Nanoblades, a new technology based on a modified murine leukemia virus, where the viral structure protein gag is fused to Cas9 and the viral particles are loaded with the Cas9 protein complexed with the gRNA and devoid of any viral genome. We showed that nanoblades were remarkably efficient for entry into human T, B and HSCs thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. Incubation of rAAV-6 vector containing 2 homologous arms to the Wiskott-Aldrich syndrome (WAS) locus flanking a GFP expression cassette with nanoblades, resulted in up to 40 % of stable expression cassette knock-in into the WAS gene locus in HSC. However, higher rAAV-6 doses increased HSC cell death. Comparing rAAV6 with rAAV2 encoding the donor DNA, we demonstrated that at high doses, AAV2 was much less toxic and gave higher transduction levels in HSCs. To improve donor template delivery at low vector doses, rAAV2 and rAAV6 were chemically bio-conjugated with a ligand, via the amino-acid residues exposed at the capsid surface, in different positions and quantities. Initial results showed high level transduction of HSCs with ligand coupled rAAV6 vectors accompanied by a remarkable lower toxicity compared to the WT-rAAV6. Bio-conjugated rAAV6 combined with nanoblades increased gene knock-in and survival of HSCs from 40% to 80% as compared to the WT-rAAV6.

Summarizing, the introduction of a specific ligand on rAAV6 increased donor mediated gene knock-in and significantly increased HSC survival, an important feature for clinical translation of HSC gene editing strategies.