High fetal hemoglobin (HbF) levels are associated with decreased severity in sickle cell disease (SCD) and beta thalassemia (BT). We developed a novel gene-edited cell therapy using autologous hematopoietic stem and progenitor cells (HSPCs) that have been genetically modified with zinc finger nucleases (ZFNs) to reactivate HbF expression. The ZFNs target the GATA1 binding motif (GATAA) within an intronic erythroid-specific enhancer (ESE) of BCL11A, which encode a major repressor of HbF. Previously, we reported successful ZFN-mediated ex vivo BCL11A gene editing and reactivation of HbF expression in dual mobilized HSPCs (peripheral stem cells mobilized with granulocyte colony-stimulating factor (G-CSF) and plerixafor). This drug product, ST 400, is in a phase 1/2a clinical trial for transfusion-dependent BT (NCT03432364). As G-CSF is not recommended for use in SCD patients due to the risk of clinical complications, an analogous gene-edited cell therapy drug product, BIVV003, was developed using autologous CD34+ HSPCs obtained from single plerixafor mobilization and apheresis (NCT03653247).
The aim is to evaluate the efficacy and specificity of plerixafor mobilized ZFN-edited HSPCs from healthy donors and compare ZFN editing outcome in single and dual-mobilized HSPC.
ZFN-mediated gene editing was measured by deep sequencing and HbF levels were assessed by reverse phase ultra-performance liquid chromatography and flow cytometry. For single cell clone analysis, individual plerixafor mobilized HSPC from four healthy donors were sorted and cultured in erythroid differentiation medium. In total, 954 single-cell derived clones were genotyped.
Here we demonstrated that ZFN-mediated gene editing in plerixafor mobilized HSPCs induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified) with high post-editing viability (77%, n = 5, research scale). Similar editing efficiencies (>70%) were obtained in HSPCs at clinical manufacturing scale (n = 2). Further, in vitro HbF protein levels and HbF+ cell frequencies within erythroid progeny of edited cells were increased by >4 and 3-fold respectively - compared to the level in non-edited cells (n = 4, research scale). Comparability of ZFN editing outcomes were observed in dual mobilized HSPCs (n = 4 and previous reports) demonstrating equivalent efficacy between BIVV003 and ST400 products.
Importantly, single cell clone analysis revealed that ZFN edited both alleles of BCL11A at high frequency (>90% of edited cells) with high levels of replicable GATAA-disrupting indels. Each edited allele contributed a 15% increase in HbF production (P = 2x10–78) with biallelic edits adding a 27–38% HbF increase to the basal level. Non-GATAA disrupting indels within BCL11A ESE independently contributed to HbF up-regulation, albeit at a lower level (β=3%, P = 0.003). Importantly, sequencing of the 12 kb surrounding the target site did not reveal any gross deletion extending outside the intronic BCL11A ESE. Overall, our data revealed that ZFN-mediated disruption of BCL11A ESE resulted in enriched biallelic editing with highly replicable and on-target small indels and increases in HbF. Further characterization of BIVV003 showed that injection of plerixafor mobilized ZFN edited HSPCs into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their progeny, including erythrocytes.
These data support the potential efficacy and specificity of plerixafor mobilized ZFN-edited HSPCs as a novel cell therapy for SCD patients.