We conducted a first-in-human phase 1 dose-finding pilot and feasibility study testing the safety of gene therapy using a lentivirus vector (LVV) expressing a microRNA embedded shRNA (shmiR) targeting BCL11A in sickle cell disease (SCD) (NCT03282656). Eleven eligible subjects with SCD (HbSS or HbS/Hbβ0) had stem cell collection; one patient withdrew prior to infusion and 10 were infused with autologous hematopoietic stem and progenitor cells transduced with the shmiR vector. Engraftment occurred in all 10 patients. With a median follow-up of 58 months (range: 35-82) after infusion, no adverse events attributed to the gene vector have occurred. Plerixafor mobilized peripheral blood hematopoietic stem cells (HSC) required for manufacturing were obtained in 1 mobilization cycle (2 consecutive days of collection) for 10/11 subjects, with one patient requiring 2 cycles (mean of 2.3 apheresis days per patient for product manufacturing). Median time from first day of apheresis to final release of product was 39 days (range: 22–151; n=11), with only one product released after >50 days. The manufacturing efficiency, defined as the number of CD34+ cells in the final product divided by the number of CD34+ cells collected was 63% (mean, range 41-82%). Transduction efficiency was 93.1% (median, range: 62%-100%, n=11) with a vector copy number of 3.7+/-1.4 (mean+/-SD, n=11) and final product cell count was 5.1 (median, range: 3.1-8.7, n=11) CD34+ x 106cells/kg. BCL11A was effectively targeted with a median of 88% (range: 67-100%) of individual erythroid colonies derived from the manufactured product containing ≥30% fetal hemoglobin (HbF) by HPLC analysis. After busulfan conditioning and infusion, all subjects engrafted promptly (neutrophils 18-30d, platelets 26-62d) and at 6 months, the median vector copy number in whole blood (WB) was 0.7 copies/diploid genome (range: 0.2-1.5) which remained stable over the entire follow-up time. The subject with the lowest in vivo VCN (BCL-010) had the lowest HbF post-infusion of 14.1% at 6 months that remained low but stable over the entire follow-up time, defining the lower dosage of effective gene modfication. Of the remaining nine infused subjects there was a robust HbF induction with WB HbF 27.8% (range: 20.8%-38.2%), F cells 71% (range: 55.3%-80.3%) and F/Fcells 11.9 pg (range: 9.4-13.6 pg) at 2 years that remained stable at 48 months (N=8), with mRNA knock-down of BCL11A stable from 42-74% and no significant change in BCL11A expression in CD19+ B cells. These data defined the manufacturing parameters required for improved overall HbF induction and further increase in F cell numbers post-infusion that were implemented in the subsequent phase 2 GRASP trial (NCT05353647). Due to the cases of leukemia and myelodysplastic syndrome in two Group A subjects with SCD in the Lentiglobin HGB-206 gene addition trial, clonal hematopoiesis was monitored in all subjects by sequencing of defined clonal hematopoiesis-associated genes, in addition to tracking insertion site analysis (ISA). No subject had ISA-defined clones larger than 3% at any time during follow-up. One subject had a clonal pathogenic DNMT3A mutation noted after infusion that was retrospectively determined to be present prior to cell manufacturing below validated detection limits. The variant allele fraction of this mutation increased after infusion to a maximum of 9.3%, and has remained stable for over 4 years of follow-up with no evidence of dysplasia on the 2-year post-GT bone marrow. All patients demonstrated sustained mitigation of SCD phenotypes. These data demonstrate first-in-human efficiency, durability, safety and efficacy of targeting BCL11A using a shmiR LVV leading to a pivotal multi-site phase 2 trial currently underway (NCT05353647).

Funding Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT03282656

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2025
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