Gene Therapy Expressing Platelet‑Derived Factor VIII for Correction of Hemophilia Α with a History of Inhibitors

Introduction: Platelet targeted gene therapy with human factor VIII has established hemostasis in murine and canine models with severe hemophilia A without eliciting inhibitory antibodies. Here-in we describe feasibility, safety and efficacy of the first subject treated on a first-in-human phase 1 trial that targets factor FVIII synthesis and storage within platelet α‑granules for delivery at the site of vascular injury for hemophilia A (NCT03818763).

Method: Eligible are severe hemophilia A patients ≥18 years with a history of inhibitors to factor VIII (≥0.6 Bethesda Units [BU]/ml). Following mobilization, autologous CD 34+ cells (≥6 x 10⁶/kg) are collected and transduced with a lentiviral vector encoding the ITGA2B gene promoter for ectopic expression of human B-domain‑deleted factor VIII within the megakaryocyte lineage. The transduced cells are infused after reduced intensity cytoreduction with fludarabine (120 mg/m²) and melphalan (120 mg/m²) followed by a washout period of ≥24 hours. The primary endpoints of feasibility and safety are defined as: 1) feasibility of the cell manufacturing procedure by the availability of ≥4 x 10⁶/kg transduced clinical grade CD34+cells, cell viability ≥70% and undetectable microbial contamination and 2) safety defined as hematopoietic recovery ≤28 days of infusion and absence of ≥ grade 3 toxicity (CTCAE version 5.0).

Outcome: Subject 1 is a 29-year-old male with severe hemophilia A who developed inhibitors to factor VIII (2.6 BU/ml) in his first year of life. Immune tolerance to factor VIII had been established with a non‑detectable inhibitor titer at enrollment. Hemostatic prophylaxis was maintained with emicizumab weekly and recombinant factor VIII for breakthrough bleeding. His annualized bleeding rate was 16 in the 12-month period preceding infusion. The cell product was the result of 1 day of collection yielding 6.73 x 10⁶ /kg total viable CD34+ cells post-transduction and vector copy number by qPCR of 1.16 copies/cell. Megakaryocyte factor VIII:C levels were 0.00 mU/10⁶ before transduction and 101.32 mU/10⁶ after transduction. The cell product satisfied release criteria. Neutrophil recovery (≥0.5 x 10⁹/L) and platelet transfusion independence (≥50 x 10⁹/L) was achieved 15 days post-infusion and sustained for >12 months. The duration of hospitalization was 21 days with no re-admissions. There was no breakthrough bleeding during collection and hospitalization. During and post-infusion there was no unexpected toxicity ≥ grade 1. He has not required immune suppression. Emicizumab was discontinued 3.6 months post‑infusion after demonstration of whole blood vector copy number at 1 and 3-months. There has been no spontaneous bleeding or a need for “on demand” factor VIII after discontinuing emicizumab. Secondary outcomes are summarized in Table 1. No replication competent lentivirus was detected through month 12 post‑infusion. Integration site analysis was carried out, and none of the samples tested through 12 months contained cell clones which exceeded 20% relative abundance. Transduced cells were highly polyclonal. Integration site analysis showed no enrichment of integration near cancer‑associated genes in the cell product nor in whole blood cell lineages post-infusion.

Conclusion: We report feasibility, safety and efficacy in the first subject with severe hemophilia A and a history of inhibitors who received lentiviral vector gene therapy directed to induce megakaryocytes to synthesize and store factor VIII within platelets. These findings extend the potential to treat severe hemophilia A patients who are not eligible for valoctcogene roxaoarvovec (AAV5-hFVIII-SQ).