Although several labs including ours have demonstrated the feasibility of gene therapy for hemophilia A in animal models, long-term expression of the transgene at therapeutic levels was not observed in the clinic. The lack of sufficient expression levels Factor VIII (FVIII) in hemophilic patients indicates that further improvements of the current gene delivery systems are needed to generate a therapeutic product. In the Phase I of this project we initiated a study to address the issues of FVIII expression level and duration based on findings showing that FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose- binding lectin LMAN1. Vectors for FVIII expression in gene therapy applications typically have used a B domain deleted (BDD) cDNA in which the coding sequence for this domain is removed to yield a shorter construct for protein production and gene transfer. Of note, the expression of BDD FVIII within the cell has the same limitations as the whole FVIII molecule. However, the inclusion of several asparagine-linked oligosaccharides within a short B-domain spacer increased ER to Golgi transport resulting in secretion of functional FVIII at levels 15- to 25-fold higher than full-length or B domain deleted FVIII both in vitro and in vivo. Phase I initiated the testing of the hypothesis that therapeutic FVIII production can be significantly enhanced by addition of parts of the B domain in hemophilic dogs, the most relevant animal model to the human disease and a prerequisite test for clinical studies, Two FIV vectors were constructed and tested which contain canine FVIII with different lengths of the B domain added. The constructs were prepared with the canine FVIII genetic sequence to reduce complicating cross-species factors when introduced into the hemophilic dog. Also, in Phase I the parameters for preparing high titer virus were optimized and the innate immune response to VSV- G and GP64 pseudotyped vectors were analyzed. The completion of the Phase I aims puts the project in position to conduct the FVIII expression level and duration studies in hemophilic mice and dogs and to select the most favorable FVIII cDNA for safe and long term expression. In this Phase II study the aims are to evaluate expression level, duration and clinical benefit from FIV delivery of canine FVIII with no B domain or with two different lengths of the B domain included. Also, additional elements that may further enhance FVIII production significantly from transduced cells will be incorporated into the transfer vector and tested. This Phase II also includes the development of methods to provide increased production of the virus vector for these and subsequent studies, which will require large amounts of the vector.

Public Health Relevance

Clinical application of Factor VIII gene therapies to hemophilia A patients have suffered from insufficient production of Factor VIII. Using an FIV lentiviral vector capable of long-term expression in host cells, Factor VIII gene sequences with improvements designed to overcome the problem of poor expression will be tested in hemophilic dogs, the animal model which closely resembles the human disease. The goal is to advance the best product as determined from this study toward application in the clinic to human patients.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1-VH-E (10))
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Pucie, Susan
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Virogenics, Inc.
Del Mar
United States
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Staber, J M; Pollpeter, M J; Anderson, C-G et al. (2017) Long-term correction of hemophilia A mice following lentiviral mediated delivery of an optimized canine factor VIII gene. Gene Ther 24:742-748
Staber, Janice M; Pollpeter, Molly J; Arensdorf, Angela et al. (2014) piggyBac-mediated phenotypic correction of factor VIII deficiency. Mol Ther Methods Clin Dev 1:14042
Burnight, Erin R; Staber, Janice M; Korsakov, Pavel et al. (2012) A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon. Mol Ther Nucleic Acids 1:e50