The Kay lab has a long history of work in the area of Gene Therapy/Genome editing and Non-coding RNA biology. A historical summary of selected accomplishments is outlined in the following sections.

Preclinical to Clinical Trials using rAAV vectors

Our early work pursued retroviral, lentiviral, and adenoviral vectors for gene therapies. In the mid 1990s we started to focus our attention on recombinant AAV vectors. We were the first to demonstrate successful rAAV mediated liver gene transfer in small and large animals. These results set the stage for the first in man systemic delivery of rAAV vectors. I was the original Sponsor (IND holder) of this first trial. In humans, unlike all animal studies including non-human primates, the AAV2 vector capsid induced an immune response that resulted in the elimination of the transduced hepatocytes. As will be mentioned our lab developed more robust AAV vectors that have entered two clinical trials.

Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D, Gown AM, Winther B, Meuse L, Cohen LK Thompson AR, Kay MA. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nature genetics 1997; 16(3): 270-276.

Snyder RO, Miao C, Meuse L, Tubb J, Donahue BA, Lin HF, Stafford DW, Patel S, Thompson AR, NicholsT, Read MS, Bellinger DA, Brinkhous KM, Kay MA. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nature medicine 1999; 5(1): 64-70.

Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D, Wright JF, Lessard RR, Sommer JM, Tigges M, Sabatino D, Luk A, Jiang H, Mingozzi F, Couto L, Ertl HC, High KA, Kay MA. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nature medicine 2006; 12(3): 342-347.

Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Pie AJ, Harrington C, O'Beirne J, Smith K, Pasi J, Glader B, Rustagi P, Ng CY, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss UM, Nienhuis AW, Davidoff AM. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. The New England journal of medicine 2011; 365(25): 2357-2365. PMCID: PMC3265081.

AAV Biology, Vector Development, and Genome Editing

Over the years we have made important contributions that involve unraveling the mechanism of AAV transduction in vivo.  We specifically focused on how the vector goes from a single-stranded DNA into a double-stranded episomal DNA and how different capsid variants transduce the same cells/tissues differently.  We were the first to establish how differential vector uncoating kinetics can affect transduction parameters.

As a participant in the more recent AAV-8 hemophilia B clinical trial, it became clear that there was a difference in predicted dose response based on animal studies.  While the AAV-2 dose response was accurately reflected in the human trial, the AAV-8 dose response was more than 10-times less effective in humans.  We used a chimeric humanized liver mouse to demonstrate that the dose response result was the result of an inherent difference in species transduction.  More importantly, we propose that this xenotransplant model may more accurately predict the dose response even when compared to non-human primates.  Recognizing early on how small sequence differences can affect species, cell and tissue transduction parameters, we constructed the first reported multi-species capsid shuffled AAV library and used mutliple selection schemes.  We used this library to isolate an AAV chimeric humanized mouse liver model.  This serotype is currently being evaluated for use in humans.  We had also previously selected an AAV capsid (DJ) that has proven to be especially robust for use in ex vivo and neurobiology applications.

Classical rAAV vectors have two major limitations: 1) episomal genomes are lost during cell division; 2) rAAV-delivery into young mice results in an elevated risk of hepatocellular carcinoma because of the selected growth of cells that have vector promoter insertion near an oncogenic locus.  To overcome these limitations, we have developed a promoterless genome targeting vector without the need for a nuclease.  In this approach, the vector DNA is designed to facilitate homologous recombination into a desired locus in such a manner that the genomic locus produces a new single mRNA that not only continues to produce the protein from the endogenous locus, but the desired protein encoded in the vector sequence.  This approach was used to cure hemophilia B mice and is currently being used in a clinical trial for the treatment of methylmalonic acidemia.

Our work continues to select improved capsids for delivery into the human CNS and pancreas for the treatment of diabetes.  We are using advanced chemical modification paradigms for targeting AAV vectors.  In addition, we are now pursuing studies showing how the capsid proteins can influence the epigenomic state of the vector and this can explain at least some of the discordance between vector transduction between various tissues and species.

Thomas CE, Storm TA, Huang Z, Kay MA. Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotyped adeno-associated virus vectors. Journal of virology 2004; 78(6): 3110-3122. PMCID: PMC353747.

Grimm D, Lee JS, Wang L, Desai T, Akache B, Storm TA, Kay MA. In vitro and in vivo gene therap vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. Journal of virology 2008; 82(12): 5887-5911. PMCID: PMC2395137.

Lisowski L, Dane AP, Chu K, Zhang Y, Cunningham SC, Wilson EM, Nygaard S, Grompe M, Alexander IE, Kay MA. Selection and evaluation of clinically relevant AAV variants in a xenograft liver model. Nature 2014; 506(7488): 382-386. PMCID: PMC3939040.

Barzel A, Paulk NK, Shi Y, Huang Y, Chu K, Zhang F, Valdmanis PN, Spector LP, Porteus MH, Gaensler KM, Kay MA. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 2015; 517(7534): 360-364. PMCID: PMC4297598.

Pekrun K, De Alencastro G, Luo QJ, Liu J, Kim Y, Nygaard S, Galivo F, Zhang F, Song R, Tiffany MR, Xu J, Hebrok M, Grompe M, Kay MA. 2019. Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors. JCI Insight.410.1172/jci.insight.131610; PMID:31723052 PMCID:PMC6948855

de Alencastro G, Puzzo F, Pavel-Dinu M, Zhang F, Pillay S, Majzoub K, Tiffany M, Jang H, Sheikali A, Cromer MK, Meetei R, Carette JE, Porteus MH, Pekrun K, Kay MA. (2021) Improved genome editing through inhibition of FANCM and members of the BTR dissolvase complex. Mol Ther 29(3):1016-1027. PMID:33678240 PMCID:PMC7934449

Spector LP, Tiffany M, Ferraro NM, Abell NS, Montgomery SB, Kay MA. (2021) Evaluating the genomic parameters governing rAAV-mediated homologous recombination. Mol Ther 29(3):1028-1046. PMID:33248247 PMCID:PMC7934627

Tsuji S, Stephens CJ, Bortuolussi G, Zhang F, Baj B, Jang H, de Alencastro G, Muro AF, Pekrun K, Kay MA. (2021) The ribonucleotide reductase inhibitor fludarabine safely increases both nuclease-free AAV and CRISPR/Cas9-mediated homologous recombination in mouse livers.  Nature Biotechnology. In press

Non-viral vectors

We were the first to develop a DNA transposon for gene therapy applications in mammals. While we continued to develop these for additional years, during our study of rAAV-vector transduction, we found that episomal DNA plasmids have the potential to last indefinitely in quiescent tissues. However, canonical plasmids are transcriptionally silenced in the liver. Over the years, we established the mechanisms responsible for silencing and designed robust non-canonical plasmid vectors that were persistently transcribed. We learned that the length (>1kb) rather than the specific sequence of DNA contained outside of the recombinant expression cassette (classically occupied by the bacterial origin of replication and selectable marker e.g., amp or kan) is responsible for silencing. To overcome the silencing effect, we have generated several new plasmid variants that are becoming more popular in the gene therapy community. Two such vectors are named minicircle and mini-intronic plasmid vectors (MIP). Both provide 10-1000 times more persistent expression when delivered into quiescent tissues compared to their canonical plasmid counterparts. Additional mechanistic findings in the epigenomic state of the vector DNAs in whole tissues are providing new general insights into general eukaryotic transcriptional paradigms.

Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay MA. Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nature genetics 2000; 25(1): 35-41.

Kay MA, He CY, Chen ZY. A robust system for production of minicircle DNA vectors. Nature biotechnology 2010; 28(12): 1287-1289. PMCID: PMC4144359.

Gracey Maniar LE, Maniar JM, Chen ZY, Lu J, Fire AZ, Kay MA. Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level. Molecular therapy: the journal of the American Society of Gene Therapy 2013; 21(1): 131-138. PMCID: PMC3538319.

Lu J, Zhang F, Kay MA. 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 2013; 21(5): 954-963. PMCID: PMC3666631.

Lu J, Williams JA, Luke J, Zhang F, Chu K, Kay MA. 2017. A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo. Hum Gene Ther.28:125-134. 10.1089/hum.2016.140; PMC5278795

Lu J, Zhang F, Fire AZ, Kay MA. 2017. Sequence-Modified Antibiotic Resistance Genes Provide Sustained Plasmid-Mediated Transgene Expression in Mammals. Mol Ther.25:1187-1198. 10.1016/j.ymthe.2017.03.003; PMC5417794

RNAi based therapies and miRNA biogenesis/function

We published the first study establishing the use of siRNA and transcriptional RNAi in whole mammals and subsequently worked towards developing a platform for delivering AAV-shRNA vectors.  Our first targets were hepatitis viruses B and C.  During our studies we found that overexpression of shRNAs can induce liver toxicity and even fatality.  From this point, we continued studies to unravel the mechanism of shRNA overexpression and hepatic toxicity.  Along the way we explored various mechanisms involved in miRNA biogenesis.  As a result, we started to investigate the mechanisms involved in miRNA-mediated gene regulation, processing, and RISC loading.  Over the years, we have provided new insights into Dicer processing of both exogenously expressed miRNAs/shRNAs and endogenous miRNAs and discovered a Dicer loop counting rule.  This rule when applied to shRNA design can drastically improve the homogenous products derived from transcriptional shRNAs increasing efficiency and decreasing the off targeting.  We recently discovered a new function for some pri/pre-miRNAs.  In this example, the precursor can bind to some target mRNAs and protect the target from the action of the mature miRNA.  Thus, in such examples, the ratio of the primary/precursor and mature miRNA is what dictates the degree of mRNA down regulation.  Furthermore, this provides an example where a single miRNA locus can regulate two mRNAs differently in the same cell.  During our studies we have discovered a new class of non-coding RNAs derived from tRNAs.  This is discussed in the next section.

Importantly, these studies have resulted in a novel finding that a long-non-coding RNA (Inc122), who's only known function is as a precursor to the hepatic specific miR122 present in 50,000 copies per hepatocyte has a separate and sometimes opposing function in regulating liver homeostasis and cancer.

McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002; 418(6893): 38-39 PMID: 12097900

Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F, Kay MA. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441(7092): 537-541. PMID: 16724069.

Gu S, Jin L, Zhang F, Sarnow P, Kay MA. Biological basis for restriction of microRNA targets to the 3' untranslated region in mammalian mRNAs. Nature structural & molecular biology 2009; 16(2): 144-150. PMCID: PMC2713750.

Gu S, Jin L, Zhang F, Huang Y, Grimm D, Rossi JJ, Kay MA. 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 2011; 108(22): 9208-9213. PMCID: PMC3107324.

Gu S, Jin L, Zhang Y, Huang Y, Zhang F, Valdmanis PN, Kay MA. The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo. Cell 2012; 151(4): 900-911. PMCID: PMC3499986.

Roy-Chaudhuri B, Valdmanis PN, Zhang Y, Wang Q, Luo QJ, Kay MA. Regulation of microRNA mediated gene silencing by microRNA precursors. Nature structural & molecular biology 2014; 21(9): 825-832. PMCID: PMC4244528.

Valdmanis PN, Gu S, Chu K, Jin L, Zhang F, Munding EM, Zhang Y, Huang Y, Kutay H, Ghoshal K, Lisowski L, Kay MA. 2016. RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice. Nat Med.22:557-62. 10.1038/nm.4079; PMC4860119

Valdmanis PN, Kim HK, Chu K, Zhang F, Xu J, Munding EM, Shen J, Kay MA. 2018. miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression. Nat Commun.9:5321. 10.1038/s41467-018-07786-7; PMC6294001

Luo QJ, Zhang J, Li P, Wang Q, Zhang Y, Roy-Chaudhuri B, Xu J, Kay MA*, Zhang QC*. RNA structure probing reveals the structural basis of Dicer binding and cleavage. Nat Commun. 2021 Jun    7;12(1):3397. doi: 10.1038/s41467-021-23607-w. PMID: 34099665; PMCID: PMC8184798.

*co-communicating authors

Gene regulation of tRNA derived small RNAs

We recently found that a 22 nucleotide (nt) 3' end of the LeuCAG transfer-RNA-derived small RNA (LeuCAG3'tsRNA) binds to the human RPS28 mRNA, unwinds the double-stranded secondary structure, which enhances RPS28 mRNA translation.  Small changes in RPS28 protein production were also shown to regulate rRNA processing and ribosome biogensis.  Inhibition of this specific tsRNA inducted apoptosis in rapidly dividing cells in culture and suppressed the growth of human hepatocellular carcinomas in vivo, making it a bona-fide target for cancer therapeutics.  A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits.

These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.  We also found that RPS28 mRNA and ribosome biogenesis were similarly regulated by the same 3' ts RNA in the mouse.  In addition, using various inhibitors of protein synthesis and polysome gradient analysis in both mouse and human cells we establish that the 3'tsRNA regulates translation at the elongation step.  We also found that the 3'tsRNAs are generated from charged tRNAs providing more insight into their regulation.  Our results suggest a conserved functional role for 3'tsRNAs to fine tune translation of mRNAs.  We propose that this may represent a feedback loop to regulate the components of protein translation and may represent new targets for treating cancer.  We are currently exploring the expression patters of the 161 3'tsRNAs in tissues including cancer.  Using various screening approaches, we have identified several 3'tsRNAs that may also regulate proteins involved in translational regulation.

Haussecker D, Huang Y, Lau A, Parameswaran P, Fire AZ, Kay MA. Human derived small tRNAs in the global regulation of RNA silencing. RNA (New York, N.Y.). 2010; 16(4):673-95. PMID: 20139967

Kim HK, Fuchs G, Wang S, Wei W, Zhang Y, Park H, Roy-Chaudhuri B, Li P, Xu J, Chu K, Zhang F, Chua MS, So S, Zhang QC, Sarnow P, Kay MA. A transfer-RNA-derived small RNA regulates ribosome biogenesis. Nature. 2017; 552(7683):57-62. PMCID: PMC6066594.

Kim HK, Xu J, Chu K, Park H, Jang H, Li P, Valdmanis PN, Zhang QC, Kay MA. A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice. Cell Reports. 2019 Dec 17;29(12):3816-3824. PMCID:31851915

Liu Z, Kim HK, Xu J, Jing Y, Kay MA. The 3'tsRNAs are aminoacylated: Implications for their biogenesis.  PLoS Genet. 2021 Jul 29;17(7):e1009675. doi: 10.1371/journal.pgen.1009675. PMID: 34324497;  PMCID: PMC8354468