Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Elevated fetal hemoglobin (HbF, α₂γ₂) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved.

We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p<0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups.

We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation.

For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×10⁶ CD34⁺ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs.

For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×10⁶ CD34⁺ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p<0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals.

In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction.