Adeno-Associated Virus Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA.
Hum Gene Ther. 2020 Oct;31(19-20):1068-1073. doi: 10.1089/hum.2019.249. Epub 2020 Apr 28.
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.
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主要载体平台，而且还是研究新型蛋白质功能基因组学的有用工具。由于大多数载体利用组成性启动子，因此在生产过程中会导致转基因表达。根据转基因产物的不同，这可能会诱导促凋亡、细胞停滞或其他未知的影响，干扰生产细胞的功能，从而降低病毒载体的产量。当试图对描述不佳的基因进行特征分析时，这可能是一个主要的限制因素。我们描述了一种新颖的用途，即在包装过程中同时转染短发夹 RNA (shRNA) 编码质粒来限制细胞毒性转基因产物的表达。这使得原本无法包装的载体得以生产。该方法简单、通用，不需要载体质粒的修饰，并且应该可以很容易地适应几乎任何具有最小成本的转基因，只需进行最小的改动。
