The goal of gene therapy is to develop gene transfer technologies and use them for 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.

We recently developed a rapid mini-circle production methodology allowing any molecular biologist to make these vectors. Our extensive work using rAAV vectors played a critical role in our human factor IX clinical trial that was the first systemic administration of rAAV into humans. Our laboratory has been a leader in developing DNA shuffling approaches for the creation of novel AAV vectors with useful transduction properties. Using gene transfer vectors, we studied the potential of using transcriptional-based RNAi to treat human disease. Some of the main RNAi accomplishments include: the first demonstration of RNAi activity in whole non-embryonic mammals, inhibition of human viral (HBV) replication in whole animals and demonstration of toxicity due to shRNA overexpression in mammals resulting in the discovery of rate-limiting processes for RNAi based therapeutics. Recently, we have started to explore how tRNA derived small RNAs regulate genes.

Preclinical to Clinical Trials using rAAV vectors

We were the first to demonstrate successful rAAVmediated 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 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.

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 b 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 and Vector Development

Over the years we 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 to me 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 model 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 multiple selection schemes. We ultimately used this library to isolate an AAV chimeric capsid (LK03) that is human selective and 10x more robust in the 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 a high 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 under evaluation for use in other disorders.

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.

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, conical plasmids are transcriptionally silenced in the liver. Over the years, we established the mechanisms responsible for silencing and designed robust non-conical 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 a number of 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 of these provide 10-1000 times more persistent expression when delivered into quiescent tissues compared to their conical plasmid counterparts. Additional mechanistic findings 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.

RNAi based therapies

We published the first study establishing the use of siRNA and transcriptional RNAi in whole mammals and subsequently worked towards delivering an AAV-shRNA against viral hepatitis. During our studies we found that overexpression of shRNAs can induce liver toxicity and even fatality. During these studies we uncovered a class of tRNA derived small RNAs that we named tsRNAs and characterized their sequences and how some of these are enzymatically generated in mammalian cells. As a result of this study, I was asked to co-chair an international committee to propose universal naming criteria of the various tRNA fragments generated in cells. The committee plans to publish these recommendations in early 2017.

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.

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

Valdmanis PN, Gu S, Chu K, Jin L, Zhang F, Munding EM, Zhang Y, Huang Y, Kutay H, Ghoshal K, Lisowski L, Kay MA.RNA interference-induced hepatotoxicity results from the first synthesized isoform Of microRNA 122 in liver. Nature medicine. 2016; 22(5):557-62. PMID: 27064447

Mechanisms of miRNA mediated gene regulation

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 the 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 new non- coding RNAs that are currently under investigation.

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 microRNAmediated gene silencing by microRNA precursors. Nature structural & molecular biology 2014; 21(9): 825-832. PMCID: PMC4244528.