Ex Vivo Gene-Edited Cell Therapy for Sickle Cell Disease: Disruption of the BCL11A Erythroid Enhancer with Zinc Finger Nucleases Increases Fetal Hemoglobin in Plerixafor Mobilized Human CD34+ Cells

Increases in fetal hemoglobin (HbF) are associated with improved clinical outcomes in the inherited hemoglobinopathies. We are developing a novel gene-edited cell therapy to treat patients with sickle cell disease (SCD) and beta-thalassemia (BT) using autologous hematopoietic stem and progenitor cells (HSPCs) genetically modified with zinc finger nucleases (ZFNs) to restore high levels of HbF expression. The ZFNs have been designed to specifically target the GATA motif within an intronic erythroid-specific enhancer (ESE) of BCL11A, the gene encoding a transcriptional regulator of the fetal-to-adult hemoglobin switch. Previously, we reported successful ZFN-mediated, ex vivoBCL11A gene editing in dual mobilized HSPCs, i.e. peripheral stem cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and plerixafor¹. The editing procedure was optimized for high on-target/low off-target modification levels and increases in HbF in erythroid progeny. This drug product, ST-400, passed extensive safety testing and is currently in a phase 1/2a clinical trial for transfusion-dependent beta-thalassemia (ClinicalTrials.gov number NCT03432364).

An analogous drug product, BIVV003, is being developed as a therapeutic for SCD. In patients with SCD, the use of G-CSF for stem cell mobilization is not recommended due to the risk of clinical complications. Therefore, peripheral HSPCs are obtained from SCD patients via single agent, plerixafor mobilization and apheresis^2-5. Understanding the effect of this change in mobilization strategy on ZFN editing efficiency and specificity is a key element in preparing for SCD gene-edited cell therapy. In the present studies, we demonstrate comparability of ZFN editing outcomes in single and dual mobilized HSPCs obtained from healthy donors. In plerixafor mobilized HSPCs from five healthy donors at research scale, ZFN-mediated gene editing induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified, as measured by MiSeq deep sequencing) with high post-editing viability (77%). Similar gene 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 non-edited cells in the same culture conditions, using reverse phase high-performance liquid chromatography and flow cytometry (n=4 healthy donors at research scale). Single cell clone analysis revealed that ZFN-mediated gene editing targeted both alleles of BCL11A at high frequency (91-94% of edited cells within erythroid progeny) with high levels of replicable GATA-disrupting indel patterns. On average, each edited allele contributed an additional 17.6% increase in HbF production in vitro, with a statistically-significant increase in HbF level for biallelic edited vs. unedited controls (3.4 fold).

Critical to BIVV003 use in clinical trials, ZFN-mediated gene editing did not impair single agent mobilized HSPC function in vitro based on measurements of colony forming unit (CFU) production and frequencies of long-term HSC (LT-HSC) and common myeloid progenitor (CMP) cells by flow cytometry. In agreement with this data, injection of BIVV003 into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their differentiated progeny. Overall, these data demonstrate potential efficacy of ZFN-edited HSPCs (BIVV003) as a novel cell therapy for SCD patients.

1. Holmes et al., 2017 (ASH abstract)

2. Fitzhugh et al., 2009

3. Lagresle-Peyrou et al., 2018

4. Hsieh and Tisdale, 2018

5. Yannaki et al., 2012