AAV-CRISPR gene editing is negated by pre-existing immunity to Cas9

Abstract Adeno-Associated Viral (AAV) vectors are a leading candidate for the delivery of CRISPR/Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T-cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR/Cas9. Although efficient genome editing occurred in mice liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8+ T-cells in the liver. This cytotoxic T-cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, complete elimination of genome-edited cells, and followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR/Cas9 based in vivo genome editing in the liver.