immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy

Protein-based therapeutics can activate the adaptive immune system and lead to the production of neutralizing antibodies and to cytotoxic-T-cell-mediated clearance of the treated cells. Here, we show that the sequential use of immune-orthogonal orthologues of the CRISPR-associated protein 9 (Cas9) and of adeno-associated viruses (AAVs) eludes adaptive immune responses and enables effective gene editing from repeated dosing. We compared total sequence similarities and predicted binding strengths to class-I and class-II major-histocompatibility-complex proteins for 284 DNA-targeting and 84 RNA-targeting CRISPR effectors, and for 167 AAV VP1-capsid-protein orthologues. We predict the absence of cross-reactive immune responses for 79% of the DNA-targeting Cas orthologs, which we validate for three Cas9 orthologs in mice, yet anticipate broad immune cross-reactivity among the AAV serotypes. We also show that efficacious in vivo gene editing is uncompromised when using multiple dosing with orthologues of AAVs and Cas9 in mice previously immunized against the AAV vector and the Cas9 payload. Multiple dosing with protein orthologues may allow for sequential regimens of protein therapeutics that circumvent pre-existing immunity or induced immunity.