PS1518 GENOME EDITING OF HBG1/2 PROMOTER LEADS TO ROBUST HBF INDUCTION IN VIVO, WHILE EDITING OF BCL11A ERYTHROID ENHANCER RESULTS IN ERYTHROID DEFECTS

Background: Induction of fetal hemoglobin (HbF) is an attractive approach to ameliorate disease symptoms in beta hemoglobinopathies. Gene editing by zinc finger or CRISPR‐Cas nucleases to disrupt BCL11A expression, a known mediator of fetal globin silencing, via targeting BCL11A erythroid‐enhancer, is one approach to reactivate fetal globin expression. Alternatively, the genetics of hereditary persistence of fetal hemoglobin (HPFH) suggest that disruption of certain cis‐regulatory elements in the beta‐globin locus may also be a viable strategy. A previous lenti‐CRISPR mediated saturated mutagenesis screen identified multiple HbF‐inducing genomic domains concentrated at the gamma‐globin ( HBG1/2 ) promoters. Aims: To compare editing of HBG1/2 promoters versus BCL11A erythroid‐enhancer in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR‐Cas technologies as potential treatments for beta hemoglobinopathies. Methods: Ribonucleoprotein (RNP) complexes were designed to target either BCL11A erythroid‐enhancer GATAA motif or several HbF‐inducing genomic domains in the HBG1/2 promoters. These RNPs were transfected into mobilized peripheral blood (mPB) CD34+ HSPCs from healthy volunteers or peripheral blood mononuclear cells (PBMCs) from patients with sickle cell disease. RNPs demonstrated to have high HbF‐inducing potentials were used to edit HSPCs which were then infused into immunocompromised NOD.Cg‐Kit W−41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ (NBSGW) mice to assess editing in SCID‐repopulating cells (SRC) and their multilineage reconstitution potential. Results: Following transfection of RNPs targeting HBG1/2 promoters into CD34+ HSPCs or sickle PBMCs, >70% editing was achieved. Erythroid progeny derived from edited HSPCs or PBMCs had increased levels of HbF compared to those derived from control cells (up to 40% vs. 10% respectively). When infused into NBSGW mice, HSPCs edited with RNPs targeting either HBG1/2 promoters or BCL11A erythroid‐enhancer both reached >70% human chimerism (hCD45+/hCD45+mCD45). Comparable monocytes, granulocytes, B lymphocytes, and HSPCs reconstitution were attained with both approaches. However, the erythroid lineage output in the BCL11A ‐edited group was approximately 80% less than that in the control group. In addition, a 20% reduction in editing levels, compounded by a 2.5‐fold higher nonproductive indel frequency, was observed in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages from the same animal in the BCL11A ‐edited group. In contrast, HBG1/2 promoter‐edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. Importantly, robust long‐term induction of HbF was observed in human erythroid cells either sorted from chimeric BM or obtained from ex vivo culture of BM from HBG1/2 promoter‐edited group compared to the control group up to 16‐weeks post‐transplant. Summary/Conclusion: Our data suggest that sickle PBMCs, bulk CD34+ cells, and long‐term hematopoietic stem cells can be successfully edited using RNPs targeting BCL11A erythroid‐enhancer or HBG1/2 promoters. However, HBG1/2 promoter‐edited cells exhibited a more favorable preclinical profile than BCL11A ‐edited cells, as BCL11A ‐edited HSPCs displayed erythroid‐specific defects in the NBSGW mouse model. Long term induction of HbF by HBG1/2 promoter‐edited cells without lineage skewing suggests that this editing strategy has favorable clinical potential for the treatment of beta hemoglobinopathies.