Priming Human Hematopoietic Stem and Progenitor Cells for Cas9/Sgrna and rAAV6-Mediated Homologous Recombination

Highly efficient engineered nuclease-mediated gene editing through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) gives rise to the opportunity of developing gene corrective therapies for hematological disorders. The β-hemoglobinopathies, such as sickle cell disease (SCD) and β-thalassemia, are inherited blood disorders that manifest because of mutations in the beta-globin gene (HBB) and afflict millions of people worldwide. The only curative treatment for these blood disorders is allogeneic-hematopoietic stem cell transplant (allo-HSCT) from a healthy donor, which comes with severe limitations, such as a shortage of immunologically matched donors, graft-versus-host disease, and graft rejection. With the lack of effective curative therapies, it is postulated that autologous HSCT of HBB gene corrected HSPCs would be a potential one-time curative treatment for patients afflicted with β-hemoglobinopathies without the risk of graft versus host diseases and graft rejection, as well as avoiding the need to find an immunologically matched donor. Our previous work demonstrated a methodology to efficiently target the beta-globin gene (HBB) in HSPCs by inducing a site-specific double strand break (DSB) with the RNA-guided (sgRNA) clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nuclease (Cas9/sgRNA) system while supplying an homologous HBB gene corrective donor template via recombinant adeno-associated virus serotype 6 (rAAV6). Here, we further build upon that work to identify the critical parameters to reproducibly achieve high levels of HR in HSPCs. By optimizing Cas9 ribonucleoprotein (RNP) concentration and electroporation conditions, we were able to induce DSBs in long-term repopulating hematopoietic stem cells (LT-HSCs), notably in serial transplants engrafting into immunodeficient non-obese diabetic (NOD)- severe combined immunodeficiency (SCID) Il2rg−/− (NSG) mice where we found ~49% of human cells had INDELS in the HBB gene, matching our input, implying the CRISPR/Cas9 system is effective at inducing double strand breaks at the HBB locus in long term repopulating stem cells. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a two-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis and subsequently higher HR levels. Perhaps the most interesting finding is that when HSPCs are cultured at low densities (100,000 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies. Building upon that work, we show that culturing HBB -targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in NSG mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies, but also as a foundation for editing HSPCs at other loci for both basic and translational research.