Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.
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.
1] Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA [2] Department of Medicine, University of Washington, Seattle, WA, USA.
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Z
Wang
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
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CL
Halbert
Department of Radiology, University of Washington, Seattle, WA, USA.
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D
Lee
Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
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T
Butts
1] Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA [2] Department of Neurology, University of Washington, Seattle, WA, USA.
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SJ
Tapscott
1] Program in Transplantation Biology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA [2] Department of Medicine, University of Washington, Seattle, WA, USA.
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R
Storb
1] Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA [2] Department of Pathology, University of Washington, Seattle, WA, USA.
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Miller
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology*; D000229:Dependovirus / Q000235:genetics / Q000276:immunology; D004285:Dogs; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D023421:Models, Animal; D009137:Muscular Dystrophy, Animal / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics* / Q000276:immunology / Q000628:therapy; D014771:Virion / Q000276:immunology
D036022:Capsid Proteins