Hemophilia is an X-linked bleeding disorder caused by a deficiency in the clotting factor protein factor VIII (FVIII) (hemophilia A, HA) or factor IX (FIX) (hemophilia B, HB) present in 1 in 5,000 males worldwide. Small amounts of clotting factor protein (>1% of normal) in the circulation provide substantial improvement of the disease phenotype. Preclinical studies of gene-based therapies for HB in dog models provided the basis for ongoing clinical trials using adeno-associated viral (AAV) vectors. These ongoing clinical trials demonstrated sustained expression of therapeutic levels of FIX. However, 80% of all hemophilia is due to FVIII deficiency, hemophilia A (HA). Gene therapy for HA presents three distinct challenges: (1) intrinsic properties of human FVIII (hFVIII) make it difficult to express compared to other proteins of similar size (2) the large size of the FVIII cDNA is prone to rearrangements which hampers AAV production and (3) high rates of anti-FVIII antibody (inhibitor) formation in patients following protein therapy. In earlier studies, AAV delivery of canine FVIII in HA dogs resulted in long-term dose-dependent expression of cFVIII that significantly improves the disease phenotype, however, the dose of AAV to achieve these goals remains relatively high. This high vector dose may be an obstacle to translating to humans since humans may have pre-existing immunity to AAV and thus may have an immune response to the AAV capsid upon AAV vector administration. We recently identified two novel human FVIII modifications that overcome the challenges of packaging the FVIII cDNA into AAV vectors and expressing hFVIII protein. First, a unique codon-optimized hFVIII DNA sequence expresses higher than wild type hFVIII but also packages more efficiently into AAV vectors. AAV packaging of this codon-optimized cDNA sequence results in a homogenous AAV vector that has a two-fold higher yield than wild type hFVIII. Second, variants of an intracellular protease cleavage recognition site in the hFVIII protein confer increased secretion and biological activity. We hypothesize that AAV delivery of these hFVIII variants will be therapeutic at lower AAV doses than native hFVIII, with no increases in immunogenicity over the currently used B-domain deleted FVIII constructs. The goal of this proposal is to determine the efficacy and safety of AAV delivery of these novel variants of hFVIII in unique hemophilia A mouse models (Specific Aim 1) and hemophilia A dog models (Specific Aim 2) that are both tolerant to human FVIII. These studies have the potential for success in reducing the AAV vector dose which will have a profound impact on avoiding the anti-AAV immune responses and on the long-term efficacy after AAV-mediated gene delivery of FVIII which will provide the necessary advances to translate this gene therapy approach for hemophilia A into the clinic.

Public Health Relevance

Gene therapy for hemophilia is a strategy for providing continuous levels of clotting factor in the circulation to prevent bleeding episodes. We have established that delivery of the defective protein (factor VIII) by gene therapy corrects the bleeding disorder in large animal models of the disease; however, it requires a relatively large viral vector dose that needs to be overcome to support clinical translation of this therapy to the clinic. The goal of this project is to use an optimized novel factor VIII variant for gene therapy or hemophilia A to correct the bleeding disorder and lower the vector dose required to achieve this therapeutic effect.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Sarkar, Rita
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Children's Hospital of Philadelphia
United States
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