In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods.

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34 mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12-14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.