Although therapeutic genome editing of autologous hematopoietic stem cells (HSCs) in principle could cure β-hemoglobinopathies, CRISPR-Cas9 mediated gene modification has demonstrated variable efficiency, specificity, and persistence in HSCs. Here we demonstrate selection-free on-target editing of the BCL11A erythroid enhancer by Cas9:sgRNA ribonucleoprotein in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. First we screened a set of 20 guide RNAs targeting the functional core of the +58 BCL11A enhancer for maximal HbF induction by RNP delivery. We used SpCas9 protein with additional NLS sequences, synthetic modified sgRNA, and optimized electroporation buffer to produce >95% on-target indels disrupting a critical GATA1 binding site within the +58 BCL11A enhancer in CD34+ HSPCs. Clonal analysis showed that even 1 bp indels around the cleavage site were sufficient for HbF reactivation. Specificity was evaluated by CIRCLE-seq, a method to define genome-wide target sequences susceptible to RNP cleavage in vitro. Amplicon deep sequencing of 24 possible off-target sites from edited CD34+ cells did not reveal any off-target editing with limit of detection 0.1% allele frequency. Despite transient induction of a p53 transcriptional response peaking at 4-8 hours after RNP electroporation, we found no evidence of selection for TP53 or 94 other hematologic malignancy associated mutations by targeted deep sequencing. Edited CD34+ HSPCs from healthy donors contributed to multilineage engraftment of primary and secondary immunodeficient mouse recipients at similar levels as unedited control cells. Likewise we found that edited CD34+ HSPCs from a plerixafor-mobilized SCD donor contributed to marrow engraftment and multilineage hematopoiesis in immunodeficient NBSGW mice after 16 weeks at similar levels as unedited cells, with ~95% indel frequency for engrafting healthy and SCD donor cells. Edited engrafting SCD cells were similarly competent for secondary transplantation as unedited controls. Erythroid progeny of edited engrafting sickle cell disease HSCs expressed therapeutic levels of fetal hemoglobin (HbF) and resisted sickling. Erythroid progeny of edited CD34+ HSPCs from 7 transfusion-dependent ß-thalassemia donors showed restored globin chain balance and amelioration of microcytosis and poikilocytosis. We found that compared to the bulk CD34+ HSPC pool, HSCs preferentially underwent nonhomologous as compared to microhomology mediated end-joining repair based on three assays: sorting of CD34+ CD38- CD90+ CD45RA- cells; isolation of cells in G0, G1, S, and G2/M cell cycle phases; and evaluation of long-term engrafting cells in immunodeficient mice. Together these data show that NHEJ-based BCL11A enhancer editing approaching complete allelic disruption is a practicable therapeutic strategy to produce durable HbF induction in SCD and ß-thalassemia.
Esrick:Bluebird Bio: Honoraria. Williams:Bluebird Bio: Research Funding.
Asterisk with author names denotes non-ASH members.