Factor VIII Expression from a Novel F8 Transgene through a Lentiviral Vector Transduced CD34+ Autologous Hematopoietic Stem Cells for Gene Therapy of Severe Hemophilia Α: Final Results from a Phase 1 Clinical Trial

We report the results of a completed single arm open label phase 1 clinical trial of lentiviral vector transduced autologous hematopoietic stem cell based gene therapy of hemophilia A (NCT 05265767; CTRI/2022/03/041304). The lentiviral vector, CD68-ET3-LV, contains a F8 transgene designed for enhanced factor VIII (FVIII) expression and secretion and a CD68 promoter that limits expression primarily to the rapidly replenishing mature monocytic compartment. We have shown previously that CD68-ET3-LV produces sustained FVIII expression in murine hemophilia A mice using transduced murine Sca+ cells and in immunodeficient mice using transduced human CD34+ cells (Doering CB et al Hum Gene Therapy 2018; 29:1183).

Adult patients with severe hemophilia A and no inhibitors after >100 exposures to exogenous FVIII were eligible for this study. They received hematopoietic stem cell transplantation (HSCT) with autologous G-CSF- and plerixafor-mobilized CD34⁺ peripheral blood cells collected by apheresis and transduced ex-vivo with the CD68-ET3-LV vector and cryo-preserved. Study participants were conditioned with myeloablative doses of treosulfan and fludarabine before infusion of a target dose of >5x10⁶ cells/kg transduced CD34⁺ cells. After the treatment of the first two participants (G1), the transduction protocol was modified to include a transduction enhancer (ASH 2023 Abstract #481) for the next three participants (G2). During the engraftment period, participants received recombinant FVIII (rFVIII) targeting a trough level of >50% FVIII activity along with standard supportive care for HSCT. As engraftment ensued and blood counts normalized, rFVIII replacement was tapered and discontinued. FVIII levels were measured by one-stage coagulant (FVIII:C) and chromogenic substrate assays. The genomic integration profile of the CD68-ET3-LV vector and vector copy number (VCN) were assessed in the peripheral blood and bone marrow post engraftment.

Five male participants aged 22-41 years, weighing 47-76 kg were included in the study. There was no Grade ≥2 toxicity associated with mobilization and collection of the CD34⁺ cells with target doses being achieved in a single apheresis. The final cell dose in the gene therapy product ranged between 5.0-6.1 x 10⁶ cells/kg with 95% or higher viability. The VCN in the final product infused was 1.02 and 0.57 in G1 and 1.49, 0.62 and 2.2 in G2 participants. The HSCT procedure was well tolerated. There were no infusion related adverse events. No Grade >2 non-hematological toxicities occurred during the conditioning or engraftment period. Neutrophil and platelet engraftment occurred at 10-12 and 12-15 days after HSCT, respectively. The duration of severe neutropenia and severe thrombocytopenia was 7-11 and 1-7 days, respectively. There were no bleeding episodes during the peri-HSCT period. The last day of exogenous rFVIII replacement ranged from 11-20 days after HSCT. The FVIII:C levels at day +60 after HSCT were 4% and 7.9% in G1 and 14.2%, 28% and 36.6% in G2. Median (range) FVIII:C level from day +60 to last follow-up were 4.6% (3-8.7%) at 25 months and 1.54% (1.1-2%) at 17 months in G1, and 37.5% (29.7-49.7%) at 12 months, 19.3% (11-31%) at 10 months and 40.2% (32.1-55.1%) at 7 months in G2 participants, with comparable one stage and chromogenic substrate assays. None of the participants reported any bleeds post-GT (ABR = 0 over 77 patient-months of combined follow-up). Peripheral blood (PB) and bone marrow (BM) VCN at 4-22 months were 0.08 to 0.17 in G1 participants compared to 2.74 to 4.82 in G2 participants, showing similar results in PB and BM and a positive correlation between plasma FVIII levels and VCN. The vector integration profile revealed no safety concerns. Analysis of FVIII mRNA levels within the cellular components of the blood showed highest expression within CD14⁺ cells, lower levels in CD15⁺ cells and none within CD3⁺ cells.

The results from this first-in-human phase 1 clinical trial of a lentiviral vector transduced HSC-based gene therapy for hemophilia A validates this novel approach with no major safety concerns identified. There is stable expression of FVIII up to 2 years and a positive correlation of VCN in PB with plasma levels of FVIII.