Enhanced Factor VIII Heavy Chain for Gene Therapy of Hemophilia A

Abstract

Hemophilia A gene therapy using recombinant adenovirus-associated virus (AAV) vectors has been hampered by the size of the factor VIII (FVIII) cDNA. Previously, splitting the FVIII coding sequence into a heavy-chain (HC) fragment and a light-chain (LC) fragment for dual recombinant AAV vector delivery has been successfully explored. However, the main disadvantage of this approach is a “chain imbalance” problem in which LC secretion is ~1–2 logs higher than that of HC, and therefore, the majority of protein synthesized is nonfunctional. To improve HC secretion, we constructed alternate FVIII HCs based on our observation that LC facilitates HC secretion. To our surprise, most of the new HC molecules exhibited enhanced expression over the traditional HC molecule (HC745). The optimized HC mutein, HCHL, including additional acidic-region-3 (ar3) sequences, exhibited three- to fivefold higher activity in both enzyme-linked immunosorbent assay (ELISA) and activated partial thromboplastin time (aPTT) assay in in vitro testing. Further characterization suggested ar3 sequences increased HC secretion, rather than promoting HC synthesis. Intravenous delivery of AAV8-HCHL+AAV8-LC or AAV8-HC745+AAV8-LC achieved phenotypic correction in hemophilia A mice. Mice receiving AAV8-HCHL+AAV8-LC achieved three- to four-fold higher HC expression than AAV8-HC745+AAV8-LC, consistent with the FVIII functional assays. HCHL should be substituted for HC745 in a dual AAV vector strategy due to its enhanced expression.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

“…However, progress has been hampered by low levels of FVIII transgene expression and the large size of the FVIII cDNA. 16,17,36 In this study, we show that a single administration of rAAV that contains a codop hFVIII construct that includes the proximal 226 aa of the hFVIII B domain resulted in therapeutic expression of hFVIII in murine and non-human primate models. For a given dose of vector, expression mediated by rAAV-HLP-codophFVIII-N6 was almost 10-fold higher than observed with rAAV containing wild-type hFVIII sequences for the BDD or N6 variants (Table 1).…”

“…13 Other innovative approaches to overcome the size constraint involve the need for simultaneous transduction with 2 AAV vectors for functional FVIII activity, which has limited their transition to the clinic. [14][15][16][17] We and others have shown that expression of FVIII can be improved in the context of lentiviral vectors by reorganization of the wildtype cDNA of hFVIII according to the codon usage of highly expressed human genes as described before for human factor IX (hFIX). 3,18,19 However, these codon-optimized (codop) FVIII variants remain hitherto untested in the setting of recombinant AAV (rAAV).…”

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors forIn VivoTransduction

“…34 The pdsAAV-Gluc (1.8 kb) and pdsAAV-EGFP (2.1 kb) plasmids were designed to carry either the Gaussia Luciferase gene (Gluc) or enhanced green fluorescent protein (EGFP) gene, driven by a human b-actin promoter with a CMV enhancer (CB). These two plasmids contained one full ITR and one mutant ITR, and were used to generate self-complementary AAV vectors (scAAV-EGFP and scAAV-Gluc).…”

Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

“…However, the secretion of the HC was inefficient but improved significantly when LC was present, much higher especially when inter-chain disulfide bond formed. Studies by Chen et al [18] have shown that the secretion of LC facilitates HC secretion through its N-terminal acidic region 3 (ar3), where a mutant HC with an additional ar3 sequences at the C-terminus exhibits three-to five-fold higher secretion in vitro. In addition, ar3 has a role in mediating interaction of FVIII with vWF [19], but unlikely to be related to this function.…”