 766-771


    Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.

    View details for DOI 10.1261/rna.5239604

    View details for Web of Science ID 000221072500002

    View details for PubMedID 15100431

    View details for PubMedCentralID PMC1370566

  • Hyperactive transposase mutants of the Sleeping Beauty transposon 7th Annual Meeting of the American-Society-of-Gene-Therapy Yant, S. R., Park, J., Huang, Y., Mikkelsen, J. G., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S310–S310
  • Toward adenoviral mediated RNA interference for the treatment of hepatitis B virus infection 7th Annual Meeting of the American-Society-of-Gene-Therapy McCaffrey, A. P., Pandey, K., Ehrhardt, A., Huang, Z., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S24–S25
  • Immune responses to AAV and to Factor IX in a phase I study of AAV-mediated, liver-directed gene transfer for hemophilia B 7th Annual Meeting of the American-Society-of-Gene-Therapy High, K., Manno, C., Sabatino, D., Hutchison, S., Dake, M., Razavi, M., Kaye, R., Aruda, V., Herzog, R., Rustagi, P., Rasko, J., Hoots, K., Blatt, P., Sommer, J., Ragni, M., Ozelo, M., Konkle, B., Lessard, R., Luk, A., Glader, B., Pierce, G., Couto, L., Kay, M. NATURE PUBLISHING GROUP. 2004: S383–S384
  • A new model to functionally measure fusion events in the liver after bone-marrow transplantation over time 7th Annual Meeting of the American-Society-of-Gene-Therapy Streetz, K. L., Jenkins, D. D., Longaker, M. T., Sylvester, K. G., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S118–S118
  • Hot spots for rAAV2 vector integration in mice 7th Annual Meeting of the American-Society-of-Gene-Therapy Nakai, H., Wu, X. L., Fuess, S., Storm, T., Burgess, S., Grompe, M., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S6–S6
  • Complete inhibition of hepatitis B virus gene expression in vivo with short hairpin RNA expressed from a novel double-stranded, bi-cistronic adeno-associated virus pseudotype 8 vector 7th Annual Meeting of the American-Society-of-Gene-Therapy Grimm, D., Streetz, K. L., Storm, T. A., Nakai, H., McCaffrey, A. P., Huang, Z., Salazar, F. H., Marion, P. L., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S141–S142
  • Comparison of adenoviral and adeno-associated viral vectors for pancreatic gene delivery in vivo HUMAN GENE THERAPY Wang, A. Y., Peng, P. D., Ehrhardt, A., Storm, T. A., Kay, M. A. 2004; 15 (4): 405-413


    Although effective gene therapy vectors have been developed for organ systems such as the liver, an effective delivery vector to the pancreas in vivo has remained elusive. Of the currently available viral vectors, adenovirus and adeno-associated virus (AAV) are two of the most efficient at transducing nondividing cells. We have constructed recombinant adenovirus (AdVLacZ), adeno-associated virus serotype 2 (AAV2LacZ), and pseudotyped adeno-associated virus serotype 5 and 8 (AAV5LacZ, AAV8LacZ) carrying the LacZ reporter, and compared the transduction efficiency of these four vectors in the pancreas of mice in vivo. We showed that adenovirus, AAV2, and AAV8 are capable of transducing the pancreas in vivo, but with different expression kinetics, efficiencies of transduction, and persistence. AdVLacZ-transduced pancreas exhibited maximum LacZ expression at 1 week postdelivery, with greater than 90% of expression lost at 4 weeks. AAV2LacZ-transduced pancreas displayed peak LacZ levels at 4 weeks postdelivery, with no significant decrease in expression for up to 8 weeks. AAV8LacZ was at least 10-fold more efficient than AAV2LacZ in transducing the pancreas in vivo, with significant levels of expression detectable at 1 week, whereas AAV5LacZ did not result in any detectable transgene expression at all tested time points. All three vectors primarily transduced pancreatic acinar cell types, with limited transduction of pancreatic endocrine cells. AdVLacZ elicited a significant leukocyte infiltration early after delivery into the pancreas, whereas none of the AAV vectors elicited a significant leukocyte response. None of the tested vectors caused significant changes in serum amylase or blood glucose levels, suggesting that they do not significantly alter pancreatic function. These vectors will be useful for studying novel gene delivery based treatments in animal models for diabetes and other pancreatic disorders.

    View details for Web of Science ID 000221606300007

    View details for PubMedID 15053865

  • Functional measurement of fusion in the liver after bone-marrow transplantation over time 39th Annual Meeting of the European-Association-for-the-Study-of-the-Liver Streetz, K. L., Jenkins, D., Longaker, G., Sylvester, K., Kay, M. A. ELSEVIER SCIENCE BV. 2004: 109–109
  • Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors JOURNAL OF VIROLOGY Thomas, C. E., Storm, T. A., Huang, Z., Kay, M. A. 2004; 78 (6): 3110-3122


    Transduction of the liver with single-stranded adeno-associated virus serotype 2 (AAV2) vectors is inefficient; less than 10% of hepatocytes are permissive for stable transduction, and transgene expression is characterized by a lag phase of up to 6 weeks. AAV2-based vector genomes packaged inside AAV6 or AAV8 capsids can transduce the liver with higher efficiency, but the molecular mechanisms underlying this phenomenon have not been determined. We now show that the primary barrier to transduction of the liver with vectors based on AAV2 capsids is uncoating of vector genomes in the nucleus. The majority of AAV2 genomes persist as encapsidated single-stranded molecules within the nucleus for as long as 6 weeks after vector administration. Double-stranded vector genomes packaged inside AAV2 capsids are at least 50-fold more active than single-stranded counterparts, but these vectors also exhibit a lag phase before maximal gene expression. Vector genomes packaged inside AAV6 or AAV8 capsids do not persist as encapsidated molecules and are more biologically active than vector genomes packaged inside AAV2 capsids. Our data suggest that the rate of uncoating of vector genomes determines the ability of complementary plus and minus single-stranded genomes to anneal together and convert to stable, biologically active double-stranded molecular forms.

    View details for DOI 10.1128/JVI.78.6.3110-3122.2004

    View details for Web of Science ID 000220043100046

    View details for PubMedID 14990730

  • Hepatic gene transfer 12th International Falk Liver Week Kay, M. A. SPRINGER. 2004: 171–171
  • Nonhomologous-end-joining factors regulate DNA repair fidelity during Sleeping Beauty element transposition in mammalian cells MOLECULAR AND CELLULAR BIOLOGY Yant, S. R., Kay, M. A. 2003; 23 (23): 8505-8518


    Herein, we report that the DNA-dependent protein kinase (DNA-PK) regulates the DNA damage introduced during Sleeping Beauty (SB) element excision and reinsertion in mammalian cells. Using both plasmid- and chromosome-based mobility assays, we analyzed the repair of transposase-induced double-stranded DNA breaks in cells deficient in either the DNA-binding subunit of DNA-PK (Ku) or its catalytic subunit (DNA-PKcs). We found that the free 3' overhangs left after SB element excision were efficiently and accurately processed by the major Ku-dependent nonhomologous-end-joining pathway. Rejoining of broken DNA molecules in the absence of Ku resulted in extensive end degradation at the donor site and greatly increased the frequency of recombination with ectopic templates. Therefore, the major DNA-PK-dependent DNA damage response predominates over more-error-prone repair pathways and thereby facilitates high-fidelity DNA repair during transposon mobilization in mammalian cells. Although transposable elements were not found to be efficiently circularized after transposase-mediated excision, DNA-PK deficiency supported more-frequent transposase-mediated element insertion than was found in wild-type controls. We conclude that, based on its ability to regulate excision site junctional diversity and transposon insertion frequency, DNA-PK serves an important protective role during transpositional recombination in mammals.

    View details for DOI 10.1128/MCB.23.23.8505-8518.2003

    View details for Web of Science ID 000186618300010

    View details for PubMedID 14612396

  • Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy BLOOD Grimm, D., Zhou, S. Z., Nakai, H., Thomas, C. E., Storm, T. A., Fuess, S., Matsushita, T., Allen, J., Surosky, R., Lochrie, M., Meuse, L., McClelland, A., Colosi, P., Kay, M. A. 2003; 102 (7): 2412-2419


    We report the generation and use of pseudotyped adeno-associated viral (AAV) vectors for the liver-specific expression of human blood coagulation factor IX (hFIX). Therefore, an AAV-2 genome encoding the hfIX gene was cross-packaged into capsids of AAV types 1 to 6 using efficient, large-scale technology for particle production and purification. In immunocompetent mice, the resultant vector particles expressed high hFIX levels ranging from 36% (AAV-4) to more than 2000% of normal (AAV-1, -2, and -6), which would exceed curative levels in patients with hemophilia. Expression was dose- and time-dependent, with AAV-6 directing the fastest and strongest onset of hFIX expression at all doses. Interestingly, systemic administration of 2 x 1012 vector particles of AAV-1, -4, or -6 resulted in hFIX levels similar to those achieved by portal vein delivery. For all other serotypes and particle doses, hepatic vector administration yielded up to 84-fold more hFIX protein than tail vein delivery, corroborated by similarly increased vector DNA copy numbers in the liver, and elicited a reduced immune response against the viral capsids. Finally, neutralization assays showed variable immunologic cross-reactions between most of the AAV serotypes. Our technology and findings should facilitate the development of AAV pseudotype-based gene therapies for hemophilia B and other liver-related diseases.

    View details for Web of Science ID 000185666500023

    View details for PubMedID 12791653

  • A gene-deleted adenoviral vector results in phenotypic correction of canine hemophilia B without liver toxicity or thrombocytopenia BLOOD Ehrhardt, A., Xu, H., Dillow, A. M., Bellinger, D. A., Nichols, T. C., Kay, M. A. 2003; 102 (7): 2403-2411


    Many approaches for treating hemophilia via gene transfer have been attempted in large animal models but all have potential drawbacks. Recombinant adenoviral vectors offer high-efficiency transfer of an episomal vector but have been plagued by the cytotoxicity/immunogenicity of early-generation vectors that contain viral genes. In our current study, we have used a nonintegrating helper-dependent (HD) adenoviral vector for liver-directed gene transfer to achieve hemostatic correction in a dog with hemophilia B. We measured plasma canine factor IX (cFIX) concentrations at a therapeutic range for up to 2.5 months and normalization of the whole blood clotting time (WBCT) for about a month. This was followed by a decrease and stabilized partial correction for 4.5 months. Hepatic gene transfer of a slightly lower dose of the HD vector resulted in WBCTs that were close to normal for 2 weeks, suggesting a dose threshold effect in dogs. In sharp contrast to other studies using first- or second-generation adenoviral vectors, we observed no vector-related elevation of liver enzymes, no fall in platelet counts, and normal liver histology. Taken together, this study demonstrates that injection of an adenoviral HD vector results in complete but transient phenotypic correction of FIX deficiency in canine models with no detectable toxicity.

    View details for Web of Science ID 000185666500022

    View details for PubMedID 12805062

  • Helper-independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo MOLECULAR THERAPY Mikkelsen, J. G., Yant, S. R., Meuse, L., Huang, Z., Xu, H., Kay, M. A. 2003; 8 (4): 654-665


    Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression in normal mice and (ii) partial phenotypic correction in a mouse model of hemophilia B. SB-based HITT vectors represent a major advance in the establishment of persistent transgene expression from nonviral gene delivery systems and should prove useful for gene transfer to tissues or cell types in which transfection efficiencies are low.

    View details for DOI 10.1016/S1525-0016(03)00216-8

    View details for Web of Science ID 000185754400021

    View details for PubMedID 14529839

  • Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo MOLECULAR THERAPY Chen, Z. Y., He, C. Y., Ehrhardt, A., Kay, M. A. 2003; 8 (3): 495-500


    The loss of transgene expression has been a major obstacle to the development of nonviral vectors for the treatment of human diseases. We previously demonstrated that bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo. To confirm these studies and develop a means to produce a robust DNA vector that is not silenced in vivo, we developed a phage phiC31 integrase-mediated intramolecular recombination technology to prepare minicircle vector DNA devoid of the bacterial backbone and then compared the transgene expression profile of the minicircle with different molecular forms of plasmid DNAs in mice. We demonstrate that minicircular DNAs devoid of bacterial sequences expressed 45- and 560-fold more serum human factor IX and alpha1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Our data suggest that minicircles are capable of expressing high and persistent levels of therapeutic products in vivo and have a great potential to serve as episomal vectors for the treatment of a wide variety of diseases.

    View details for DOI 10.1016/S1525-0016(03)00168-0

    View details for Web of Science ID 000185197100019

    View details for PubMedID 12946323

  • Hepatocyte targeting for quantifiable and functional cellular transplantation 89th Annual Clinical Congress of the American-College-of-Surgeons Jenkins, D. D., Streetz, K., Kay, M., Longaker, M., Sylvester, K. ELSEVIER SCIENCE INC. 2003: S15–S15
  • System for simultaneous tissue-specific and disease-specific regulation of therapeutic gene expression HUMAN GENE THERAPY Chyung, Y. H., Peng, P. D., Kay, M. A. 2003; 14 (13): 1255-1264


    Gene therapy has been proposed as an alternative strategy for treating nongenetic disorders, such as cancer and coronary artery disease. However, for many of these types of diseases, the therapeutic genes must be tightly regulated, as extensive toxicity and pathology can result if their expression is not adequately controlled. Toward this end, we have developed a regulatory system in which the expression of a therapeutic transgene is controlled simultaneously by both a tissue-specific promoter and a disease-specific promoter. Thus, the transgene of interest will be expressed in a given cell only if both of these promoters are active. Unlike many other transgene-regulatory systems that have been previously developed, this system does not require the persistent expression of any foreign genes that could provoke an immune response or lead to toxicity. As proof of concept, we synthesized a construct harboring the lacZ transgene that is under the control of both the hepatocyte-specific human alpha(1)-antitrypsin promoter and the zinc-inducible mouse metallothionein promoter. We show that reporter gene expression from this construct is regulated in both a hepatocyte-specific and zinc-regulated manner, as reporter gene expression occurs only in hepatocyte-derived cells that have been exposed to zinc. The improved regulation offered by our system would facilitate the targeting of transgene expression to sites of disease in the body and spare healthy tissue, thereby considerably enhancing the therapeutic window of gene therapy.

    View details for Web of Science ID 000184794500005

    View details for PubMedID 12952597

  • A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice HEPATOLOGY McCaffrey, A. P., Meuse, L., Karimi, M., Contag, C. H., Kay, M. A. 2003; 38 (2): 503-508


    Hepatitis C virus (HCV) is an RNA virus infecting one in every 40 people worldwide. Current treatments are ineffective and HCV is the leading cause of liver failure leading to transplantation in the United States and Europe. Translational control of HCV is a prime therapeutic target. We assessed the inhibitory potential of morpholino phosphoramidate antisense oligonucleotides (morpholinos) on HCV translation by codelivering them with reporter plasmids expressing firefly luciferase under the translational control of the HCV internal ribosome entry site (IRES) into the livers of mice. Real-time imaging of HCV IRES luciferase reporter messenger RNA (mRNA) translation in living mice showed that a 20-mer complementary to nucleotides 345-365 of the IRES inhibited translation by greater than 95% for at least 6 days and showed mismatch specificity. No significant nonspecific inhibition of a cap-dependent luciferase or encephalomyocarditis virus (EMCV) IRES luciferase reporter translation was observed. Inhibition by the 20-mer morpholino was dose dependent, with 1 nmol/mouse giving the highest inhibition. In conclusion, morpholino antisense oligonucleotides are potent inhibitors of HCV IRES translation in a preclinical mouse model; morpholinos have potential as molecular therapeutics for treating HCV and other viral infections. The in vivo model described is a broadly applicable, straightforward, and rapid readout for inhibitor efficacy. As such, it will greatly facilitate the development of novel therapeutic strategies for viral hepatitis. Notably, the level of antisense inhibition observed in this in vivo model is similar to the maximal inhibition we have obtained previously with RNA interference in mice.

    View details for DOI 10.1053/jhep.2003.50330

    View details for Web of Science ID 000184531300026

    View details for PubMedID 12883495

  • The effect of age on hepatic gene transfer with self-inactivating lentiviral vectors in vivo MOLECULAR THERAPY Park, F., Ohashi, K., Kay, M. A. 2003; 8 (2): 314-323


    It is known that cellular proliferation, by either compensatory regeneration or direct hyperplasia, can augment lentiviral vector transduction into hepatocytes in vivo. For this reason, the present study was designed to determine if adolescent mice (312 weeks of age), which still have relatively proliferating livers, would have differential transduction compared to older (7 weeks of age) mice. Self-inactivating lentiviral vectors containing the human alpha(1)-antitrypsin (hAAT) promoter driving the expression of either the bacterial lacZ gene or the hAAT cDNA were generated for these studies. We found that adolescent mice given lentiviral vectors expressing lacZ (50 micro g p24/mouse) via intravenous administration had a significantly higher level of hepatocyte transduction as measured by X-gal staining of liver sections compared to the 7-week-old mice. In addition, serum hAAT levels were nearly 40-fold higher in 312-week-old mice administered lentiviral vectors expressing hAAT (50 micro g p24/mouse) compared to the 7-week-old mice. Moreover, the incorporation of a matrix attachment region from immunoglobulin kappa significantly increased transduction of hepatocytes in vivo. Although there was a small reduction in the circulating levels of hAAT, likely due to an immune response against the transgene product, gene expression was sustained for the duration of the study (30 weeks in total). In conclusion, the present study strongly demonstrates that lentiviral vector transduction efficiency and transgene expression were significantly enhanced in adolescent compared to older mice.

    View details for DOI 10.1016/S1525-0016(03)00169-2

    View details for Web of Science ID 000184767300016

    View details for PubMedID 12907154

  • From virus evolution to vector revolution: use of naturally occurring serotypes of adeno-associated virus (AAV) as novel vectors for human gene therapy. Current gene therapy Grimm, D., Kay, M. A. 2003; 3 (4): 281-304


    Gene transfer vectors based on the human adeno-associated virus serotype 2 (AAV-2) have been developed and tested in pre-clinical studies for almost 20 years, and are currently being evaluated in clinical trials. So far, all these studies have provided evidence that AAV-2 vectors possess many properties making them very attractive for therapeutic gene delivery to humans, such as a lack of pathogenicity or toxicity, and the ability to confer long-term gene expression. However, there is concern that two restrictions of AAV-2 vectors might limit their clinical use in humans. First, these vectors are rather inefficient at transducing some cells of therapeutic interest, such as liver and muscle cells. Second, gene transfer might be hampered by neutralizing anti-AAV-2 antibodies, which are highly prevalent in the human population. In efforts to overcome both limitations, an increasing number of researchers are now focusing on the seven other naturally occurring serotypes of AAV (AAV-1 and AAV-3 to -8), which are structurally and functionally different from AAV-2. To this end, several strategies have been devised to cross-package an AAV-2 vector genome into the capsids of the other AAV serotypes, resulting in a new generation of "pseudotyped" AAV vectors. In vitro and in vivo, these novel vectors were shown to have a host range different from AAV-2, and to escape the anti-AAV-2 immune response, thus underscoring the great potential of this approach. Here the biology of the eight AAV serotypes is summarized, existing technology for pseudotyped AAV vector production is described, initial results from pre-clinical evaluation of the vectors are reviewed, and finally, the prospects of these promising novel tools for human gene therapy are discussed.

    View details for PubMedID 12871018

  • In vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus JOURNAL OF CLINICAL INVESTIGATION Bordier, B. B., Ohkanda, J., Liu, P., Lee, S. Y., Salazar, F. H., Marion, P. L., Ohashi, K., Meuse, L., Kay, M. A., Casey, J. L., Sebti, S. M., Hamilton, A. D., Glenn, J. S. 2003; 112 (3): 407-414


    Hepatitis delta virus (HDV) can dramatically worsen liver disease in patients coinfected with hepatitis B virus (HBV). No effective medical therapy exists for HDV. The HDV envelope requires HBV surface antigen proteins provided by HBV. Once inside a cell, however, HDV can replicate its genome in the absence of any HBV gene products. In vitro, HDV virion assembly is critically dependent on prenyl lipid modification, or prenylation, of its nucleocapsid-like protein large delta antigen. To overcome limitations of current animal models and to test the hypothesis that pharmacologic prenylation inhibition can prevent the production of HDV virions in vivo, we established a convenient mouse-based model of HDV infection capable of yielding viremia. Such mice were then treated with the prenylation inhibitors FTI-277 and FTI-2153. Both agents were highly effective at clearing HDV viremia. As expected, HDV inhibition exhibited duration-of-treatment dependence. These results provide the first preclinical data supporting the in vivo efficacy of prenylation inhibition as a novel antiviral therapy with potential application to HDV and a wide variety of other viruses.

    View details for DOI 10.1172/JCI200317704

    View details for Web of Science ID 000184569100016

    View details for PubMedID 12897208

    View details for PubMedCentralID PMC166292

  • Episomal persistence of recombinant adenoviral vector Genomes during the cell cycle in vivo JOURNAL OF VIROLOGY Ehrhardt, A., Xu, H., Kay, M. A. 2003; 77 (13): 7689-7695


    Previously we showed that recombinant adenoviral helper-dependent (HD) vectors result in long-term transgene expression levels in vivo which slowly declined by 95% over a period of 1 year. In this study, we further establish that this was not predominantly immune mediated. To determine if cell turnover was responsible for the loss of transgene expression, we induced rapid hepatocyte cell cycling in mouse liver, by performing a surgical two-thirds partial hepatectomy. We observed a 55 and 65% reduction in transgene expression levels and a 50 and 71% loss of vector genomes for the HD vector and the first-generation adenoviral vector. In sharp contrast, in nonviral, episomal plasmid DNA-injected mice, transgene expression levels and DNA copy numbers decreased by 95 and 99%, respectively. These findings suggest that cell division alone was not the primary reason for the slow decrease in transgene expression levels and that recombinant adenoviral vectors have a more robust mechanism for maintaining persistence during cell cycling. Several potential mechanisms are proposed.

    View details for DOI 10.1128/JVI.77.13.7689-7695.2003

    View details for Web of Science ID 000183598600058

    View details for PubMedID 12805471

  • AAV serotype 2 vectors preferentially integrate into active genes in mice NATURE GENETICS Nakai, H., Montini, E., Fuess, S., Storm, T. A., Grompe, M., Kay, M. A. 2003; 34 (3): 297-302


    Recombinant adeno-associated virus serotype 2 (rAAV2) is a promising vector for gene therapy because it can achieve long-term stable transgene expression in animals and human subjects after direct administration of vectors into various target tissues. In the liver, although stable transgene expression primarily results from extrachromosomal vector genomes, a series of experiments has shown that vector genomes integrate into host chromosomes in hepatocytes at a low frequency. Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice and two human subjects have raised concerns about the potential for rAAV2-mediated insertional mutagenesis. Here we characterize rAAV2-targeted chromosomal integration sites isolated from selected or non-selected hepatocytes in vector-injected mouse livers. We document frequent chromosomal deletions of up to 2 kb at integration sites (14 of 14 integrations, 100%; most of the deletions were <0.3 kb) and preferred integration into genes (21 of 29 integrations, 72%). In addition, all of the targeted genes analyzed (20 of 20 targeted genes, 100%) were expressed in the liver. This is the first report to our knowledge on host chromosomal effects of rAAV2 integration in animals, and it provides insights into the nature of rAAV2 vector integration into chromosomes in quiescent somatic cells in animals and human subjects.

    View details for DOI 10.1038/ng1179

    View details for Web of Science ID 000183815300017

    View details for PubMedID 12778174

  • Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer GENE THERAPY Ortiz-Urda, S., Lin, Q., Yant, S. R., Keene, D., Kay, M. A., Khavari, P. A. 2003; 10 (13): 1099-1104


    Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

    View details for DOI 10.1038/

    View details for Web of Science ID 000183524000004

    View details for PubMedID 12808440

  • Inhibition of hepatitis B virus in mice by RNA interference NATURE BIOTECHNOLOGY McCaffrey, A. P., Nakai, H., Pandey, K., Huang, Z., Salazar, F. H., Xu, H., Wieland, S. F., Marion, P. L., Kay, M. A. 2003; 21 (6): 639-644


    Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.

    View details for DOI 10.1038/nbt824

    View details for Web of Science ID 000183220800021

    View details for PubMedID 12740585

  • Pathways of removal of free DNA vector ends in normal and DNA-PKcs-deficient SCID mouse Hepatocytes transduced with rAAV vectors HUMAN GENE THERAPY Nakai, H., Storm, T. A., Fuess, S., Kay, M. A. 2003; 14 (9): 871-881


    Elucidation of the mechanisms of transformation of single-stranded (ss) recombinant adeno-associated virus (rAAV) vector genomes into a variety of stable double-stranded (ds) forms is key to a complete understanding of rAAV vector transduction in vivo. Ds monomer genome formation and cellular ds DNA break (DSB) repair pathways that remove free vector ends toxic to cells, presumably play a central role in this process. By delivering rAAV and naked ds linear DNA vectors into livers of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-deficient severe combined immunodeficiency (SCID) and wild-type mice, we demonstrate the presence of three major pathways for free ds vector end removal: (1) DNA-PKcs-dependent self-circularization, (2) DNA-PKcs-independent self-circularization, and (3) DNA-PKcs-independent concatemerization. By using the DNA-PKcs-independent pathways, mouse hepatocytes efficiently removed free ds rAAV vector ends even in the absence of DNA-PKcs. Our studies suggest a hierarchical organization of these processes; self-circularization is the preferred pathway over concatemerization, although the former has a limited capacity to remove free vector ends. These studies shed new light on the molecular mechanisms of rAAV vector transduction in vivo.

    View details for Web of Science ID 000183528700003

    View details for PubMedID 12828858

  • Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6 MOLECULAR THERAPY Grimm, D., Kay, M. A., Kleinschmidt, J. A. 2003; 7 (6): 839-850


    We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as "HOT," is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.

    View details for DOI 10.1016/S1525-0016(03)00095-9

    View details for Web of Science ID 000183385100017

    View details for PubMedID 12788658

  • Progress and problems with the use of viral vectors for gene therapy NATURE REVIEWS GENETICS Thomas, C. E., Ehrhardt, A., Kay, M. A. 2003; 4 (5): 346-358


    Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.

    View details for DOI 10.1038/nrg1066

    View details for Web of Science ID 000182664800015

    View details for PubMedID 12728277

  • Assessing the risk of inadvertant germline transmision of recombinant AAV-2 vector 6th Annual Meeting of the American-Society-of-Gene-Therapy Arruda, V. R., Schuettrumpf, J., Liu, J. H., Addya, K., Leonard, D., Couto, L., Chew, A., Zhen, Z., Sommer, J., Herzog, R. W., Kay, M. A., Bert, G., Manno, C. S., High, K. A. NATURE PUBLISHING GROUP. 2003: S161–S162
  • Cleavage/excision of plasmid DNA in vivo leads to increased maintenance and persistence of transgenes expression in mouse 6th Annual Meeting of the American-Society-of-Gene-Therapy Riu, E., Huang, Z., Kay, M. A. NATURE PUBLISHING GROUP. 2003: S9–S10
  • Australian patients in a multi-centre phase I/II trial of AAV-mediated gene transfer to the liver for severe hemophilia B 3rd Meeting of the Australasian-Gene-Therapy-Society Rasko, J. E., High, K., Kay, M. A., Glader, B., Manno, C. S., Hutchinson, S., Dake, M., Razavi, M., Kaye, R., Arruda, V. R., Herzog, R., McClelland, A., Pearce, G., Rustagi, P., Johnson, F., Hoots, K., Blatt, P., Leonard, D. G., Addya, K., Konkle, B., Chew, A., Larsen, S., Couto, L. WILEY-BLACKWELL. 2003: S3–S4
  • Sleeping Beauty transposase-transposon cis-vectors for efficient nonviral gene delivery and persistent gene expression in vivo 6th Annual Meeting of the American-Society-of-Gene-Therapy Mikkelsen, J. G., Yant, S. R., Meuse, L., Huang, Z., Xu, H., Kay, M. A. NATURE PUBLISHING GROUP. 2003: S74–S74
  • Pre-clinical in vivo evaluation of pseudotyped adeno-associated virus (AAV) vectors for liver gene therapy 6th Annual Meeting of the American-Society-of-Gene-Therapy Grimm, D., Zhou, S. Z., Nakai, H., Thomas, C. E., Storm, T. A., Fuess, S., Matsushita, T., Surosky, R., Lochrie, M., Meuse, L., McClelland, A., Colosi, P., Kay, M. A. NATURE PUBLISHING GROUP. 2003: S27–S27
  • AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B BLOOD Manno, C. S., Chew, A. J., Hutchison, S., Larson, P. J., Herzog, R. W., Arruda, V. P., Tai, S. J., Ragni, M. V., Thompson, A., Ozelo, M., Couto, L. B., Leonard, D. G., Johnson, F. A., McClelland, A., Scallan, C., Skarsgard, E., Flake, A. W., Kay, M. A., High, K. A., Glader, B. 2003; 101 (8): 2963-2972


    Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 x 10(11) vector genomes (vg)/kg to 1.8 x 10(12) vg/kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Pre-existing high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals.

    View details for DOI 10.1182/blood-2002-10-3296

    View details for Web of Science ID 000182101400015

    View details for PubMedID 12515715

  • Advancing molecular therapies through in vivo bioluminescent imaging. Molecular imaging McCaffrey, A., Kay, M. A., Contag, C. H. 2003; 2 (2): 75-86


    Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLi is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

    View details for PubMedID 12964305

  • Optimization of cis-acting elements for gene expression from nonviral vectors in vivo HUMAN GENE THERAPY Ehrhardt, A., Peng, P. D., Xu, H., Meuse, L., Kay, M. A. 2003; 14 (3): 215-225


    While naked DNA gene transfer in vivo usually results in transient gene expression, in some cases long-term transgene expression can be achieved. Here we demonstrate that cis-acting DNA elements flanking the transgene expression cassette and components in the plasmid backbone can significantly influence expression levels from nonviral vectors. To demonstrate this, we administered our most robust human coagulation factor IX (hFIX) expression cassette placed in two different plasmid backbones, into the livers of mice, by hydrodynamic transfection. We found that placing the expression cassette within a minimal plasmid vector pHM5, a modified version of pUC19, resulted in 10 times higher serum hFIX expression levels (up to 20000 ng/ml, 400% of normal hFIX serum levels), compared to a pBluescript backbone. To optimally increase expression levels from a nonviral vector, we added matrix attachment regions (MARs) as cis-acting DNA elements flanking the hFIX expression cassette. We detected five fold higher hFIX expression levels in vivo for up to 1-year posttransfection from a vector that contained the chicken MAR from the lysozyme locus. Together, the present work demonstrates that in addition to the transgene expression cassette, cis-acting DNA elements within and outside of the plasmid backbone need to be evaluated to achieve optimal expression levels in a nonviral gene therapy approach.

    View details for Web of Science ID 000181231200003

    View details for PubMedID 12639302

  • Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice MOLECULAR THERAPY Nakai, H., Montini, E., Fuess, S., Storm, T. A., Meuse, L., Finegold, M., Grompe, M., Kay, M. A. 2003; 7 (1): 101-111


    Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Here we describe the use of naked double-stranded (ds) linear DNA as a way to insert exogenous DNA sequences into chromosomes of mouse hepatocytes in vivo, without helper components such as integrase or transposase. We constructed ds linear DNA vectors with or without adeno-associated virus inverted terminal repeats (AAV-ITRs), introduced them into mouse hepatocytes in vivo using a hydrodynamics-based transfection technique, and analyzed for vector genome integration in various ways. Surprisingly, these linear DNA molecules integrated in mouse hepatocytes in vivo at a level of 0.3-0.5 vector genome, or more, per diploid genomic equivalent irrespective of the AAV-ITR sequences. Our results establish a novel and simple way to engineer chromosomes in vivo and provide further insights into the mechanisms of recombinant AAV vector integration in vivo. In addition, they may provide a clue for developing new nonviral integrating gene delivery vector systems.

    View details for DOI 10.1016/S1525-0016(02)00023-0

    View details for Web of Science ID 000180967900015

    View details for PubMedID 12573623

  • Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo MOLECULAR THERAPY Nakai, H., Fuess, S., Storm, T. A., Meuse, L. A., Kay, M. A. 2003; 7 (1): 112-121


    Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C- and L-, which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.

    View details for DOI 10.1016/S1525-0016(02)00034-5

    View details for Web of Science ID 000180967900016

    View details for PubMedID 12573624

  • Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice BIOTECHNOLOGY PROGRESS Sclimenti, C. R., Neviaser, A. S., Baba, E. J., Meuse, L., Kay, M. A., Calos, M. P. 2003; 19 (1): 144-151


    We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo. We demonstrated that addition of the EBV EBNA1 gene and its family of repeats binding sites provided a 10- to 100-fold increase in prolonged hFIX expression in mouse liver. A single 25-microg dose of vector DNA generated normal (>5 microg/mL) levels of hFIX throughout the 8 month duration of the experiment. Vector DNA with or without the EBV sequences was retained in liver cells, and vector replication was not a factor in these nondividing liver cells. Instead, it appears that enhancement of stable hFIX expression by the EBV components was responsible for the increased level and duration of therapeutic gene expression. The EBV sequences also significantly enhanced stable expression of a vector carrying the full genomic hFIX gene delivered to mouse liver. These results underline the crucial importance of appropriate gene expression signals on gene therapy vectors and the utility of EBV sequences in particular for increasing stable gene expression.

    View details for DOI 10.1021/bp0200907

    View details for Web of Science ID 000180973100020

    View details for PubMedID 12573017

  • In vivo correction of murine tyrosinemia type I by DNA-mediated transposition MOLECULAR THERAPY Montini, E., Held, P. K., Noll, M., Morcinek, N., Al-Dhalimy, M., Finegold, M., Yant, S. R., Kay, M. A., Grompe, M. 2002; 6 (6): 759-769


    Gene therapy applications of naked DNA constructs for genetic disorders have been limited because of lack of permanent transgene expression. This limitation, however, can be overcome by the Sleeping Beauty (SB) transposable element, which can achieve permanent transgene expression through genomic integration from plasmid DNA. To date, only one example of an in vivo gene therapy application of this system has been reported. In this report, we have further defined the activity of the SB transposon in vivo by analyzing the expression and integration of a fumarylacetoacetate hydrolase (FAH) transposon in FAH-deficient mice. In this model, stably corrected FAH(+) hepatocytes are clonally selected and stable integration events can therefore be quantified and characterized at the molecular level. Herein, we demonstrate that SB-transposon-transfected hepatocytes can support significant repopulation of the liver, resulting in long-lasting correction of the FAH-deficiency phenotype. A single, combined injection of an FAH-expressing transposon plasmid and a transposase expression construct resulted in stable FAH expression in approximately 1% of transfected hepatocytes. The average transposon copy number was determined to be approximately 1/diploid genome and expression was not silenced during serial transplantation. Molecular analysis indicated that high-efficiency DNA-mediated transposition into the mouse genome was strictly dependent on the expression of wild-type transposase.

    View details for DOI 10.1006/mthe.2002.0812

    View details for Web of Science ID 000180026100010

    View details for PubMedID 12498772

  • A story of mice and men. Gene therapy McCaffrey, A. P., Kay, M. A. 2002; 9 (23): 1563-?

    View details for PubMedID 12424608

  • A phase I/II clinical trial for liver directed AAV-mediated gene transfer for severe hemophilia B. 44th Annual Meeting of the American-Society-of-Hematology Kay, M. A., High, K., Glader, B., Manno, C. S., Hutchinson, S., Dake, M., Razavi, M., Kaye, R., Arruda, V. R., Herzog, R., McClelland, A., Rustagi, P., Johnson, F., Rasko, J. E., Hoots, K., Blatt, P., Leonard, D. G., Addya, K., Konkle, B., Chew, A., Couto, L. AMER SOC HEMATOLOGY. 2002: 115A–115A
  • Assessing the risk of inadvertent germline transmission of vector DNA following intravascular delivery of rAAV vector. 44th Annual Meeting of the American-Society-of-Hematology Arruda, V. R., Schuettrumpf, J., Couto, L., Leonard, D., Addya, K., Liu, J. H., Sommer, J., Herzog, R. W., Kay, M. A., Glader, B., Manno, C. S., Chew, A., High, K. A. AMER SOC HEMATOLOGY. 2002: 869A–869A
  • Site-specific genomic integration produces therapeutic Factor IX levels in mice NATURE BIOTECHNOLOGY Olivares, E. C., Hollis, R. P., Chalberg, T. W., Meuse, L., Kay, M. A., Calos, M. P. 2002; 20 (11): 1124-1128


    We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.

    View details for DOI 10.1038/nbt753

    View details for Web of Science ID 000179041500023

    View details for PubMedID 12379870

  • A limited number of transducible hepatocytes restricts a wide-range linear vector dose response in recombinant adeno-associated virus-mediated liver transduction JOURNAL OF VIROLOGY Nakai, H., Thomas, C. E., Storm, T. A., Fuess, S., Powell, S., Wright, J. F., Kay, M. A. 2002; 76 (22): 11343-11349


    Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 x 10(8) and 1.1 x 10(13) vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 x 10(9) to 3.0 x 10(11) vg/mouse. Vector doses above 3.0 x 10(11) vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 x 10(12) vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 x 10(12) vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

    View details for DOI 10.1128/JVI.76.22.11343-11349.2002

    View details for Web of Science ID 000178822400018

    View details for PubMedID 12388694

  • A prenylation inhibitor prevents production of infectious hepatitis delta virus particles JOURNAL OF VIROLOGY Bordier, B. B., Marion, P. L., Ohashi, K., Kay, M. A., Greenberg, H. B., Casey, J. L., Glenn, J. S. 2002; 76 (20): 10465-10472


    Hepatitis delta virus (HDV) causes both acute and chronic liver disease throughout the world. Effective medical therapy is lacking. Previous work has shown that the assembly of HDV virus-like particles (VLPs) could be abolished by BZA-5B, a compound with farnesyltransferase inhibitory activity. Here we show that FTI-277, another farnesyltransferase inhibitor, prevented the production of complete, infectious HDV virions of two different genotypes. Thus, in spite of the added complexity and assembly determinants of infectious HDV virions compared to VLPs, the former are also sensitive to pharmacological prenylation inhibition. Moreover, production of HDV genotype III virions, which is associated with particularly severe clinical disease, was as sensitive to prenylation inhibition as was that of HDV genotype I virions. Farnesyltransferase inhibitors thus represent an attractive potential class of novel antiviral agents for use against HDV, including the genotypes associated with most severe disease.

    View details for DOI 10.1128/JVI.76.20.10465-10472.2002

    View details for Web of Science ID 000178319600041

    View details for PubMedID 12239323

  • Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo NATURE BIOTECHNOLOGY Yant, S. R., Ehrhardt, A., Mikkelsen, J. G., Meuse, L., Pham, T., Kay, M. A. 2002; 20 (10): 999-1005


    A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

    View details for DOI 10.1038/nbt738

    View details for Web of Science ID 000178313100016

    View details for PubMedID 12244327

  • Efficient gene transduction to cultured hepatocytes by HIV-1 derived lentiviral vector 2nd International Congress on Immunosuppression Ohashi, K., Park, F., Schwall, R. H., Kay, M. A. ELSEVIER SCIENCE INC. 2002: 1431–33

    View details for Web of Science ID 000177369700028

    View details for PubMedID 12176427

  • RNA interference in adult mice. Nature McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., Kay, M. A. 2002; 418 (6893): 38-39


    RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

    View details for PubMedID 12097900

  • Gene expression - RNA interference in adult mice NATURE McCaffrey, A. P., Meuse, L., Pham, T. T., Conklin, D. S., Hannon, G. J., Kay, M. A. 2002; 418 (6893): 38-39

    View details for DOI 10.1038/418038a

    View details for Web of Science ID 000176599200030

  • A new adenoviral helper-dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo BLOOD Ehrhardt, A., Kay, M. A. 2002; 99 (11): 3923-3930


    We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus-treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 x 10(9) transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor-alpha serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that the cis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.

    View details for Web of Science ID 000175804700009

    View details for PubMedID 12010790

  • Determinants of hepatitis C translational initiation in vitro, in cultured cells and mice MOLECULAR THERAPY McCaffrey, A. P., Hashi, K., Meuse, L., Shen, S. L., Lancaster, A. M., Lukavsky, P. J., Sarnow, P., Kay, M. A. 2002; 5 (6): 676-684


    Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice. We compared translation by several mutant HCV IRES variants in cell lysates, cultured cells, and mouse liver. We measured the contribution to translation of a cap, HCV 3'-untranslated region (UTR), poly(A) tail, domains II, IIIb, IIIabc, IIIabcd, IIId, and the initiator codon. Efficient translation required a 3'-UTR in mice and HeLa cells, but not in rabbit reticulocyte lysates. Translational regulation of transfected RNAs was stringent in mice. The method we describe could be useful for studies in mice of antisense or ribozyme inhibitors targeting the IRES as well as other RNA biochemical studies in vivo.

    View details for DOI 10.1006/mthe.2002.0600

    View details for Web of Science ID 000176082700007

    View details for PubMedID 12027551

  • Role of hepatocyte direct hyperplasia in lentivirus-mediated liver transduction in vivo HUMAN GENE THERAPY Ohashi, K., Park, F., Kay, M. A. 2002; 13 (5): 653-663


    Lentiviral vectors have been used for gene transfer into the liver, but the ability of these vectors to efficiently transduce quiescent hepatocytes remains controversial. Regardless, lentivirus-mediated gene transfer is greatly enhanced when delivered during hepatocellular cycling. For this reason, the present study was designed to determine the role of hepatocyte proliferation in the enhancement of lentiviral transduction by using three different modes of liver regeneration: (1) compensatory regeneration stimulated by two-thirds partial hepatectomy, (2) direct hyperplasia after intragastric administration of the primary mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), and (3) a combination of modes 1 and 2. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentiviral vector expressing beta-galactosidase was administered to mice via the peripheral circulation after a regeneration stimulus. Gene transfer as measured by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) staining showed 30-fold higher levels of liver transduction in groups 1 and 2 as compared with the non-liver-manipulated control group (p < 0.005). The combination of TCPOBOP and partial hepatectomy (group 3) resulted in an ~80-fold increase in transduction efficiency compared with the control animals. The enhanced transduction was consistent with higher levels of hepatocellular proliferation observed in animals that received both treatments compared with either single treatment alone. Importantly, the hepatocytes were the predominant cell type transduced, although transgene expression was observed in a low number of nonparenchymal cells regardless of which liver stimulus was received. Biodistribution studies confirmed that most of the gene transfer was limited to the liver and spleen. Taken together, this study suggests that disease-induced cellular proliferation in the liver will enhance the utility of this vector in treating diseases such as viral hepatitis, liver cirrhosis, and cancer.

    View details for Web of Science ID 000174531000006

    View details for PubMedID 11916488

  • Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males MOLECULAR THERAPY Arruda, V. R., Fields, P. A., Milner, R., Wainwright, L., De Miguel, M. P., Donovan, P. J., Herzog, R. W., Nichols, T. C., Biegel, J. A., Razavi, M., Dake, M., Huff, D., Flake, A. W., Couto, L., Kay, M. A., High, K. A. 2001; 4 (6): 586-592


    A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

    View details for DOI 10.1006/mthe.2001.0491

    View details for Web of Science ID 000172632300014

    View details for PubMedID 11735343

  • Approaches for generating recombinant adenovirus vectors ADVANCED DRUG DELIVERY REVIEWS Mizuguchi, H., Kay, M. A., Hayakawa, T. 2001; 52 (3): 165-176


    Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.

    View details for Web of Science ID 000172766200003

    View details for PubMedID 11718941

  • Hepatocyte transplantation: clinical and experimental application JOURNAL OF MOLECULAR MEDICINE-JMM Ohashi, K., Park, F., Kay, M. A. 2001; 79 (11): 617-630


    Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency. Based on a significant body of work, the technique of hepatocyte transplantation has recently moved into the clinic in order to reestablish liver function without organ transplantation or to bridge the time between whole-organ liver transplantation. In addition, hepatocyte transplantation has also been proposed as a liver-directed gene therapy for a number of inherited hepatic disorders by transplanting either freshly isolated hepatocytes or genetically altered hepatocytes. To establish a research system based on the developing technology of hepatocyte transplantation, chimeric small animal models using human hepatocytes have recently been established, which would allow the study of human hepatocyte-specific functions, such as hepatitis viral infection and replication in vivo. Various aspects related to the recent progress and existing obstacles in the area of hepatocyte transplantation are summarized in this report.

    View details for Web of Science ID 000172518700003

    View details for PubMedID 11715065

  • Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vollrath, D., Feng, W., Duncan, J. L., Yasumura, D., D'Cruz, P. M., Chappelow, A., Matthes, M. T., Kay, M. A., LaVail, M. M. 2001; 98 (22): 12584-12589


    The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the retinal pigment epithelium (RPE) to phagocytize shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. We recently used positional cloning to demonstrate that the gene Mertk likely corresponds to the retinal dystrophy (rdy) locus of the RCS rat. In the present study, we sought to determine whether gene transfer of Mertk to a RCS rat retina would result in correction of the RPE phagocytosis defect and preservation of photoreceptors. We used subretinal injection of a recombinant replication-deficient adenovirus encoding rat Mertk to deliver the gene to the eyes of young RCS rats. Electrophysiological assessment of animals 30 days after injection revealed an increased sensitivity of treated eyes to low-intensity light. Histologic and ultrastructural assessment demonstrated substantial sparing of photoreceptors, preservation of outer segment structure, and correction of the RPE phagocytosis defect in areas surrounding the injection site. Our results provide definitive evidence that mutation of Mertk underlies the RCS retinal dystrophy phenotype, and that the phenotype can be corrected by treatment of juvenile animals. To our knowledge, this is the first demonstration of complementation of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration by gene transfer to the RPE. These results, together with the recent discovery of MERTK mutations in individuals with retinitis pigmentosa, emphasize the importance of the RCS rat as a model for gene therapy of diseases that arise from RPE dysfunction.

    View details for Web of Science ID 000171806100054

    View details for PubMedID 11592982

  • Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo MOLECULAR THERAPY Park, F., Kay, M. A. 2001; 4 (3): 164-173


    Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

    View details for DOI 10.1006/mthe.2001.0450

    View details for Web of Science ID 000170743800003

    View details for PubMedID 11545606

  • Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo JOURNAL OF VIROLOGY Nakai, H., Yant, S. R., Storm, T. A., Fuess, S., Meuse, L., Kay, M. A. 2001; 75 (15): 6969-6976


    Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.

    View details for Web of Science ID 000169870700026

    View details for PubMedID 11435577

  • Epstein-Barr virus/human vector provides high-level, long-term expression of alpha(1)-antitrypsin in mice MOLECULAR THERAPY Stoll, S. M., Sclimenti, C. R., Baba, E. J., Meuse, L., Kay, M. A., Calos, M. P. 2001; 4 (2): 122-129


    We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.

    View details for Web of Science ID 000170371100007

    View details for PubMedID 11482983

  • A simplified system for constructing recombinant adenoviral vectors containing heterologous peptides in the Hl loop of their fiber knob GENE THERAPY Mizuguchi, H., Koizumi, N., Hosono, T., Utoguchi, N., Watanabe, Y., Kay, M. A., Hayakawa, T. 2001; 8 (9): 730-735


    The use of recombinant adenovirus (Ad) vectors containing genetically modified capsid proteins is an attractive strategy for achieving targeted gene transfer. The HI loop of the fiber knob is a promising candidate location for the incorporation of foreign ligands for achieving this goal. However, the method of constructing an Ad vector containing a foreign ligand in the HI loop of the fiber knob has proved difficult. In this study, we developed a simple system to construct fiber-modified vectors. To do this, a vector plasmid containing a complete E1/E3-deleted Ad type 5 genome and a unique Csp45I and/or ClaI site between positions 32679 and 32680 of the Ad genome (residues threonine-546 and proline-547 of the fiber protein) was constructed. Oligonucleotides corresponding to the Arg-Gly-Asp (RGD) or Asn-Gly-Arg (NGR)-containing peptide motif (as a model) and containing a Csp45I and/or ClaI recognition site, were ligated into the Csp45I and/or ClaI-digested plasmid. The foreign transgene expression cassette was inserted into the E1 deletion site of the vector plasmid and the fiber-mutant Ad vector was produced by transfection of the PacI-digested plasmid into 293 cells. The virus containing the RGD or NGR peptide on the fiber knob was able to infect human glioma cells, which do not express coxsackievirus and adenovirus receptor (CAR), one of the Ad virus receptors, about 100-1000 times more efficient than the virus containing wild-type fiber. This suggested that the mutant virus mediated CAR-independent cell entry pathway. The simplicity of this method allows not only for easy construction of fiber-mutant Ad vectors, but also for screening of the peptides that target the vector to the desired cells and tissues.

    View details for Web of Science ID 000168638700009

    View details for PubMedID 11406768

  • In vitro ligation-based cloning of foreign DNAs into the E3 and E1 deletion regions for generation of recombinant adenovirus vectors BIOTECHNIQUES Mizuguchi, H., Kay, M. A., Hayakawa, T. 2001; 30 (5): 1112-?

    View details for Web of Science ID 000168636500033

    View details for PubMedID 11355346

  • Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver MOLECULAR THERAPY Chen, Z. Y., Yant, S. R., He, C. Y., Meuse, L., Shen, S., Kay, M. A. 2001; 3 (3): 403-410


    The short duration of transgene expression remains a major obstacle for the implementation of nonviral DNA vectors in clinical gene therapy trials. Here, we demonstrate stable, long-term transgene expression in vivo by transfecting a linear DNA expression cassette (LDNA) into mouse liver. Interestingly, despite similar quantities and cellular distribution of injected DNAs in their livers, mice receiving LDNA encoding human alpha1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular (cc) DNA for a period of 9 months (length of study). Furthermore, when a linear human factor IX expression cassette was delivered to factor IX-deficient mice, sustained serum concentrations of more than 4 microg/ml (80% of normal) of the human clotting factor and correction of the bleeding diathesis were obtained. Southern blot analyses indicate that, unlike ccDNA, LDNA rapidly formed large, unintegrated concatemers in vivo, suggesting that transgene persistence from plasmid-based vectors was influenced by the structure of the vector in transfected cells. No differences in transgene expression or DNA molecular structures were observed when AAV ITRs were included to flank the hAAT expression cassette in both ccDNA- and LDNA-treated animals. Linear DNA transfection provides an approach for achieving long-term expression of a transgene in vivo.

    View details for Web of Science ID 000167967900017

    View details for PubMedID 11273783

  • cMet activation allows persistent engraftment of ectopically transplanted xenogenic human hepatocytes in mice 18th World Congress of the Transplantation-Society Ohashi, K., Meuse, L., Schwall, R., Kay, M. A. ELSEVIER SCIENCE INC. 2001: 587–88

    View details for Web of Science ID 000167629900277

    View details for PubMedID 11266970

  • Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics NATURE MEDICINE Kay, M. A., Glorioso, J. C., Naldini, L. 2001; 7 (1): 33-40


    Considered by some to be among the simpler forms of life, viruses represent highly evolved natural vectors for the transfer of foreign genetic information into cells. This attribute has led to extensive attempts to engineer recombinant viral vectors for the delivery of therapeutic genes into diseased tissues. While substantial progress has been made, and some clinical successes are over the horizon, further vector refinement and/or development is required before gene therapy will become standard care for any individual disorder.

    View details for Web of Science ID 000166243100032

    View details for PubMedID 11135613

  • A phase I trial of AAV-mediated muscle directed gene transfer for hemophilia B. Manno, C. S., Glader, B., Ragni, M. V., Thompson, A., Costa, F. F., Chew, A. J., Herzog, R. W., Arruda, V. R., Couto, L. B., Clelland, A. M., Johnson, F., Flake, A., Skarsgard, E., Armstrong, E., Kay, M., High, K. A. AMER SOC HEMATOLOGY. 2000: 801A–801A
  • Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo JOURNAL OF VIROLOGY Nakai, H., Storm, T. A., Kay, M. A. 2000; 74 (20): 9451-9463


    Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

    View details for Web of Science ID 000089503400014

    View details for PubMedID 11000214

  • Therapeutic levels of human factor VIII and IX using HIV-1-based lentiviral vectors in mouse liver BLOOD Park, F., Ohashi, K., Kay, M. A. 2000; 96 (3): 1173-1176


    Lentiviral vectors have the potential to play an important role in hemophilia gene therapy. The present study used human immunodeficiency virus (HIV)-based lentiviral vectors containing an EF1alpha enhancer/promoter driving human factors VIII (hFVIII) or IX (hFIX) complementary DNA expression for portal vein injection into C57Bl/6 mice. Increasing doses of hFIX-expressing lentivirus resulted in a dose-dependent, sustained increase in serum hFIX levels up to approximately 50-60 ng/mL. Partial hepatectomy resulted in a 4- to 6-fold increase (P < 0.005) in serum hFIX of up to 350 ng/mL compared with the nonhepatectomized counterparts. The expression of plasma hFVIII reached 30 ng/mL (15% of normal) but was transient as the plasma levels fell concomitant with the formation of anti-hFVIII antibodies. However, hFVIII levels were persistent in immunodeficient C57Bl/6 scid mice, suggesting humoral immunity-limited gene expression in immunocompetent mice. This study demonstrates that lentiviral vectors can produce therapeutic levels of coagulation factors in vivo, which can be enhanced with hepatocellular proliferation.

    View details for Web of Science ID 000088394000055

    View details for PubMedID 10910939

  • Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro MOLECULAR THERAPY Miao, C. H., Ohashi, K., Patijn, G. A., Meuse, L., Ye, X., Thompson, A. R., Kay, M. A. 2000; 1 (6): 522-532


    We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the in vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 microg/ml (normal = 5 microg/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 microg/ml (10 to 40% of normal) for 225 days (length of experiment). These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.

    View details for Web of Science ID 000090048200007

    View details for PubMedID 10933977

  • Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system NATURE GENETICS Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z., Kay, M. A. 2000; 25 (1): 35-41


    The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications.

    View details for Web of Science ID 000086884000013

    View details for PubMedID 10802653

  • Increasing the size of rAAV-mediated expression cassettes in vivo by intermolecular joining of two complementary vectors NATURE BIOTECHNOLOGY Nakai, H., Storm, T. A., Kay, M. A. 2000; 18 (5): 527-532


    A major shortcoming to the use of adeno-associated virus (rAAV) vectors is their limited packaging size. To overcome this hurdle, we split an expression cassette and cloned it into two separate vectors. The vectors contained either a nuclear localizing Escherichia coli lacZ transgene (nlslacZ) with a splice acceptor, or the human elongation factor 1alpha ( EF1alpha) gene enhancer/promoter(s) (EF1alphaEP) with a splice donor. We co-injected a promoter-less nlslacZ vector with a vector containing either a single EF1alphaEP or a double copy of the EF1alphaEP in a head-to-head orientation, into the portal vein of mice. Gene expression, measured by both transduction efficiency and quantitation of the recombinant protein, was as much as 60-70% of that obtained from mice that received a single vector containing a complete EFalphaEP/nlslacZ expression cassette. This two-vector approach may allow development of gene therapy strategies that will carry exogenous DNA sequences with large therapeutic cDNAs and/or regulatory elements.

    View details for Web of Science ID 000087017500026

    View details for PubMedID 10802620

  • Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: Cell cycling does not influence hepatocyte transduction JOURNAL OF VIROLOGY Miao, C. H., Nakai, H., Thompson, A. R., Storm, T. A., Chiu, W., Snyder, R. O., Kay, M. A. 2000; 74 (8): 3793-3803


    Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about approximately 5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only approximately 5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5'-bromo-2'-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic beta-galactosidase. Colabeling for beta-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

    View details for Web of Science ID 000086048000039

    View details for PubMedID 10729154

    View details for PubMedCentralID PMC111888

  • Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector NATURE GENETICS Kay, M. A., Manno, C. S., Ragni, M. V., Larson, P. J., Couto, L. B., McClelland, A., Glader, B., Chew, A. J., Tai, S. J., Herzog, R. W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A. W., High, K. A. 2000; 24 (3): 257-261


    Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

    View details for Web of Science ID 000085590600015

    View details for PubMedID 10700178

  • Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses NATURE MEDICINE Ohashi, K., Marion, P. L., Nakai, H., Meuse, L., Cullen, J. M., Bordier, B. B., Schwall, R., Greenberg, H. B., Glenn, J. S., Kay, M. A. 2000; 6 (3): 327-331


    Persistence of hepatocytes transplanted into the same or related species has been established. The long-term engraftment of human hepatocytes into rodents would be useful for the study of human viral hepatitis, where it might allow the species, technical and size limitations of the current animal models to be overcome. Although transgenic mice expressing the hepatitis B virus (HBV) genome produce infectious virus in their serum, the viral life cycle is not complete, in that the early stages of viral binding and entry into hepatocytes and production of an episomal transcriptional DNA template do not occur. As for hepatitis delta virus (HDV), another cause of liver disease, no effective therapy exists to eradicate infection, and it remains resistant even to recent regimens that have considerably changed the treatment of HBV (ref. 13). Here, we demonstrate long-term engraftment of primary human hepatocytes transplanted in a matrix under the kidney capsule of mice with administration of an agonistic antibody against c-Met. These mice were susceptible to HBV infection and completion of the viral life cycle. In addition, we demonstrate super-infection of the HBV-infected mice with HDV. Our results describe a new xenotransplant model that allows study of multiple aspects of human hepatitis viral infections, and may enhance studies of human liver diseases.

    View details for Web of Science ID 000085580500044

    View details for PubMedID 10700236

  • Efficient lentiviral transduction of liver requires cell cycling in vivo NATURE GENETICS Park, F., Ohashi, K., Chiu, W., Naldini, L., Kay, M. A. 2000; 24 (1): 49-52


    Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.

    View details for Web of Science ID 000084609200014

    View details for PubMedID 10615126

  • Nuclear import of moloney murine leukemia virus DNA mediated by adenovirus preterminal protein is not sufficient for efficient retroviral transduction in nondividing cells JOURNAL OF VIROLOGY Lieber, A., Kay, M. A., Li, Z. Y. 2000; 74 (2): 721-734


    Moloney murine leukemia virus (MoMLV)-derived vectors require cell division for efficient transduction, which may be related to an inability of the viral DNA-protein complex to cross the nuclear membrane. In contrast, adenoviruses (Ad) can efficiently infect nondividing cells. This property may be due to the presence of multiple nuclear translocation signals in a number of Ad proteins, which are associated with the incoming viral genomes. Of particular interest is the Ad preterminal protein (pTP), which binds alone or in complex with the Ad polymerase to specific sequences in the Ad inverted terminal repeat. The goal of this study was to test whether coexpression of pTP with retroviral DNA carrying pTP-binding sites would facilitate nuclear import of the viral preintegration complex and transduction of quiescent cells. In preliminary experiments, we demonstrated that the karyophylic pTP can coimport plasmid DNA into the nuclei of growth-arrested cells. Retroviral transduction studies were performed with G(1)/S-arrested LTA cells or stationary-phase human primary fibroblasts. These studies demonstrated that pTP or pTP-Ad polymerase conferred nuclear import of retroviral DNA upon arrested cells when the retrovirus vector contained the corresponding binding motifs. However, pTP-mediated nuclear translocation of MoMLV DNA in nondividing cells was not sufficient for stable transduction. Additional cellular factors activated during S phase or DNA repair synthesis were required for efficient retroviral integration.

    View details for Web of Science ID 000084421700015

    View details for PubMedID 10623734

  • A phase I trial of AAV-mediated muscle-directed gene therapy for hemophilia B. Manno, C. S., Herzog, R. W., Arruda, V. R., Couto, L. B., Tai, S. J., McClelland, A., Flake, A. W., Chew, A. J., Fields, P. A., Armstrong, A. E., Leonard, D., Skarsgard, E. D., Glader, B. E., Ragni, M. V., Larson, P. J., Kay, M. A., High, K. A. AMER SOC HEMATOLOGY. 1999: 642A–642A
  • Optimization of factor IX gene expression in the liver of mice using cis-DNA elements. Miao, C. H., Patijn, G. A., Meuse, L., Ohashi, K., Thompson, A. R., Kay, M. A. AMER SOC HEMATOLOGY. 1999: 454A–455A
  • Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes JOURNAL OF VIROLOGY Lieber, A., Steinwaerder, D. S., Carlson, C. A., Kay, M. A. 1999; 73 (11): 9314-9324


    Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus-adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (DeltaAd.AAV) and stimulate transgene integration. We demonstrate here that DeltaAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. DeltaAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. DeltaAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The DeltaAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with DeltaAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that DeltaAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. DeltaAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

    View details for Web of Science ID 000083071200045

    View details for PubMedID 10516040

  • NF-kappa B activation is required for human endothelial survival during exposure to tumor necrosis factor-alpha but not to interleukin-1 beta or lipopolysaccharide JOURNAL OF BIOLOGICAL CHEMISTRY Zen, K., Karsan, A., Stempien-Otero, A., Yee, E., Tupper, J., Li, X. W., Eunson, T., Kay, M. A., Wilson, C. B., Winn, R. K., Harlan, J. M. 1999; 274 (40): 28808-28815


    In the presence of a protein synthesis inhibitor, cycloheximide, tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), or lipopolysaccharide (LPS) induces human umbilical vein endothelial cells (HUVECs) to undergo apoptosis, suggesting that constitutive or inducible cytoprotective pathways are required for cell survival. We studied the correlation between nuclear factor-kappaB (NF-kappaB) activation and cell death induced by TNF-alpha, IL-1beta, or LPS. Adenovirus-mediated overexpression of a dominant-negative IkappaBalpha (inhibitor of kappaB) mutant blocked NF-kappaB activation by gel shift assay and blocked induction of vascular cell adhesion molecule-1 protein by TNF-alpha, IL-1beta, and LPS, a NF-kappaB-dependent response. In cells overexpressing the IkappaBalpha mutant, TNF-alpha induced cell death, whereas IL-1beta or LPS did not. We conclude that cell survival following TNF-alpha stimulation is NF-kappaB-dependent but that a constitutive or inducible NF-kappaB-independent pathway(s) protects IL-1beta- or LPS-treated HUVECs from cell death.

    View details for Web of Science ID 000082912700103

    View details for PubMedID 10497254

  • A simple method for constructing E1-and E1/E4-deleted recombinant adenoviral vectors HUMAN GENE THERAPY Mizuguchi, H., Kay, M. A. 1999; 10 (12): 2013-2017


    We previously developed a two-plasmid in vitro ligation method that did not require a recombination step to produce new recombinant E1- or E1/E3-deleted adenoviral vectors. In this study, we have modified the system to improve the simplicity of vector construction and, in addition, to allow for production of an E1/E4-deleted vector.

    View details for Web of Science ID 000082062900012

    View details for PubMedID 10466635

  • Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver JOURNAL OF VIROLOGY Nakai, H., Iwaki, Y., Kay, M. A., Couto, L. B. 1999; 73 (7): 5438-5447


    Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1alpha-F.IX (hF.IX expression was controlled by the human elongation factor 1alpha [EF1alpha] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the alpha1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.

    View details for Web of Science ID 000080813500022

    View details for PubMedID 10364291

  • Implication of interfering antibody formation and apoptosis as two different mechanisms leading to variable duration of adenovirus-mediated transgene expression in immune-competent mice JOURNAL OF VIROLOGY Schowalter, D. B., Himeda, C. L., Winther, B. L., Wilson, C. B., Kay, M. A. 1999; 73 (6): 4755-4766


    This study explores the genetic and immunologic factors involved in the differences in duration of transgene expression following in vivo transduction with recombinant adenoviruses. Different strains of mice (C3H/HeJ [C3H], C57BL/6J [B6], BALB/cJ [Balb/c], C. B10-H2(b)/LiMcdJ [Balb.B], CB6F1/J [(Balb/c x B6)F1], B6C3F1/J [(B6 x C3H)F1], and BALB/cj SCID) received 5 x 10(9) PFU of the first-generation adenovirus, which expresses human alpha1-antitrypsin (Ad/RSVhAAT). While all strains studied showed similar patterns of anti-adenovirus antibody formation, only Balb/c and C3H mice developed significant levels of anti-hAAT antibodies by 8 weeks posttransduction. In addition, while all strains had quantitatively comparable amounts of adenovirus genomes and hAAT mRNA transcripts in the liver 9 days posttransduction, only Balb/c mice had undetectable adenovirus vector genomes and hAAT mRNA in the liver 40 days posttransduction. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of liver sections from control and Ad/RSVhAAT-infected mice 5, 9, and 40 days posttransduction suggested that apoptosis was involved in the rapid elimination of transduced hepatocytes in Balb/c mice. Persistent expression of hAAT protein observed in BALB/cj SCID mice suggests that antigen-dependent immunity was essential for this apoptotic process in transduced Balb/c hepatocytes. In contrast to Balb/c mice, the loss of expression in C3H mice did not correlate with the loss of vector genomes or hAAT mRNA. Instead, the anti-hAAT antibodies in C3H but not Balb/c mice were found to interfere with detection of hAAT in the serum. In Balb. B and B6 mice, vector genome, hAAT mRNA transcripts, and hAAT protein levels persisted for at least 40 days posttransduction. This persistence correlated with poor anti-hAAT antibody formation and minimal hepatocyte toxicity. The expression of hAAT in (Balb/c x B6)F1 pups was found to be intermediate between the duration observed in the parental strains, while in (C3H x B6)F1 pups hAAT expression was similar to that seen in the B6 parents, which together support polygenic control of the immune responses in these mice. In summary, these findings suggest that there are three different profiles and at least two defined immune system-mediated mechanisms resulting in the loss of hAAT expression in mice and that different strains differ in the capacity to utilize these mechanisms.

    View details for Web of Science ID 000080271400027

    View details for PubMedID 10233936

  • Mechanisms of hypoxia-induced endothelial cell death - Role of p53 in apoptosis JOURNAL OF BIOLOGICAL CHEMISTRY Stempien-Otero, A., Karsan, A., Cornejo, C. J., Xiang, H., Eunson, T., Morrison, R. S., Kay, M., Winn, R., Harlan, J. 1999; 274 (12): 8039-8045


    Endothelial cell death may contribute to tissue injury from ischemia. Little is known, however, about the characteristics of endothelial cell death in response to hypoxia. Using an in vitro model, we found that human umbilical vein endothelial cells were resistant to hypoxia-induced cell death with only a 2% reduction in viability at 24 h and 45% reduction in viability at 48 h. Overexpression of a mutant, IkappaBalpha, via adenoviral vector did not potentiate cell death in hypoxia, indicating that nuclear factor-kappaB activation was not involved in cytoprotection. Cell death in hypoxia was determined to be apoptotic by 3' labeling of DNA using terminal deoxynucleotidyl transferase staining and reversibility of cell death with a caspase inhibitor. Exposure of endothelial cells to hypoxia did not alter levels of proapoptotic and antiapoptotic Bcl-2 family members Bax and Bcl-XL by immunoblot analysis. In contrast, changes in p53 protein levels correlated with the induction of apoptosis in hypoxic endothelial cells. Inhibition of the proteasome increased p53 protein levels and accelerated cell death in hypoxia. Overexpression of p53 by adenoviral transduction was sufficient to initiate apoptosis of normoxic endothelial cells. These data provide a framework for the study of factors regulating endothelial cell survival and death in hypoxia.

    View details for Web of Science ID 000079268100062

    View details for PubMedID 10075703

  • Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors NATURE MEDICINE Snyder, R. O., Miao, C., Meuse, L., Tubb, J., Donahue, B. A., Lin, H. F., Stafford, D. W., Patel, S., Thompson, A. R., Nichols, T., Read, M. S., Bellinger, D. A., Brinkhous, K. M., Kay, M. A. 1999; 5 (1): 64-70


    Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

    View details for Web of Science ID 000077885000031

    View details for PubMedID 9883841

  • Hepatic gene therapy using adeno-associated virus vectors SEMINARS IN LIVER DISEASE Patijn, G. A., Kay, M. A. 1999; 19 (1): 61-69


    Recombinant adeno-associated virus vectors have been shown to safely transduce a number of tissues in preclinical animal studies. The level of gene transfer is sufficient to successfully treat a large number of medical disorders. Moreover, the long-term persistence of the vector sequences in animals makes it likely that this vector will be useful for genetic diseases requiring life-long therapy. This review outlines the biological principles of the vector, as well as its advantages and current limitations as it relates to use in hepatic gene therapy.

    View details for Web of Science ID 000080225400007

    View details for PubMedID 10349684

  • Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method HUMAN GENE THERAPY Mizuguchi, H., Kay, M. A. 1998; 9 (17): 2577-2583


    An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the E1 deletion site of the vector plasmid, which contains a complete E1, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9-10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CeuI and PI-SceI sites, was constructed. After the gene of interest was inserted into this shuttle plasmid, the plasmid for E1-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector). After transformation into E. coli, more than 90% of the transformants had the correct insert. To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus. The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone.

    View details for Web of Science ID 000077221800013

    View details for PubMedID 9853524

  • Inhibition of NF-kappa B activation in combination with Bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver JOURNAL OF VIROLOGY Lieber, A., He, C. Y., Meuse, L., Himeda, C., Wilson, C., Kay, M. A. 1998; 72 (11): 9267-9277


    NF-kappaB is a key regulator of the innate antiviral immune response, due in part to its transcriptional activation of cytokines and adhesion molecules, which, in turn, function in chemotaxis and activation of inflammatory cells. We reported earlier that viral gene expression in hepatocytes transduced with first-generation (E1-deleted) adenoviruses induced NF-kappaB activation, elevation of serum cytokines, and hepatocellular apoptosis during the first days postinfusion. These events did not occur in mice infused with an adenovirus vector deleted for E1, E2, E3, and late gene expression. In the present study, we used an adenovirus expressing an IkappaBalpha supersuppressor (Ad.IkappaBM) and bcl-2 transgenic mice to unravel the role of virus-induced NF-kappaB activation and apoptosis in the clearance of recombinant adenovirus vectors from the liver. The combined action of IkappaBM and Bcl-2 allowed for vector persistence in livers of C57BL/6 x C3H mice. In the absence of Bcl-2, IkappaBM expression in mouse livers significantly reduced NF-kappaB activation, cytokine expression, leukocyte infiltration, and the humoral immune response against the transgene product; however, this was not sufficient to prevent the decline of vector DNA in transduced cells. Infusion of Ad.IkappaBM caused extended apoptosis predominantly in periportal liver regions, indicating that NF-kappaB activation may protect transduced hepatocytes from apoptosis induced by adenovirus gene products. To confer vector persistence, bcl-2 transgene expression was required to block virus-induced apoptosis if NF-kappaB protection was inactivated by IkappaBM. Expression of gene products involved in early stages of apoptotic pathways was up-regulated in response to virus infusion in bcl-2 transgenic mice, which may represent a compensatory effect. Our study supports the idea that the suppression of innate defense mechanisms improves vector persistence.

    View details for Web of Science ID 000076373700093

    View details for PubMedID 9765474

  • Transient inhibition of CD28 and CD40 ligand interactions prolongs adenovirus-mediated transgene expression in the lung and facilitates expression after secondary vector administration JOURNAL OF VIROLOGY Wilson, C. B., Embree, L. J., Schowalter, D., Albert, R., Aruffo, A., Hollenbaugh, D., Linsley, P., Kay, M. A. 1998; 72 (9): 7542-7550


    Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced beta-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.

    View details for Web of Science ID 000075328600064

    View details for PubMedID 9696851

  • Method for continuous infusion into the portal vein of mice LABORATORY ANIMAL SCIENCE Patijn, G. A., Terpstra, O. T., Kay, M. A. 1998; 48 (4): 379-383


    Recombinant retroviral vectors are attractive for in vivo gene transfer into the liver because they integrate into the host-cell genome, resulting in permanent gene expression. Gene-transfer efficiency can be improved by increasing the number of retroviral particles delivered to hepatocytes. For this purpose, we report a mouse model for continuous infusion into the portal circulation permitting large-volume vector administration, which will allow marked increase in gene-transfer efficiency. Continuous saline infusion was evaluated, using various parameters, and an infusion rate of 6 ml/24 h was found safe and well tolerated for at least 2 weeks. No significant changes in liver and kidney function and electrolyte balance were observed during the infusion. In addition to providing a valuable method for in vivo hepatic gene therapy, this model has a number of other potential applications, including mouse studies of hepatic tumor therapy, pharmacology, toxicology, and liver biology.

    View details for Web of Science ID 000075659200014

    View details for PubMedID 10090047

  • High-efficiency retrovirus-mediated gene transfer into the livers of mice HUMAN GENE THERAPY Patijn, G. A., Lieber, A., Meuse, L., Winther, B., Kay, M. A. 1998; 9 (10): 1449-1456


    Recombinant retroviral vectors represent an attractive means of transferring genes into the liver because they integrate in the host cell genome and result in permanent gene expression. However, efficient gene transfer is limited by the requirement of active cell division for integration. Surgical partial hepatectomy has been the traditional method of inducing hepatocellular proliferation, but this invasive approach would be difficult to justify in clinical gene therapy. As an alternative, we used a recombinant adenovirus expressing a nonsecreted form of urokinase plaminogen activator (Ad.PGKmuPA), which results in liver regeneration over a period of 8 days. When a high-titer retroviral vector was continuously infused into the portal vein of mice during this period of hepatocyte proliferation, 33.5% of hepatocytes were stably transduced. In addition, high-level expression of a secreted transgene reporter was sustained for at least 48 weeks (length of experiment). We investigated the influence of vector titer on the in vivo transduction efficiency in our system, and found that the total number of infectious retroviral particles delivered per target cell is a critical factor. The results presented here demonstrate the ability to obtain a high gene transfer efficiency and long-term gene expression in hepatocytes in vivo without the need for surgical hepatectomy. The two-vector system described here may be of clinical relevance, as the level of hepatic gene transfer achieved has potential to be curative for a large number of genetic liver diseases.

    View details for Web of Science ID 000074706800008

    View details for PubMedID 9681416

  • Adenoviral preterminal protein stabilizes mini-adenoviral genomes in vitro and in vivo NATURE BIOTECHNOLOGY Lieber, A., He, C. Y., Kay, M. A. 1997; 15 (13): 1383-1387


    In the absence of host immunity, nonintegrating, first-generation adenoviral vectors remain stable in the nucleus of quiescent transduced cells in mice. A mini-adenoviral genome (9 kb) deleted for viral E1, E2, E3, and late genes, but containing the viral inverted terminal repeats (ITRs), transgene expression cassette (human alpha 1-antitrypsin), and the viral E4 genes was equally efficient at transducing cells in vitro or in vivo as first generation, E1-deleted vectors. In contrast to a first generation vector, gene expression as well as vector DNA was short-lived in cells transduced with the deleted adenoviral genome. We demonstrate that coexpression of the adenoviral E2-preterminal protein from the vector or in trans stabilizes the mini-genome in vitro and in vivo without evidence of cellular toxicity.

    View details for Web of Science ID A1997YK36100030

    View details for PubMedID 9415891

  • The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors JOURNAL OF VIROLOGY Lieber, A., He, C. Y., Meuse, L., Schowalter, D., Kirillova, I., Winther, B., Kay, M. A. 1997; 71 (11): 8798-8807


    Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.

    View details for Web of Science ID A1997YB14300084

    View details for PubMedID 9343240

  • Gene therapy: A status report PEDIATRIC ANNALS Schowalter, D. B., Kay, M. A. 1997; 26 (9): 562-568

    View details for Web of Science ID A1997XV78300009

    View details for PubMedID 9302719

  • Constitutive expression of murine CTLA4Ig from a recombinant adenovirus vector results in prolonged transgene expression GENE THERAPY Schowalter, D. B., Meuse, L., Wilson, C. B., Linsley, P. S., Kay, M. A. 1997; 4 (8): 853-860


    The administration of soluble muCTLA4Ig around the time of adenovirus vector mediated gene transfer into murine hepatocytes has been shown to markedly prolong transgene expression, diminish the formation of adenovirus neutralizing antibody, decrease T cell proliferative response and infiltration into the liver without causing irreversible systemic immunosuppression. In this study, an E1/E3-deleted adenovirus vector constitutively expressing murine CTLA4Ig (Ad.RSV-muCTLA4Ig) was constructed in order to determine if production of muCTLA4Ig from within transduced cells (i.e. hepatocytes) would provide a more specific/localized interference with the CD28/B7-1 and B7-2 signaling pathways, and thus result in prolonged transgene expression in vivo at nonimmunosuppressive serum concentrations. In contrast to C3H mice receiving a control adenovirus, transduction with 6 x 10(9) p.f.u. of Ad.RSV-muCTLA4Ig and a reporter adenovirus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) resulted in prolonged reporter gene expression, reduced anti-adenovirus and anti-hAAT antibody production, and attenuated T cell proliferation and IFN-gamma production in response to adenoviral vector. Mice given a constant total amount of adenovirus with diminishing amounts of Ad.RSV-muCTLA4Ig and a constant amount of reporter virus (2 x 10(9) p.f.u. of Ad.PGK-hAAT) demonstrated prolonged reporter gene expression and decreased anti-adenovirus and anti-hAAT antibody production only when high serum levels of muCTLA4Ig were produced. Taken together, these findings suggest that a certain threshold of muCTLA4Ig must be achieved to alter the immune responses and prolong transgene expression from adenoviral vectors.

    View details for Web of Science ID A1997XP16200015

    View details for PubMedID 9338015

  • Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors NATURE GENETICS Snyder, R. O., Miao, C. H., Patijn, G. A., Spratt, S. K., Danos, O., Nagy, D., GOWN, A. M., Winther, B., Meuse, L., Cohen, L. K., Thompson, A. R., Kay, M. A. 1997; 16 (3): 270-276


    Haemophilia B, or factor IX deficiency, is a X-linked recessive disorder that occurs in about one in 25,000 males, and severely affected people are at risk for spontaneous bleeding into numerous organs. Bleeding can be life-threatening or lead to chronic disabilities with haemophilic arthropathy. The severity of the bleeding tendency varies among patients and is related to the concentration of functional plasma factor IX. Patients with 5-30% of the normal factor IX have mild haemophilia that may not be recognized until adulthood or after heavy trauma or surgery. Therapy for acute bleeding consists of the transfusion of clotting-factor concentrates prepared from human blood and recombinant clotting factors that are currently in clinical trials. Both recombinant retroviral and adenoviral vectors have successfully transferred factor IX cDNA into the livers of dogs with haemophilia B. Recombinant retroviral-mediated gene transfer results in persistent yet subtherapeutic concentrations of factor IX and requires the stimulation of hepatocyte replication before vector administration. Recombinant adenoviral vectors can temporarily cure the coagulation defect in the canine haemophilia B model; however, an immune response directed against viral gene products made by the vector results in toxicity and limited gene expression. The use of recombinant adeno-associated virus (rAAV) vectors is promising because the vector contains no viral genes and can transduce non-dividing cells. The efficacy of in vivo transduction of non-dividing cells has been demonstrated in a wide variety of tissues. In this report, we describe the successful transduction of the liver in vivo using r-AAV vectors delivered as a single administration to mice and demonstrate that persistent, curative concentrations of functional human factor IX can be achieved using wild-type-free and adenovirus-free rAAV vectors. This demonstrates the potential of treating haemophilia B by gene therapy at the natural site of factor IX production.

    View details for Web of Science ID A1997XG60900023

    View details for PubMedID 9207793

  • Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kay, M. A., Meuse, L., GOWN, A. M., Linsley, P., Hollenbaugh, D., Aruffo, A., Ochs, H. D., Wilson, C. B. 1997; 94 (9): 4686-4691


    Although recombinant adenovirus vectors offer a very efficient means by which to transfer genetic information into cells in vivo, antigen-dependent immunity limits the duration of gene expression and prevents retreatment. Recombinant murine CTLA4Ig and anti-CD40 ligand antibody block costimulatory interactions between T cells and antigen presenting cells. We previously reported that murine CTLA4Ig prolongs adenoviral-mediated gene transfer, but does not allow for secondary expression after readministration of the vector. In studies described here, when anti-CD40 ligand and recombinant murine CTLA4Ig were coadministered around the time of primary vector administration (i) prolonged adenovirus-mediated gene expression (length of experiment up to 1 year) from the livers of >90% of treated mice was observed, and (ii) secondary adenovirus-mediated gene transfer was achieved in >50% of the mice even after the immunosuppressive effects of these agents were no longer present. Nearly two-thirds of these mice had persistent secondary gene expression lasting for at least 200-300 days. Neither agent alone allowed transduction after secondary vector administration. Treated mice had decreased immune responses to the vector as shown by markedly decreased production of neutralizing antibodies, diminished spleen proliferation responses and IFN-gamma production in vitro, and reduced T cell infiltrates in the liver. These results suggest that it may be possible to obtain persistence as well as secondary adenoviral-mediated gene transfer with transient immunosuppressive therapies.

    View details for Web of Science ID A1997WX36200082

    View details for PubMedID 9114052

  • Expansion of donor hepatocytes after recombinant adenovirus-induced liver regeneration in mice HEPATOLOGY Peeters, M. J., Patijn, G. A., Lieber, A., Perkins, J., Kay, M. A. 1997; 25 (4): 884-888


    Hepatocyte transplantation is a potential form of therapy for patients with genetic hepatodeficiency disorders. Unfortunately, hepatocellular transplantation has been limited because of the relatively low numbers of donor cells that can ultimately take up residence in the host liver. To give the donor cells a proliferative stimulus, a recombinant adenovirus vector that expresses a nonsecreted urokinase (urokinase-type plasminogen activator) was transduced into the livers of recipient animals before transplantation. Because urokinase production in hepatocytes causes the slow turnover of hepatocytes, 2 days after adenovirus-mediated gene transfer into the livers of recipient mice, 2 x 10(6) congenic donor cells tagged with beta-galactosidase (beta-Gal) reporter were implanted via the portal vein. As a result, on average, 8.6% of the recipient hepatocytes in the livers were derived from donor cells--a 20-fold increase compared with control animals in which no proliferative stimulus was present.

    View details for Web of Science ID A1997WT54000016

    View details for PubMedID 9096592

  • Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo GENE THERAPY Schowalter, D. B., Tubb, J. C., Liu, M., Wilson, C. B., Kay, M. A. 1997; 4 (4): 351-360


    An E1a-deleted adenovirus vector constitutively expressing native adenovirus E3-gp19K (Ad.RSV-gp19K) was constructed in order to determine whether or not E3-gp19K mediated interference with antigen presentation would result in prolonged transgene expression in vivo. Cultured fibroblasts infected with Ad.RSV-gp19K produced a native size gp19K protein and had decreased cell surface levels of MHC I as shown by immunoprecipitation and flow cytometry. The congenic mouse strains Balb/b (H-2b MHC I with high gp19K affinity), Balb/k (H-2k MHC I with no gp19K affinity), and Balb/c (H-2d MHC I with moderate gp19K affinity) were chosen for in vivo experiments because of their range of gp19K affinities. Following transduction of mice form each strain with Ad.RSV-gp19K and AD/RSV-hAAT (a reporter adenovirus), or Ad/RSV-cFIX (control adenovirus) and Ad/RSV-hAAT, the level and duration of serum hAAT protein were unrelated to gp19K protein expression. Evaluation of MHC I abundance on hepatocytes following in vivo transduction demonstrated that recombinant adenovirus rapidly increased the abundance of surface MHC I molecules on hepatocytes, and surface MHC I molecules were reduced earlier and to a greater extent following wild-type adenovirus infection compared with hepatocytes transduced with control or Ad.RSV-gp19K recombinant adenovirus. This difference in surface MHC I down-regulation may be related to the different promoters (RSV-LTR versus the native E3 promoter) and will be an important consideration in the development of newer generation adenovirus vectors designed to evade host immune responses.

    View details for Web of Science ID A1997WT21800013

    View details for PubMedID 9176522

  • Adenovirus-mediated gene therapy in a mouse model of hereditary tyrosinemia type I HUMAN GENE THERAPY Overturf, K., ALDHALIMY, M., Ou, C. N., Finegold, M., Tanguay, R., Lieber, A., Kay, M., Grompe, M. 1997; 8 (5): 513-521


    Mice lacking the enzyme fumarylacetoacetate hydrolase (FAH) have symptoms similar to humans with the disease hereditary tyrosinemia type I (HT1). FAH-deficient mice were injected with a first-generation adenoviral vector expressing the human FAH gene and followed for up to 9 months. Nontreated FAH mutant control mice died within 6 weeks from fulminant liver failure, whereas FAH adenovirus-infected animals survived until sacrifice at 2-9 months. Nine of 13 virus-treated animals developed hepatocellular cancer. Immunohistochemical analysis revealed a mosaic of FAH-deficient and FAH-positive cells in all animals and liver function tests were improved compared to controls. Even mice harvested 9 months after viral infection had > 50% FAH-positive cells. These results demonstrate the strong selective advantage of FAH-expressing cells in an FAH-deficient liver but also illustrate the danger of carcinomas arising from FAH-deficient hepatocytes in HT1.

    View details for Web of Science ID A1997WT55000002

    View details for PubMedID 9095403

  • Liver regeneration: Prospects for therapy based on new technologies MOLECULAR MEDICINE TODAY Kay, M. A., Fausto, N. 1997; 3 (3): 108-115


    The liver is an amazing organ because it can regenerate. The differentiated parenchymal cells, which do not normally divide, can undergo multiple rounds of cellular division. This brings into question the exact role of the liver stem-cell, which has not been fully characterized. The knowledge gained from the dissection of the basic molecular and cellular events that occur during hepatic regeneration will be useful for advancing therapeutic interventions for individuals with liver disease or genetic deficiencies. This article reviews the basic principles of liver regeneration, experimental manipulations in animal models, and human clinical applications including cellular transplantation, gene therapy and artificial livers.

    View details for Web of Science ID A1997WQ32500010

    View details for PubMedID 9095485

  • Development of a high-performance liquid chromatographic assay for G418 sulfate (Geneticin) ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Bethune, C., Bui, T., Liu, M. L., Kay, M. A., Ho, R. J. 1997; 41 (3): 661-664


    We have developed a chromatographic assay with high sensitivity and specificity to quantify G418 sulfate (Geneticin), an antibiotic used routinely in molecular genetics experiments for selecting eukaryotic transformants. With this method, G418 in tissues and plasma samples can be quantitated without the confounding factors often associated with biological assays. After removal of proteins in homogenized tissue or plasma samples with methanol (2:1, vol/vol), the amino group of G418 was derivatized with 1-fluoro-2,4-dinitrobenzene (DNFB) to form the UV-visible G418-DNFB product. The DNFB-derivatized G418 was separated on a reversed-phase C18 column with an acetonitrile and water gradient as the mobile phase. Under these assay conditions, the detection limit for G418 sulfate in buffer, plasma, and tissues was recorded at 78 ng/ml and the linearity was recorded for concentrations up to 100 micrograms/ml. The data obtained from this analysis indicate that this assay can be used for the quantitative determination of G418 sulfate in plasma and tissue samples.

    View details for Web of Science ID A1997WL11100029

    View details for PubMedID 9056010

  • Liver-associated toxicity of the HSV-tk/GCV approach and adenoviral vectors CANCER GENE THERAPY Brand, K., Arnold, W., Bartels, T., Lieber, A., Kay, M. A., Strauss, M., Dorken, B. 1997; 4 (1): 9-16


    Intravenous injection of immunodeficient mice with adenoviral vectors carrying the HSV-tk gene led to preferential transduction of liver tissue. Subsequent application of ganciclovir (GCV) resulted in extremely high toxicity with negligible survival rates. This toxicity was only seen when GCV was applied in addition to the adenoviral vector and not if combined with the Ad-beta gal control vector. This indicates a specific Ad-tk/GCV-related toxicity. Low survival rates were seen not only after intravenous administration but also after injection of the vectors into the portal vein, the liver tissue and liver tumors, but not during the treatment of subcutaneous tumors. This, together with extensive signs of liver degeneration occurring as early as one day after the initiation of GCV treatment, strongly suggests a specific liver-associated toxicity. Severe toxicity was observed when animals received GCV treatment as late as 7 weeks after vector administration. Moreover, in vitro survival rates of resting primary hepatocytes treated with Ad-tk and GCV were very low. We therefore suppose a mechanism of toxicity of phosphorylated GCV which is independent from cellular proliferation. Since our results indicate that the Ad-HSV-tk/GCV approach is toxic for the resting liver, additional safety features such as tumor-restricted transgene expression should be included in adenoviral vectors.

    View details for Web of Science ID A1997WE47000002

    View details for PubMedID 9012446

  • Methods for delivery of genes to hepatocytes in vivo using recombinant adenovirus vectors. Methods in molecular medicine Barr, D., Kay, M. A. 1997; 7: 205-212


    In many ways, the liver represents an ideal target organ for gene delivery. Anatomically, the sheer bulk cof its tissue mass and its dual blood supply are advantageous for intravascular injection of virus into either portal or systemic circulation. The portal vein provides a direct iv route into the liver. It also theoretically provides an indirect route by oral administration since the portal system drains the gut.

    View details for DOI 10.1385/0-89603-484-4:205

    View details for PubMedID 24493428

  • Recombinant adenoviruses with large deletions generated by cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo JOURNAL OF VIROLOGY Lieber, A., He, C. Y., Kirillova, I., Kay, M. A. 1996; 70 (12): 8944-8960


    In vivo gene transfer of recombinant E1-deficient adenoviruses results in early and late viral gene expression that elicits a host immune response, limiting the duration of transgene expression and the use of adenoviruses for gene therapy. The prokaryotic Cre-lox P recombination system was adapted to generate recombinant adenoviruses with extended deletions in the viral genome (referred to here as deleted viruses) in order to minimize expression of immunogenic and/or cytotoxic viral proteins. As an example, an adenovirus with a 25-kb deletion that lacked E1, E2, E3, and late gene expression with viral titers similar to those achieved with first-generation vectors and less than 0.5% contamination with E1-deficient virus was produced. Gene transfer was similar in HeLa cells, mouse hepatoma cells, and primary mouse hepatocytes in vitro and in vivo as determined by measuring reporter gene expression and DNA transfer. However, transgene expression and deleted viral DNA concentrations were not stable and declined to undetectable levels much more rapidly than those found for first-generation vectors. Intravenous administration of deleted vectors in mice resulted in no hepatocellular injury relative to that seen with first-generation vectors. The mechanism for stability of first-generation adenovirus vectors (E1a deleted) appeared to be linked in part to their ability to replicate in transduced cells in vivo and in vitro. Furthermore, the deleted vectors were stabilized in the presence of undeleted first-generation adenovirus vectors. These results have important consequences for the development of these and other nonintegrating vectors for gene therapy.

    View details for Web of Science ID A1996VT70400081

    View details for PubMedID 8971024

  • Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes JOURNAL OF VIROLOGY Lieber, A., He, C. Y., Polyak, S. J., Gretch, D. R., Barr, D., Kay, M. A. 1996; 70 (12): 8782-8791


    Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.

    View details for Web of Science ID A1996VT70400064

    View details for PubMedID 8971007

  • HBV-derived promoters direct liver-specific expression of an adenovirally transduced LDL receptor gene GENE THERAPY Sandig, V., Loser, P., Lieber, A., Kay, M. A., Strauss, M. 1996; 3 (11): 1002-1009


    In vivo approaches to liver gene therapy will require restriction of transgene expression to hepatocytes. Since targeting of viral vectors exclusively to the liver is not easy to achieve, use of liver-specific promoters for driving expression of therapeutic genes is an interesting alternative. We have shown previously that regulatory elements of the hepatotrophic hepatitis B virus (HBV) are strong and liver-specific in vitro and therefore might be useful in hepatic gene therapy. Here we describe recombinant adenoviruses in which the human LDL receptor gene is under the transcriptional control of the HBV core promoter, the core promoter linked directly to HBV enhancer I, or a HBV-CMV hybrid promoter, respectively. These viruses allowed for a moderate to strong expression of the LDL receptor gene in vitro in a hepatocyte-specific manner when compared with the CMV immediate-early promoter. In vivo experiments demonstrated that the promoter gave rise to an expression level comparable to that from the CMV promoter in mouse liver, but was very weak in lung and skeletal muscle. Thus, the HBV-CMV hybrid promoter is strong and hepatocyte specific both in vitro and in vivo even in the adenoviral context and would be a good choice for driving a therapeutic gene in liver gene therapy.

    View details for Web of Science ID A1996VR64300009

    View details for PubMedID 9044740

  • Adenovirus-mediated hepatic gene transfer in mice: Comparison of intravascular and biliary administration HUMAN GENE THERAPY Peeters, M. J., Patijn, G. A., Lieber, A., Meuse, L., Kay, M. A. 1996; 7 (14): 1693-1699


    Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues. In an attempt to restrict adenovirus-mediated gene transfer to the liver, we developed a microsurgical method that allowed for vector administration directly into the biliary tract of a mouse. We demonstrate that gene transfer was 4- to 10-fold more restricted to the liver after biliary tract infusion than after intravascular infusion. Intravascular infusion of recombinant adenovirus elicits a powerful immune response that limits gene expression and the ability to readminister the vector. Biliary infusion resulted in a slightly lesser immune response as determined by the lower neutralizing antibody titers directed against the vector compared with animals treated by intravascular infusion. There was no difference in the persistence of gene expression, suggesting a similar cell-mediated immune response against the vector containing cells in animals administered vector by either method. As future-generation adenovirus vectors that are safer and less immunogenic become available, the more liver specific gene transfer via the biliary tract may offer advantages over intravenous infusion for hepatic gene therapy.

    View details for Web of Science ID A1996WD32500004

    View details for PubMedID 8886840

  • erbB-2 knockout employing an intracellular single-chain antibody (sFv) accomplishes specific toxicity in erbB-2-expressing lung cancer cells AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY Grim, J., Deshane, J., Feng, M. Z., Lieber, A., Kay, M., Curiel, D. T. 1996; 15 (3): 348-354


    erbB-2 is known to be overexpressed in several human malignancies including lung cancer. Because of its role in neoplastic transformation as well as its association with poor prognosis, this oncogene has been targeted through various anti-cancer methodologies. In this regard, we have recently demonstrated that erbB-2-overexpressing ovarian tumor cell lines transfected with an endoplasmic reticulum form of an anti-erbB-2 single-chain antibody undergo a specific cytotoxicity through the induction of apoptosis. Since certain forms of lung cancer are also associated with overexpression of erbB-2, we evaluated the use of this novel therapeutic in this context. For these studies, several human lung adenocarcinoma cell lines were stably and transiently transfected with the anti-erbB-2 sFv gene. We demonstrate here that the anti-erbB-2 sFv can cause specific cytotoxicity in lung cancer cells. As a first step toward clinical translation of this strategy, we constructed a replication-deficient recombinant adenoviral vector expressing the anti-erbB-2 sFv construct. We further demonstrate that our anti-erbB-2 sFv-encoding adenoviral vector can accomplish high levels of cytotoxicity in lung cancer cells. Based on these results, it is proposed that this strategy of oncoprotein ablation may have use in the treatment of some forms of human lung cancer.

    View details for Web of Science ID A1996VH50900007

    View details for PubMedID 8810638

  • Adenovirus-mediated expression of ribozymes in mice JOURNAL OF VIROLOGY Lieber, A., Kay, M. A. 1996; 70 (5): 3153-3158


    Ribozymes are a new pharmaceutical class of reagents that offer potential in treating a number of different medical disorders, including infectious diseases and cancer. As a first step towards using ribozymes for the treatment of liver disorders such as viral hepatitis, adenovirus vectors that contain a ribozyme expression cassette under the control of different promoters directed against human growth hormone (hGH) were constructed and infused into transgenic mice that produce hGH from the gastrointestinal tract and liver. Adenovirus-mediated transfer of expressed ribozymes resulted in up to a 96% reduction of hepatic hGH mRNA over a period of several weeks in the transgenic mouse model. Furthermore, the concentration of ribozyme RNA correlated with the degree of hGH mRNA reduction. This study clearly demonstrates that ribozymes can function during the period of expression in an intact organ after somatic gene transfer.

    View details for Web of Science ID A1996UF24700056

    View details for PubMedID 8627795

  • Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: Comparison of VSV-G and amphotropic vectors for hepatic gene transfer JOURNAL OF VIROLOGY Liu, M. L., Winther, B. L., Kay, M. A. 1996; 70 (4): 2497-2502


    Recombinant retrovirus vectors are widely used for gene transfer studies. The recent development of a pseudotyped Moloney murine leukemia virus vector that contains the G envelope protein from the vesicular stomatitis virus allows for efficient concentration of vector and offers hope for potential use of these vectors for gene expression in vivo. A standard amphotropic vector expressing a serum marker protein, human alpha 1-antitrypsin, was infused into regenerating mouse liver and was 10-fold more efficient at achieving stable gene expression than was an equivalent pseudotyped vector. Discrepant results were obtained with cultured hepatocytes infected with an Escherichia coli beta-galactosidase-producing pseudotype and amphotropic vector. High rates of beta-galactosidase-positive cells were detected with the vesicular stomatitis virus G glycoprotein vector under culture conditions known to be relatively nonpermissive for retrovirus-mediated gene transfer. Subsequent studies demonstrated that beta-galactosidase protein was concentrated and copurified during pseudotype vector preparation, resulting in high rates of protein transfer rather than stable gene transfer, a process referred to as pseudotransduction. The cotransfer of protein with concentrated pseudotyped retroviruses indicates that caution must be used when interpreting gene transduction efficiencies in gene therapy experiments.

    View details for Web of Science ID A1996UA39700052

    View details for PubMedID 8642678

  • Method for multiple portal vein infusions in mice: Quantitation of adenovirus-mediated hepatic gene transfer BIOTECHNIQUES Peeters, M. J., Lieber, A., Perkins, J., Kay, M. A. 1996; 20 (2): 278-?


    For many preclinical studies, the mouse has been an invaluable model. For hepatic studies, including gene therapy, the use of the mouse has been limited because of the inability to obtain long-term portal vein access. In this study, we have developed a surgical cannula model that allows for repeat portal vein infusion in a noninvasive manner. We have used this model to establish that the tissue distribution of recombinant adenoviral vectors is similar after portal vein or peripheral vein infusion. The majority of the vector was present in the liver, ranging from 14 to 28 copies per hepatocyte. The second most prevalent tissues were the spleen and lung with 1/10 less adenoviral DNA. The brain and ovaries had the least DNA, 1/1000 less than the liver. Additional studies were performed to study the effects of secondary adenovirus infusion through the portal vein cannula. Permanent portal vein access in a mouse model will be invaluable for a large number of medical studies, including the development of new technologies for hepatic gene transfer.

    View details for Web of Science ID A1996TT95500022

    View details for PubMedID 8825158

  • LONG-TERM HEPATIC ADENOVIRUS-MEDIATED GENE-EXPRESSION IN MICE FOLLOWING CTLA4LG ADMINISTRATION NATURE GENETICS Kay, M. A., Holterman, A. X., Meuse, L., Gown, A., Ochs, H. D., Linsley, P. S., Wilson, C. B. 1995; 11 (2): 191-197


    Recombinant adenovirus vectors are efficient at transferring genes into somatic tissues but are limited for use in clinical gene therapy by immunologic factors that result in the rapid loss of gene expression and inhibit secondary gene transfer. This study demonstrates that systemic coadministration of recombinant adenovirus with soluble CTLA4Ig, which is known to block co-stimulatory signals between T cells and antigen presenting cells, leads to persistent adenoviral gene expression in mice without long-term immunosuppression. This form of immunotherapy greatly enhances the likelihood that recombinant adenovirus vectors will be useful for human gene therapy.

    View details for Web of Science ID A1995RX80600022

    View details for PubMedID 7550348

  • GENE-THERAPY FOR HEMOPHILIA-B - HOST IMMUNOSUPPRESSION PROLONGS THE THERAPEUTIC EFFECT OF ADENOVIRUS-MEDIATED FACTOR-IX EXPRESSION HUMAN GENE THERAPY Fang, B., Eisensmith, R. C., Wang, H., Kay, M. A., Cross, R. E., Landen, C. N., Gordon, G., Bellinger, D. A., Read, M. S., Hu, P. C., Brinkhous, K. M., Woo, S. L. 1995; 6 (8): 1039-1044


    Hemophilia B is caused by a deficiency of blood clotting factor IX (FIX). Previous studies have shown that the delivery of a recombinant adenoviral vector expressing canine FIX (cFIX) resulted in a complete correction of hemophilia B in FIX-deficient dogs, but that cFIX expression decreased to only about 1-2% of normal levels 3 weeks after treatment. In the present study, therapeutic levels of cFIX expression capable of producing a partial correction of hemophilia B were maintained for at least 6 months after the coadministration of the cFIX-expressing adenovirus and the immunosuppressive agent cyclosporin A (CsA). These findings support a recent report (Yang et al., 1994) that host T-cell-mediated immunity against virally transduced cells is a major contributing factor to the transient nature of adenovirus-mediated gene expression in immunocompetent animals. Although a second administration of the cFIX-expressing adenovirus 6 months after the first infusion had only a minimal effect on plasma FIX levels in a dog that had been continuously treated with CsA, the prolonged expression of the transgene indicates that immunosuppression may be applicable in attaining long-term treatment of clinically relevant disorders.

    View details for Web of Science ID A1995RP88200007

    View details for PubMedID 7578416



    Direct retrovirus-mediated hepatic gene transfer results in permanent gene expression; however, gene transfer requires surgical hepatectomy (to stimulate cell division) and has been inefficient. We recently used recombinant adenovirus vectors that transiently expressed urokinase from mouse hepatocytes to induce hepatocellular regeneration in place of a partial hepatectomy. The adenovirus method allowed for five-fold more efficient retrovirus transduction in vivo compared to the conventional partial hepatectomy approach. The major problem with the urokinase-mediated hepatic regeneration was the transient secretion of urokinase into the bloodstream that led to hypocoagulation. To circumvent this side-effect, the urokinase protein was modified by adding amino-terminal and carboxy-terminal endoplasmic reticulum retention signals. The recombinant urokinase molecules expressed from adenoviral vectors remained in hepatocytes, were enzymatically active, and resulted in similar rates of hepatic regeneration as found with the secreted urokinase. Modified urokinase-mediated liver regeneration was equally capable of allowing retrovirus-mediated gene transfer in vivo. Thus, the method of direct retrovirus transduction of hepatocytes becomes clinically relevant as the technology becomes safer.

    View details for Web of Science ID A1995RP88200006

    View details for PubMedID 7578415



    Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.

    View details for Web of Science ID A1995RF05000093

    View details for PubMedID 7597103



    High efficiency gene transfer and gene expression in hepatocytes in vivo can be achieved using recombinant adenoviral vectors. However, the persistence of gene expression in different experimental animal models has been variable. To determine if similar differences could be observed in a single species, persistence of gene expression was studied in inbred strains of mice using a recombinant adenoviral vector that expresses human alpha 1-antitrypsin. Marked variability in the persistence of gene expression ranging from several weeks (C3H/HeJ and Balb/c) to more than 3 months [C57Bl/6, B10.A(2R) and B10.BR] was observed when this vector was transduced in different strains of inbred mice. This variability did not correlate with H-2 type. To evaluate the role of T and B cell immunity in the persistence of gene expression, congenic C3H-scid and Balb/c-scid mice were studied and found to have indefinite gene expression from transduced hepatocytes. These animals unlike their immunocompetent counter-parts were able to undergo secondary transduction of hepatocytes with a different recombinant adenoviral vector. These findings suggest that as yet unidentified genetic loci influence the persistence of adenovirus-mediated hepatic gene expression in vivo, and these effects are mediated at least in part, by the antigen specific immune system.

    View details for Web of Science ID A1995QK78200010

    View details for PubMedID 7719932



    Alpha-1-antitrypsin is a relatively common genetic deficiency that results in early emphysema. The liver as the natural source of most alpha-1-antitrypsin synthesis was the target organ selected for gene replacement therapy studies. Previous work used recombinant retroviral vectors that encode the human alpha-1-antitrypsin cDNA for ex vivo and direct in vivo transduction of hepatocytes in dogs and rodents. This approach led to low levels of human protein in the serum of recipients. In this study, recombinant adenoviral vectors that express the human alpha-1-antitrypsin cDNA under the transcriptional control of the phosphoglycerate kinase (PGK) or RSV-LTR promoters have been constructed and used for the direct transduction of mouse hepatocytes in vivo. The animals transduced with the recombinant adenoviral vectors had therapeutic serum levels of human alpha-1-antitrypsin of up to 700 micrograms/mL. Thus, adenovirus-mediated gene transfer of the hAAT cDNA into the liver was able to produce therapeutic serum concentrations of protein.

    View details for Web of Science ID A1995RB79000030

    View details for PubMedID 7875680



    The presence of functional amphotropic receptors on the cell surface is necessary for amphotropic retrovirus-mediated gene transfer. A recombinant adenoviral vector that expresses the receptor for amphotropic retrovirus (RAM) was constructed and used to express the receptor cDNA in different cell types in culture. Transfer of the RAM cDNA increased amphotropic retroviral-mediated transfer from 0 to 60% in Chinese hamster ovary cells. RAM expression increased retroviral transduction four- to eight-fold from 2-4% to 18%-35% in HeLa, Namalva, and X63 cells, but had no effect on 208F and HepG2 cells which have high baseline retroviral transduction rates of about 50%. For the purpose of application to ex vivo gene therapy, primary mouse hepatocytes were studied in a similar manner. Hepatocytes had a baseline transduction efficiency of about 40% and did not have increased rates of retroviral-mediated gene transfer with expression of recombinant RAM. This recombinant adenoviral vector conferred infection of amphotropic retrovirus into cells that were relatively resistant to infection, thus offering a rapid and easy method to stably introduce genes into these cell lines.

    View details for Web of Science ID A1995QC65300003

    View details for PubMedID 7703287



    Most diseases caused by genetic deficiencies could, in theory, be treated by the introduction and expression of a normal gene into an appropriate target tissue. It seems likely that gene therapy strategies for most metabolic disorders will not require strict gene regulation, as a fraction of the normal levels of gene activity could result in amelioration or significant improvement in the clinical outcome. Gene therapy is making rapid progress towards the goal of treating various disorders: here, we summarize the state of gene therapy for metabolic disorders.

    View details for Web of Science ID A1994NV10200010

    View details for PubMedID 8091506



    Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.

    View details for Web of Science ID A1994NC04300080

    View details for PubMedID 8134398



    Receptor-mediated endocytosis is an effective method for gene delivery into target cells. We have previously shown that DNA molecules complexed with asialoglycoprotein can be efficiently endocytosed by primary hepatocytes and the internalized DNA can be released from endosomes by the use of a replication-defective adenovirus. Because the DNA and virus enter target cells independently, activity enhancement requires high concentrations of adenoviral particles. In this study, adenoviral particles were chemically conjugated to poly(L-lysine) and bound ionically to DNA molecules. Quantitative delivery to primary hepatocytes was achieved with significantly reduced viral titer when the asialoorosomucoid-poly(L-lysine) conjugate was included in the complex. The conjugated adenovirus was used to deliver a DNA vector containing canine factor IX to mouse hepatocytes, resulting in the expression of significant concentrations of canine factor IX in the culture medium. The results suggest that receptor-mediated endocytosis coupled with an efficient endosomal lysis vector should permit the application of targeted and efficient gene delivery into the liver for gene therapy of hepatic deficiencies.

    View details for Web of Science ID A1993MM51500024

    View details for PubMedID 8265587

  • IN-VIVO GENE-THERAPY OF HEMOPHILIA-B - SUSTAINED PARTIAL CORRECTION IN FACTOR-IX-DEFICIENT DOGS SCIENCE Kay, M. A., Rothenberg, S., Landen, C. N., Bellinger, D. A., LELAND, F., Toman, C., Finegold, M., Thompson, A. R., Read, M. S., Brinkhous, K. M., Woo, S. L. 1993; 262 (5130): 117-119


    The liver represents a model organ for gene therapy. A method has been developed for hepatic gene transfer in vivo by the direct infusion of recombinant retroviral vectors into the portal vasculature, which results in the persistent expression of exogenous genes. To determine if these technologies are applicable for the treatment of hemophilia B patients, preclinical efficacy studies were done in a hemophilia B dog model. When the canine factor IX complementary DNA was transduced directly into the hepatocytes of affected dogs in vivo, the animals constitutively expressed low levels of canine factor IX for more than 5 months. Persistent expression of the clotting factor resulted in reductions of whole blood clotting and partial thromboplastin times of the treated animals. Thus, long-term treatment of hemophilia B patients may be feasible by direct hepatic gene therapy in vivo.

    View details for Web of Science ID A1993LZ63500037

    View details for PubMedID 8211118



    Recombinant adenoviral vectors have recently been used to transfer genes into a number of different cell types in vitro and in vivo. A recombinant adenoviral vector bearing the Escherichia coli beta-galactosidase (beta-gal) gene was used to quantitate the frequency of hepatocyte transduction in the mouse after direct viral infusion into the portal vein. When 10(10) adenoviral particles were infused, over 95% of the hepatocytes were transduced in vivo as determined by x-gal staining. The transduction protocol is relatively safe in that there is no detectable helper virus production in transduced animals and that very few extrahepatic cells are transduced by this method. There is also no evidence of significant liver pathology unless substantially greater quantities of virus are used. However, the transduced hepatocytes do not appear to persist in vivo because the percentage of hepatocytes expressing beta-gal declined over time. Four months after the procedure, 0.5-10% of the hepatocytes contain detectable beta-gal activity in vivo. The change in beta-gal-positive cells correlates with decreasing amounts of adenoviral DNA. Thus, current recombinant adenoviral vectors may have clinical applications in gene therapy for acute hepatic disorders.

    View details for Web of Science ID A1993LU38700002

    View details for PubMedID 8399487

  • DEVELOPMENT OF A CLINICAL PROTOCOL FOR HEPATIC GENE-TRANSFER - LESSONS LEARNED IN PRECLINICAL STUDIES PEDIATRIC RESEARCH Ledley, F. D., ADAMS, R. M., Soriano, H. E., Darlington, G., Finegold, M., Lanford, R., Carey, D., Lewis, D., BALEY, P. A., Rothenberg, S., Kay, M., Brandt, M., Moen, R., Anderson, W. F., Whitington, P., POKORNY, W., Woo, S. L. 1993; 33 (4): 313-320


    Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1993KV79100001

    View details for PubMedID 8386832



    The liver represents an excellent target organ for gene therapy. The current strategy for hepatic gene therapy involves the isolation of primary hepatocytes from a resected liver lobe, transduction of therapeutic genes in vitro followed by autologous hepatocellular transplantation. This ex vivo approach is a rather complex procedure in its entirety; thus, a simple method for direct gene delivery into hepatocytes in vivo has been developed. The procedure involves partial hepatectomy followed by the portal vein infusion of recombinant retroviral vectors. Histological analysis of hepatocytes after in vivo delivery of a recombinant retrovirus bearing the E. coli beta-galactosidase gene showed that 1-2% of the parenchymal cells were transduced. Direct hepatic transfer of human alpha 1-antitrypsin cDNA under the transcriptional direction of the albumin promoter-enhancer led to constitutive expression of the human protein in the sera of recipients at concentrations of 30-1,400 ng/ml for at least 6 months. The experimental animals showed no signs of illness and histologic analysis of the liver revealed no evidence of pathologic abnormalities. The results suggest that the in vivo approach is an attractive alternative for hepatic gene therapy.

    View details for Web of Science ID A1992KE78600005

    View details for PubMedID 1482704



    The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. We have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here we demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. At least 1 x 10(9) hepatocytes or 5% of the liver mass can be transplanted by the portal vasculature. In two animals we have transplanted hepatocytes transduced with a retroviral vector containing the human alpha 1-antitrypsin cDNA under transcriptional control of the cytomegalovirus promoter. Both animals had significant human alpha 1-antitrypsin in the serum for 1 month. Although the serum levels of human alpha 1-antitrypsin eventually fell due to inactivation of the cytomegalovirus promoter, PCR analysis demonstrated that a significant fraction of transduced hepatocytes migrated to the liver and continued to survive in vivo. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstitution with the use of promoters of cellular genes that are active in the normal liver.

    View details for Web of Science ID A1992GY04700020

    View details for PubMedID 1729724



    The hematopoietic system and the liver are two primary target organs for attempting somatic gene therapy of hereditary deficiencies. Several leading laboratories have recently been able to demonstrate that bone marrow cells from rodents and non-human primates can be successfully transduced with foreign genes, resulting in the functional expression of these genes in culture. The genetically reconstituted cells can subsequently be transplanted into X-irradiated recipients, and expression of the transduced genes is observed in the recipients for more than 6 months. Subsequently, gene transfer into peripheral T-lymphocytes in humans has been attempted, and the clinical trials are currently in progress. The liver is the other major organ under intensive investigation. Primary hepatocytes can be isolated from rodents, rabbits, and dogs, and successfully transduced with recombinant retroviruses. After autologous transplantation, long term survival of the engrafted cells in vivo has been observed. More recently, it has been shown that human hepatocytes can also be efficiently transduced with recombinant retroviruses. These experimental results have laid the foundation for somatic gene therapy of hereditary deficiencies in humans in the future.

    View details for Web of Science ID A1992HZ93600001

    View details for PubMedID 1627818



    Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). The recent creation of a mouse strain for PAH deficiency has provided an excellent model system to explore the possibility of its phenotypic correction by hepatic gene therapy. A recombinant retrovirus containing the mouse PAH cDNA under the transcriptional control of the human CMV promoter was constructed and used to transduce hepatocytes isolated from PAH-deficient mice. Viral-transduced hepatocytes produced dramatically higher levels of mouse PAH mRNA as compared to control mock-infected hepatocytes. The PAH mRNA was translated efficiently into PAH protein that is capable of converting phenylalanine to tyrosine in vitro. These results demonstrate that the PAH-deficient mouse hepatocytes can be readily reconstituted by retroviral-mediated gene transduction, which is a crucial step towards somatic gene therapy for PKU.

    View details for Web of Science ID A1992HJ94800008

    View details for PubMedID 1312261