Abstract
Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.
Highlights
Hemophilia A is a congenital bleeding disorder caused by a deficiency of procoagulation Factor VIII (FVIII)
The transduction efficiencies were reciprocally correlated with the vector sizes, the full-length FVIII cDNA (10.6 kb) could be transduced as efficiently as the BDD FVIII cDNA (Figure S3)
When we transfected piggyBac vectors expressing EGFP, BDD FVIII, or full-length FVIII (Figure 1A), we observed that full-length FVIII vector could integrate into host chromosomes as efficiently as BDD FVIII vector (Figure 1B)
Summary
Hemophilia A is a congenital bleeding disorder caused by a deficiency of procoagulation Factor VIII (FVIII). Repeated intravenous injection of recombinant FVIII protein can prevent bleeding events, but alternative treatments could potentially improve patients’ quality of life; hemophilia A is an attractive target disease for the application of gene therapy. Significant advances in both preclinical animal models and phase I/II human clinical trials have been reported [1]. Most preclinical studies of hemophilia A gene therapy have focused on the use of viral vectors, such as adenovirus [2,3], adeno-associated virus (AAV) [4,5,6], gammaretrovirus [7], and lentivirus [8,9,10,11,12]. Whether the B-domain is beneficial for FVIII expression in the context of gene therapy remains to be determined
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