Ligand-modified rAAV6 vectors with nanoblades allow high-level gene knockin in HSPCs without compromising cell survival.

Nanoblades are viral particles loaded with the Cas9 protein complexed with gRNA, which allowed efficient gene editing in hematopoietic stem and progenitor cells (HSPCs). Combined with recombinant adeno-associated vector (rAAV) 6 containing two homologous arms to a gene locus resulted in 50% of expression cassette knockin into HSPCs. However, high effective doses of rAAV6 induced HSPC cell death. Here, we demonstrated that, at high doses, rAAV2 was much less toxic for template DNA delivery and allowed transduction levels in HSPCs equivalent to rAAV6. To improve donor template delivery, rAAV2 and rAAV6 were chemically bio-conjugated with a mannose ligand, via the lysine or tyrosine amino acid residues exposed at the adeno-associated vector (AAV) capsid surface. High-level transduction of HSPCs with mannose-coupled rAAV6 vectors accompanied by a remarkable lower toxicity was achieved as compared to control rAAV6 in correlation with highly reduced p53 pathway activation. Mannose-conjugated rAAV6 combined with nanoblades allowed efficient gene knockin and increased survival of HSPCs from 10% to 80% as compared to the unmodified rAAV6 even in the most immature CD34CD38lowCD90 hematopoietic stem cell (HSC) population. Summarizing, mannose-conjugated rAAV6 maintained high-level donor mediated gene knockin when combined with nanoblades without inducing significant toxicity for the HSPCs, an important feature for clinical translation of HSPC gene-editing strategies.