Abstract
Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.
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Acknowledgements
We thank E Zellmer, P Stroup, Jennifer Totman and Baocheng Chu, MD, PhD for technical assistance, A Joslyn, J Duncan, DVM, M Spector, DVM and their team for their care of the dogs. We further thank S Carbonneau, H Crawford, B Larson, K Carbonneau, J Vermeulen, and D Gayle for administrative assistance and help with manuscript preparation. We are grateful for research funding from the National Institutes of Health Grants R01AR056949 and R21EB008166 and Career Development Grant MDA 114979 from the Muscular Dystrophy Association to ZW.
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CLH and ADM are inventors on a patent application covering the captron method for AAV production. The other authors declare no conflict of interest.
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Wang, Z., Halbert, C., Lee, D. et al. Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model. Gene Ther 21, 363–370 (2014). https://doi.org/10.1038/gt.2014.4
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DOI: https://doi.org/10.1038/gt.2014.4
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