Abstract
Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding β-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of β-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-γ release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.
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Acknowledgements
We are grateful to Dr Katsujiro Sato, Dr Yasushi Mochizuki, Dr Naoko Yugeta, Dr Akiyo Nishiyama, Dr Madoka Ikemoto, Dr Michiko Wada, Ms Kazue Kinoshita, Ms Ryoko Nakagawa, Mr Satoru Masuda, Dr Masayuki Tomohiro and Dr Yoshiki Shimatsu for technical assistance. We also thank Mr Hideki Kita and Mr Shinichi Ichikawa and other staff of JAC Co. for care and management of experimental animals. This work is supported by Grants-in-Aid for Scientific Research for Research on Nervous and Mental Disorders (10B-1, 13B-1, 16B-3) and Health Sciences Research Grants for Research on Human Genome and Gene Therapy (H10-genome-015, H13-genome-001, H16-genome-003) from the Ministry of Heath, Labor and Welfare of Japan, and Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). A part of this work in Musashino University is supported by High-Tech Research Center Project for Private Universities: matching fund subsidy from MEXT, 2004.
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Yuasa, K., Yoshimura, M., Urasawa, N. et al. Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther 14, 1249–1260 (2007). https://doi.org/10.1038/sj.gt.3302984
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DOI: https://doi.org/10.1038/sj.gt.3302984
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