Title: 543. In Vivo Beta-Cell-Targeted Gene Editing by AAV Vectors
Abstract: The CRISPR/Cas9 and guide RNA system has emerged as a promising genome editing platform, due to its simplicity, versatility and efficiency, compared to prior techniques for genome modification. Although the original Cas9 gene is too long to be packaged by AAV vectors, Dr. Zhang and colleagues have identified a shorter Cas9 protein from S. aureus (saCas9), which allows generation of a single AAV vector carrying both saCas9 and a guide RNA. Previously, we and others have demonstrated that AAV serotypes 8 and 9-based vectors are highly pancreas-tropic. Our long-term goal is to develop efficient beta-cell- and acinar-cell-targeted gene editing systems based on the AAV-saCas9 system. We first assessed on-target effects of the AAV-saCas-gRNA vector system in vitro. We generated two GFP sequence-targeted gRNA vectors, and tested their genome editing effects using two HT1080 cell lines carrying a single copy of GFP. Infection of HT1080 lines with an multiplicity of infection (MOI) of 2×104 resulted in 40-55% GFP knockout by two AAV-gRNA constructs. Gene editing efficiency was comparable in two HT1080 lines, and dose-dependent. Consecutive, multiple high-dose infections for 3 days achieved up to 80% GFP knockdown. To test whether a subset of cells are more resistant to the AAV-saCas9-mediated genome editing, we sorted the remaining 20% of GFP-positive cells and re-infected recovered GFP-positive cells with the two saCas9 vectors. Our data showed those cells were equally permissive to the saCas9 vectors, when compared to their parental line. To assess the off-target effects of the AAV-saCas9-gRNA system, we compared the exome sequences of the vector-treated cells. Our preliminary result identified multiple off-target deletions upon AAV-saCas9 treatments. We are currently analyzing whether those off-target effects were due to their similarity to the GFP targeting gRNA sequences, or independent from the gRNA sequences. We then tested the feasibility of the AAV-saCas9-gRNA vector system for beta-cell-targeted gene editing in vivo. We generated AAV8 vectors carrying Pdx1-targeting gRNA, with saCas9 driven by CMV promoter. Single intraperitoneal injection of AAV saCas9 vectors led to efficient knockout of Pdx1 expression in the pancreatic beta-cells. To limit potential off-target effects, we need to restrict saCas9 expression in beta-cells. However, our original beta-cell targeted vectors carry a relatively long, 1.1kb insulin promoter sequence. We therefore optimized the insulin promoter sequence and determined the minimum, essential promoter sequence, which facilitates robust transgene expression with high tissue-specificity. We are in the process of testing the AAV-saCas9-gRNA vectors with the optimized insulin promoter sequence for in vivo beta-cell gene editing.