New Approaches for Therapeutic Gene Transfer and Genome Editing In Vivo
My laboratory has long been interested in the development of gene transfer vectors for gene therapy as well as manipulating non-coding RNAs for therapeutic purposes. A major interest has been in unraveling the mechanism of viral vector transduction in vivo. Our work during the last 15 years has focused on two vector systems, mini-circles and recombinant AAVs (rAAV). We have shown that DNA vectors consisting of a circularized eukaryotic expression cassette (lacking plasmid DNA backbone sequences) provide more persistent levels of transgene expression from quiescent tissues compared to the same plasmid vectors. We recently developed a rapid mini-circle production methodology allowing any molecular biologist to make these vectors. Moreover, we have evidence that differential chromatinization of these DNAs plays a role in their expression profiles. 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. My 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. Our work uncovered key mechanistic insights into how non-coding miRNAs are loaded into active RISC complexes in mammals as well as the discovery of new classes of small RNAs derived from tRNA, and non-coding RNAs that may be generated by RNA-directed RNA transcription in vertebrates. 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.