Carol H. Miao

The most significant complication in hemophilia A treatment is the formation of inhibitors against factor VIII (FVIII) protein. Glycans and glycan-binding proteins are central to a properly functioning immune system. This study focuses on whether glycosylation of FVIII plays an important role in induction and regulation of anti-FVIII immune responses. We investigated the potential roles of 4 N-glycosylation sites, including N41 and N239 in the A1 domain, N1810 in the A3 domain, and N2118 in the C1 domain of FVIII, in moderating its immunogenicity. Glycomics analysis of plasma-derived FVIII revealed that sites N41, N239, and N1810 contain mostly sialylated complex glycoforms, while high mannose glycans dominate at site N2118. A missense variant that substitutes asparagine (N) to glutamine (Q) was introduced to eliminate glycosylation on each of these sites. Following gene transfer of plasmids encoding B domain deleted FVIII (BDD-FVIII) and each of these 4 FVIII variants, it was found that specific activity of FVIII in plasma remained similar among all treatment groups. Slightly increased or comparable immune responses in N41Q, N239Q, and N1810Q FVIII variant plasmid-treated mice and significantly decreased immune responses in N2118Q FVIII plasmid-treated mice were observed when compared with BDD-FVIII plasmid-treated mice. The reduction of inhibitor response by N2118Q FVIII variant was also demonstrated in AAV-mediated gene transfer experiments. Furthermore, a specific glycopeptide epitope surrounding the N2118 glycosylation site was identified and characterized to activate T cells in an FVIII-specific proliferation assay. These results indicate that N-glycosylation of FVIII can have significant impact on its immunogenicity.

We previously showed that intraosseous (IO) delivery of factor VIII (FVIII, gene F8) lentiviral vector (LV) driven by the megakaryocyte-specific promoter Gp1bα (G-F8-LV) partially corrected the bleeding phenotype in hemophilia A (HemA) mice for up to 5 months. In this study, we further characterized and confirmed the successful transduction of self-regenerating hematopoietic stem and progenitor cells (HSPCs) in treated mice. In addition, secondary transplant of HSPCs isolated from G-F8-LV–treated mice corrected the bleeding phenotype of the recipient HemA mice, indicating the potential of long-term transgene expression following IO-LV therapy. To facilitate the translation of this technology to human applications, we evaluated the safety and efficacy of this gene transfer therapy into human HSPCs. In vitro transduction of human HSPCs by the platelet-targeted G-F8-LV confirmed megakaryocyte-specific gene expression after preferential differentiation of HSPCs to megakaryocyte lineages. Lentiviral integration analysis detected a polyclonal integration pattern in G-F8-LV–transduced human cells, profiling the clinical safety of hemophilia treatment. Most importantly, IO delivery of G-F8-LV to humanized NBSGW mice produced persistent FVIII expression in human platelets after gene therapy, and the megakaryocytes differentiated from human CD34+ HSPCs isolated from LV-treated humanized mice showed up to 10.2% FVIII expression, indicating efficient transduction of self-regenerating human HSPCs. Collectively, these results indicate the long-term safety and efficacy of the IO-LV gene therapy strategy for HemA in a humanized model, adding further evidence to the feasibility of translating this method for clinical applications.