Expanding CRISPR Genome Editing Strategies in Hematopoietic Stem and Progenitor Cells for the Treatment of Hematologic Diseases

We have previously shown that CRISPR/Cas9 mediates high levels of gene editing in adult human CD34⁺ hematopoietic stem and progenitor cells (HSPCs). We now demonstrated that high levels of gene editing can also be achieved in HSPCs using Cpf1. In addition to NHEJ-mediated gene editing, we explored targeted integration at the ß-hemoglobin gene (HBB) with Cas9, toward gene correction for sickle cell disease (SCD). For targeted integration strategies at HBB, we designed AAV6 DNA donor repair templates flanked by either DNA sequences homologous to the cut site in the genomic location of the SCD mutation or DNA sequences non-homologous to the human genome. To evaluate targeted integration in HSPCs, adult mobilized peripheral blood (mPB) CD34⁺ cells were electroporated with Cas9 ribonucleoprotein (RNP) and were then transduced with AAV6 containing DNA repair templates with or without homology arms. We analyzed targeted integration at the HBB target site by digital droplet PCR (ddPCR) analysis 7 days after transduction of mPB CD34⁺ cells. The ddPCR results show ~30% targeted integration in HBB in mPB CD34⁺ cells with no impact on cell viability. DNA sequencing analysis supported the ddPCR results indicating that perfect homology directed repair-driven integration of the DNA donor template was the preferential repair mechanism. Less than 1% of targeted integration was detected by ddPCR in mPB CD34⁺ cells transduced with AAV6 DNA repair template that lacked homology arms, suggesting that the mechanism of integration is homology repair dependent. These integrations were maintained in the Glycophorin A⁺ erythroid progeny of genome-edited mPB CD34⁺ cells, paving the way toward gene correction for SCD. The combination of CRISPR nucleases (Cpf1, Cas9) and AAV6 DNA repair templates form a powerful genome editing platform that enables broader applications for the treatment of hematologic diseases beyond hemoglobinopathies.