Genomic profile of gene edited hematopoietic stem cells

Sickle cell disease (SCD) and β-thalassemia patients with elevated gamma
globin (HBG1/G2) levels exhibit mild or no symptoms. To recapitulate this
natural phenomenon, the most coveted gene therapy approach is to edit the
regulatory sequences of HBG1/G2 to reactivate them, with a magnified
effect by simultaneous targeting of multiple sequences. Here, we used Cas9
RNP-ssODN-mediated homology-directed gene editing to mimic two naturally
occurring HBG promoter point mutations, namely, -175T>C, which is
linked to high HbF levels, and -158C>T, the most common
polymorphism in the Indian population that induces HbF under
erythropoietic stress, in HSPCs. We observed high complete HDR
conversions, with at least 30% of HSPCs exhibiting both -175T>C and
-158C>T mutations, which increased to over 50% under optimized
conditions. In NBSGW mice, up to 30% of long-term engrafted human HSPCs
showed both -175T>C and -158C>T HDR conversions, with the
efficiency peaking with up to 57% of HSPCs containing at least one of the
beneficial mutations. Cas9 RNP-ssODN-based nucleotide conversion at the
HBG promoter offers a promising gene therapy approach to ameliorate the
disease phenotypes of both β-thalassemia and SCD. The developed approach
can simplify and broaden applications that require multiple nucleotide
modifications in HSPCs.