Clinical-Scale Genome Editing of the Human BCL11A Erythroid Enhancer for Treatment of the Hemoglobinopathies

We describe here a fundamentally novel way to develop a disease therapeutic: combining genome-wide association studies (GWAS) with targeted genome editing to create, in a clinically compliant setting, a disease-ameliorating genotype in the patient's own cells. In β-thalassemia, elevated levels of fetal hemoglobin (HbF) lessen or eliminate disease symptoms, thus making a reversal of HbF silencing in patients an appealing therapeutic strategy. Loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, elevate HbF; rare individuals carrying a monoallelic knockout of BCL11A exhibit no known hematologic abnormality and up to 30% circulating HbF. We previously reported de novo knockout of BCL11A using targeted genome editing with engineered zinc finger nucleases (ZFNs) yielding up to 40% HbF in erythroid progeny of edited human CD34 cells in vitro. We now find that the targeted ablation of a single, specific GATAA motif in the BCL11A intronic enhancer does not affect in vitro erythroid differentiation, but reproducibly (n=6) activates fetal globin transcription in erythroid progeny of modified CD34 cells; importantly, at similar levels of on-target marking in CD34+ cells, these effects on fetal globin mRNA are comparable to those resulting from ZFN-driven coding knockout of BCL11A itself. We demonstrate reproducible (n=8), high-efficiency (up to 82%; average, 69%) ZFN-driven marking at the enhancer in peripheral blood mobilized human CD34 cells at clinical production scale (>1e8 cells) in a GMP-compliant setting for which we use a clinical-grade electroporation device to deliver nuclease-encoding transcribed mRNA ex vivo. Using erythroid colony assay genotyping we find that up to 70% of the cells in the resulting population are biallelically modified at the target locus, while ~10% remain wild-type, and find comparably high levels of marking in research-scale preparations of CD34 cells from patients with β-thalassemia. We observe robust long-term (18-24 week) engraftment and multilineage differentiation of genome-edited cells in immunodeficient mice, similar to control cells, and equivalent modification at the targeted enhancer locus at all timepoints in both differentiated (CD19+, CD3+, CD33+) and more primitive progenitor (CD34+CD38low) cells of human origin purified from bone marrow of long-term-engrafted animals. Our findings support clinical development of enhancer editing as a treatment of the β hemoglobinopathies with autologous hematopoietic stem cell transplant.