Intravenous Lentiviral Gene Therapy for Hemophilia a: Endothelial-Specific Expression and Increased Glycosylation for Improved FVIII Function

Abstract

Background: Hemophilia A (HA) is an X-linked monogenic coagulation disorder in the intrinsic coagulation cascade resulting from deficiency of the factor VIII (FVIII, F8) gene. The current treatment of HA is based on protein replacement therapy (PRT) with limitations of short half-life, high cost, and life-time requirement of the treatment. Gene therapy has become a promising treatment option for HA. Methods: An advanced lentiviral vector (LV) system for intravenous (iv) F8 gene therapy has been established based on a novel B domain deleted (BDD) F8 gene, which restored all N-glycosylation sites (F8-299) in the B domain with increased coagulation function (DOI:10.1016/j.jbc.2021.101397). LV-F8-299 containing a universal (EF1a) or endothelial-specific (VEC) promoter was investigated in vitro using a human endothelial cell line, EA-hy926, and biologically and immunologically characterized in vivo in F8 knockout (KO) mice and in a non-human primate. Results: We found that LV-VEC-F8-299 (VEC-299) exhibited higher virus packaging (100 folds) and transduction efficiencies than EF1a-299. The F8 protein expression was verified in EA-hy926 by RT-PCR, Western blot and ELISA. We detected F8 activities close to 4 folds above the normal plasma level from the EF1a-299 transduced cells, whereas the VEC-299 expressed only 0.1 fold of the normal plasma F8 level. In vivo studies in F8 KO mice via iv injection of LVs after non-myeloablative radiation conditioning illustrated variable phenotypic corrections as well as anti-F8 immune responses from the two LVs. The iv gene therapy of EF1a-299 and VEC-299 LVs illustrated therapeutic F8 activities over time, in the range of 90% and 40%, respectively, in 60 days. Importantly, VEC-299 steadily reached a very high level (180%) of F8 activities, whereas EF-299 dropped to 3% after 120 days (Figure A). Kinetic analyses of anti-F8 IgG and inhibitor titer showed that the VEC-299 treated mice exhibited the lowest F8 inhibitory immune response over time. We next assessed the distribution of LV F8 gene in a macaque monkey (Macaca fascicularis). The monkey was chemo-conditioned with methylprednisolone (10 mg/kg) and prednisone (2 mg/kg) and iv injected with 1x109 TU/kg LV-VEC-299. The vector copy number (VCN) in body fluids was closely monitored by qPCR. From day 1 to week 14 after injection, the VCN in the blood remained stable (~1%), while in feces and saliva was high on day 1 (~3%), and in urine reached peak (~5%) on day 7, but all of which decreased with time. The VCN in body fluids (blood, urine, faces, saliva and tear) became nearly undetectable after 14 weeks. A second LV injection of 1x109TU/kg LV-VEC-299 was performed using a modified non-myeloablative conditioning with busulfan (2 mg/kg), cyclophosphamide (40 mg/kg) and dexamethasone (3 mg/kg). The VCN in the blood reached 20% from day 1 to day 7, then reduced to 5% and remained stable to week 6 (Figure B). The blood biochemical analyses including Alanine Transaminase (ALT), Aspartate Transaminase (AST), Total Protein (TP) and Albumin (ALB) remained in the normal range before and after treatment. Conclusion: These results demonstrated that the LV-VEC-299 exhibited delivery consistency, high coagulation function and low immunogenicity. The in vivo study supported that iv LV-VEC-299 gene therapy could be a safe, convenient and effective HA gene therapy strategy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal