Highly Recombinogenic AAV for Genome Editing of Somatic and ES Cells
An explosion in the ability to edit the human genome, in conjunction with the identification of human tissue and embryonic stem cells allows for the first time in history the ability for “definitive genetic therapy” through the correction of genetic mutations in stem cells. We investigated the efficiency of genome editing tools with an eye for scalability and cGMP compatibility. We have explored conventional targeting, zinc finger nucleases, transcriptional activator-like effector nucleases (TALENs), and CRISPR/Cas9-based editing. In addition, in collaboration with Mark Kay at Stanford, we have found a novel adeno-associated viral AAV variant (AAV-DJ) that has high recombinogenic activity. AAV-DJ–mediated targeting (AT) has the advantage that AAVs have previously been used in human gene therapy trials, have low off-target rates, and are easily detectable. In our initial paper, we demonstrated the ability of AAV-DJ to directly correct the LamA3 mutation in Junctional Epidermolysis Bullosa patient keratinocytes. This provided proof-of-principle that direct correction, without the use of selectable markers, could be used clinically. Moreover, in a followup paper, we showed that an AAV-DJ variant could correct the COL7A1 locus in Dystrophic Epidermolysis bullosa induced –pleuripotent cells, demonstrating its usefulness in manufacturing protocols for the Therapeutic Reprogramming of skin. Because AAV-DJ–mediated homologous recombination rates were comparable to those of CRISPR/Cas9, and potentially safer and easier to deliver, we concluded that AAV-DJ–mediated genome editing would provide the ideal genome-editing system for correction during therapeutic iPSC manufacturing.