Clonal Analysis of Human Bone Marrow CD34+ Cells Edited By BCL11A-Targeting Zinc Finger Nucleases Reveals Clinically Relevant Levels of Fetal Globin Expression in Edited Erythroid Progeny

Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms.

Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34⁺ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34⁺ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD.