Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease

Liwen Xu; Premanjali Lahiri; Jason Skowronski; Neehar Bhatia; Annalisa Lattanzi; Matthew H Porteus

doi:10.1016/j.omtm.2023.07.009

Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease

Mol Ther Methods Clin Dev. 2023 Jul 27:30:317-331. doi: 10.1016/j.omtm.2023.07.009. eCollection 2023 Sep 14.

Authors

Liwen Xu¹, Premanjali Lahiri², Jason Skowronski², Neehar Bhatia², Annalisa Lattanzi¹, Matthew H Porteus¹

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
² Stanford Laboratory for Cell and Gene Medicine, Stanford University, Stanford, CA 94304, USA.

Abstract

Ex vivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48 h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.

Keywords: CRISPR; Cas9; HBB gene; SCD; genome editing; hematopoietic stem and progenitor cell; p53 activation; rAAV6.