Hematopoietic stem cell transplant is curative of sickle cell disease (SCD) but limited by donor availability. Searches for 85 patients with SCD without matched sibling donors from 2009-2013 using modern techniques (allele-level HLA matching for unrelated donors and higher total nucleated cell doses for umbilical cord blood (UCB)) showed potential match probabilities of 20% for 8/8 HLA-matched unrelated donors, 84% for 7/8 donors, and 97% for two 4-6/6 UCBs suitable for ex-vivo expanded/double cord transplant. Searches performed by age 43 months would have a 90% chance of finding a suitable 5-6/6 UCB.

No abstract

No abstract

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on-target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.