A Universal Approach to Target Various HBB Gene Mutations in Hematopoietic Stem/Progenitor Cells for Beta-Thalassemia Gene Therapy by CRISPR/Cas9 and the rAAV6 Vector

Background: Engineered nuclease-mediated gene targeting through homology-directed repair (HDR) in autologous hematopoietic stem and progenitor cells (HSPCs) has the potential to cure β-thalassemia (β-thal). Although previous studies have precisely corrected site-specific HBB mutations by HDR in vitro and in vivo, targeting the various HBB mutations in β-thal is still challenging. Here, we devised a universal strategy to achieve repaired most types of HBB mutations through the CRISPR/Cas9 and the rAAV6 donor. Methods: Using cord blood-derived HSPCs from health donors, we tested the strategy to achieved highly efficient targeted integration by optimizing design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and modified single guide RNA, together with a rAAV6 donor. We assessed the edited HSPCs function in vitro by methylcellulose colonies assay, CFU assay, differentiation experiment and Wright-Giemsa staining. Edited HSPCs transplanted into NSI mice to assess the long-term reconstitution in vivo. Whole-genome sequencing was used to analysis the off-target mutagenesis of edited HSPCs. Results: Edited HSPCs exhibited normal multilineage formation and erythroid differentiation abilities without off-target mutagenesis and retained the ability to engraft. Moreover, we used the strategy to efficiently correct the β-CD41/42 mutation of patient-derived HSPCs, erythrocytes differentiation from which expressed more HBB mRNA than uncorrected cells. Conclusion: This strategy demonstrated a universal approach to correct most types of HBB gene mutations in β-thal.