Rescue of Adeno-Associated Virus Production by shRNA Cotransfection
Rescue of Adeno-Associated Virus Production by shRNA Cotransfection
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DOI：
10.1089/hum.2019.249
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发表时间：
2020-04-28
影响因子：
4.2
通讯作者：
Chiorini, John A.
中科院分区：
文献类型：
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作者：
Guimaro, Maria C.;Afione, Sandra A.;Chiorini, John A.
腺相关病毒（AAV）载体技术正在迅速发展，不仅成为基因治疗领域的领先载体平台，而且也是新蛋白质功能基因组研究的有用工具。由于大多数载体利用组成型启动子，这导致生产期间的转基因表达。取决于转基因产物，这可能诱导促凋亡、细胞生长抑制或其他干扰生产细胞功能的未知效应，从而降低病毒载体产量。这可能是一个主要的限制，当试图描述不好的基因。我们描述了包装过程中共转染的shRNA编码质粒的新用途，以限制细胞毒性转基因产物的表达。这允许产生否则无法包装的载体。该方法简单，通用，不需要修饰载体质粒，并且应该容易地以最小的成本适应几乎任何转基因。
Adeno-associated virus (AAV) vector technology is rapidly advancing and becoming not only the leading vector platform in the field of gene therapy but also a useful tool for functional genomic studies of novel proteins. As most vectors utilize constitutive promoters, this results in transgene expression during production. Depending on the transgene product, this could induce proapoptotic, cytostatic, or other unknown effects that interfere with producer cell function and, therefore, reduce viral vector yield. This can be a major limitation when trying to characterize poorly described genes. We describe the novel use of shRNA encoding plasmids cotransfected during packaging to limit the expression of the cytotoxic transgene product. This allowed the production of an otherwise unpackageable vector. The approach is simple, versatile, does not require modification of the vector plasmid, and should be easily adaptable to almost any transgene with minimal cost.