OC 11.1 - CRISPR-based gene editing for hemophilia: comprehensive analysis of off-target and on-target effects

Background: Liver-directed gene therapy in adult patients with hemophilia A and B using adeno-associated viral vectors (AAV) results in multi-year therapeutic factor VIII (FVIII) and factor IX (FIX) expression levels. Nevertheless, questions remain about the expression durability in the face of hepatocyte proliferation, particularly in newborn or juvenile patients. To achieve life-long correction, stable targeted genomic integration would be desired which could be achieved using CRISPR/Cas9.

Aims: The genomic consequences of using AAV vectors for in vivo delivery of CRISPR-Cas9 components are not fully understood. In this study, we therefore evaluated CRISPR-Cas9-mediated on-target and off-target effects and examined the AAV genomic integration events.

Methods: AAV vectors were employed to deliver the CRISPR-Cas components to the liver of neonatal mice. Guide RNAs (gRNA) were specifically designed to target the coagulation factor IX gene (F9) whereas Cas9 was expressed from a hepatocyte-specific promoter (HSP). Whole-genome sequencing (WGS) was conducted to examine the on-target and off-target insertions/deletions (indels). Genome-wide AAV integration site analyses was conducted by Shearing Extension Primer Tag Selection Ligation-Mediated PCR (S-EPTS/LM-PCR).

Results: Efficient CRISPR targeting was apparent on the on-target site in the F9 locus (36.45 ± 18.29%). In contrast, off-target effects were rare or below the WGS detection limit. WGS and S-EPTS/LM-PCR analysis demonstrated that AAV integrations occurred preferentially in the CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside this locus were not in proximity to any of more than 5000 putative computationally predicted off-target sites (median distance of 70 kb).

Conclusion(s): Our study indicates that it is possible to specifically edit the F9 gene with near-zero levels of off-target effects. The propensity of AAV to integrate into CRISPR-Cas9–induced double-strand breaks could be exploited to identify potential CRISPR-Cas9 off-target effects with high sensitivity. Future clinical applications should consider the efficacy and safety implications of these on-target and off-target effects.