Bio

Administrative Appointments


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

Honors & Awards


  • Sam Rosenthal Prize for Excellence in Pediatrics, Rosenthal Foundation (2011-2013)
  • Outstanding Achievement/Investigator Award, American Society for Cell and Gene Therapy (2013)
  • Elected Member, AAP (2010)
  • Researcher of the Year, National Hemophilia Foundation (2000)
  • Pediatric Researcher of the Year, E. Mead Johnson Award (2000)
  • Elected Member, American Society for Clinical Investigation (1997)
  • Arosenius Swedish Honorary Lectureship, - (1997)
  • Young Investigator Award, Western Society for Clinical Investigation (1996)

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.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • 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

  • 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

    Abstract

    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

    Abstract

    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

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

    Abstract

    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

  • 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-?

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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; ClinicalTrials.gov number, NCT00979238.).

    View details for DOI 10.1056/NEJMoa1108046

    View details for Web of Science ID 000298320200002

    View details for PubMedID 22149959

  • 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

    Abstract

    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

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

    Abstract

    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

  • 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

    Abstract

    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

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

    Abstract

    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, ?C31 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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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)

    Abstract

    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

  • 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

    Abstract

    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

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

    Abstract

    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

    Abstract

    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 CIRCULATION Huang, M., Chen, Z., Hu, S., Jia, F., Li, Z., Hoyt, G., Robbins, R. C., Kay, M. A., Wu, J. C. 2009; 120 (11): S230-S237

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    : 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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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/sj.mt.6300399

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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/sj.mt.6300293

    View details for Web of Science ID 000250382400015

    View details for PubMedID 17726459

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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/sj.mt.6300177

    View details for Web of Science ID 000247646400022

    View details for PubMedID 17457320

  • Combinatorial RNAi: A winning strategy for the race against evolving targets? MOLECULAR THERAPY Grimm, D., Kay, M. A. 2007; 15 (5): 878-888

    Abstract

    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/mt.sj.6300116

    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)

    Abstract

    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

    Abstract

    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/sj.mt.6300032

    View details for Web of Science ID 000244405200007

    View details for PubMedID 17235302

  • 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

    Abstract

    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/sj.mt.6300053

    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

    Abstract

    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

    Abstract

    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/sj.mt.6300011

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

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

    Abstract

    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/sj.gt.3302727

    View details for Web of Science ID 000235836000014

    View details for PubMedID 16453009

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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 MOLECULAR APPROACHES TO CONTROLLING CANCER Tward, A. D., Jones, K. D., Yant, S., Kay, M. A., Wang, R., BISHOP, J. M. 2005; 70: 217-224

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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 TRANSPLANTATION PROCEEDINGS Ohashi, K., Kay, M. A. 2004; 36 (8): 2469-2470
  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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/sj.gt.3302231

    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 HUMAN GENE THERAPY Wang, A. Y., Peng, P. D., Ehrhardt, A., Storm, T. A., Kay, M. A. 2004; 15 (4): 405-413

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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/sj.gt.3301978

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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 TRANSPLANTATION PROCEEDINGS Ohashi, K., Park, F., Schwall, R. H., Kay, M. A. 2002; 34 (5): 1431-1433

    View details for Web of Science ID 000177369700028

    View details for PubMedID 12176427

  • 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

    Abstract

    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 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • IN-VIVO HEPATIC GENE-THERAPY - COMPLETE ALBEIT TRANSIENT CORRECTION OF FACTOR-IX DEFICIENCY IN HEMOPHILIA-B DOGS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kay, M. A., Landen, C. N., ROTHENBERG, S. R., TAYLOR, L. A., LELAND, F., Wiehle, S., Fang, B. L., Bellinger, D., Finegold, M., Thompson, A. R., Read, M., Brinkhous, K. M., Woo, S. L. 1994; 91 (6): 2353-2357

    Abstract

    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

  • 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

    Abstract

    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

Conference Proceedings


  • Stable Factor IX Activity Following AAV-Mediated Gene Transfer in Patients with Severe Hemophilia B 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
  • Adeno-Associated Viral Vector Mediated Gene Transfer for Hemophilia B 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
  • 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 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

    Abstract

    Under normoxic conditions, hypoxia-inducible factor (HIF)-1? is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1? 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-1? protein and angiogenesis genes, respectively.We demonstrated that HIF-1? 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

  • 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 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: 114-114
  • 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 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: 1362-1362
  • Liver directed AAV-mediated homologous recombination is largely independent of serotype Wursthorn, K., Paulk, N., Storm, T., Finegold, M. J., Kay, M., Grompe, M. WILEY-BLACKWELL. 2007: 887A-887A
  • Analysis of vector genome integration sites in various tissues following systemic administration of AAV serotype 8 vector in mice Inagaki, K., Wu, X., Fuess, S., Storm, T. A., Kay, M. A., Nakai, H. WILEY-BLACKWELL. 2006: 391-391
  • In vivo selection of primary and bone-marrow-derived hepatocytes after allogeneic transplantation in mice 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 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 Sylvester, K. G., Jenkins, D., Streetz, K., Doyannis, R., Perryman, S., Kay, M., Blau, H. ELSEVIER SCIENCE INC. 2005: S47-S48
  • Allogeneic bone-marrow transplantation in mice provides tolerance and promotes massive in vivo selection of subsequently transplanted hepatocytes Streetz, K. L., Doyonnas, R., Jenkins, D., Sylvester, K., Kay, M. A. ELSEVIER SCIENCE BV. 2005: 6-6
  • 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 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
  • A new model to functionally measure fusion events in the liver after bone-marrow transplantation over time 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 Nakai, H., Wu, X. L., Fuess, S., Storm, T., Burgess, S., Grompe, M., Kay, M. A. NATURE PUBLISHING GROUP. 2004: S6-S6
  • Functional measurement of fusion in the liver after bone-marrow transplantation over time Streetz, K. L., Jenkins, D., Longaker, G., Sylvester, K., Kay, M. A. ELSEVIER SCIENCE BV. 2004: 109-109
  • Hepatocyte targeting for quantifiable and functional cellular transplantation Jenkins, D. D., Streetz, K., Kay, M., Longaker, M., Sylvester, K. ELSEVIER SCIENCE INC. 2003: S15-S15
  • Pre-clinical in vivo evaluation of pseudotyped adeno-associated virus (AAV) vectors for liver 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
  • Sleeping Beauty transposase-transposon cis-vectors for efficient nonviral gene delivery and persistent gene expression in vivo Mikkelsen, J. G., Yant, S. R., Meuse, L., Huang, Z., Xu, H., Kay, M. A. NATURE PUBLISHING GROUP. 2003: S74-S74
  • cMet activation allows persistent engraftment of ectopically transplanted xenogenic human hepatocytes in mice Ohashi, K., Meuse, L., Schwall, R., Kay, M. A. ELSEVIER SCIENCE INC. 2001: 587-588

    View details for Web of Science ID 000167629900277

    View details for PubMedID 11266970

  • 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
  • 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

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