Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.
Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.
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DOI：
10.1038/gt.2014.4
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发表时间：
2014-04
期刊：
影响因子：
5.1
通讯作者：
Miller, A. D.
中科院分区：
文献类型：
--
作者：
Wang, Z.;Halbert, C. L.;Lee, D.;Butts, T.;Tapscott, S. J.;Storb, R.;Miller, A. D.
利用AAV载体的动物和人类基因治疗研究表明，对AAV衣壳蛋白的免疫应答可严重限制转基因表达。衣壳抗原的主要来源是与AAV载体相关的衣壳抗原，其可以通过严格的载体纯化来减少。AAV衣壳蛋白的第二个来源是在载体生产期间从异常包装到AAV病毒体中的cap基因表达的。该抗原来源可通过使用太大而不能掺入AAV衣壳的cap基因来消除，例如含有大内含子的cap基因（captron基因）。在这里，我们研究了消除帽基因转移和载体纯化CsCl梯度离心AAV载体免疫原性和表达肌内注射后的狗。我们发现这两种方法都降低了载体的免疫原性，并且将两者结合产生了最低的免疫应答和最高的转基因表达。这种组合方法使得能够使用相对温和的免疫抑制方案来促进杜氏肌营养不良症影响的狗中的稳健的微小肌营养不良蛋白基因表达。我们的研究显示了最小化AAV cap基因杂质的重要性，并表明AAV载体生产的这种改进可能有益于人类应用。
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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