Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Gene Ther. 2009 Nov;16(11):1314-9. doi: 10.1038/gt.2009.106. Epub 2009 Sep 3.
For certain gene therapy applications, the simultaneous delivery of multiple genes would allow for novel therapies. In the case of adeno-associated virus (AAV) vectors, the limited packaging capacity greatly restricts current methods of carrying multiple transgene cassettes. To address this issue, a recombinant AAV (rAAV) vector was designed such that a furin proteolytic cleavage site (RKRRKR) was placed between the coding sequences of two genes (green fluorescent protein (GFP) and galanin), to allow cleavage of the chimeric protein into two fragments. In addition, these constructs contained the fibronectin secretory signal sequence that causes the gene products to be constitutively secreted from transduced cells. In vitro studies show that after transfection of HEK293 cells, the appropriate cleavage and constitutive secretion occurred regardless of the order of the genes in the transgene cassette. In vivo, infusion of rAAV vectors into the piriform cortex resulted in both GFP expression and significant galanin attenuation of kainic acid-induced seizure activity. Thus, the present results establish the utility of a proteolytic approach for the expression and secretion of multiple gene products from a single AAV vector transgene cassette.
对于某些基因治疗应用，同时递送多个基因可以实现新的治疗方法。在腺相关病毒 (AAV) 载体的情况下，有限的包装容量极大地限制了目前携带多个转基因盒的方法。为了解决这个问题，设计了一种重组 AAV(rAAV) 载体，使得在两个基因（绿色荧光蛋白 (GFP) 和甘丙肽）的编码序列之间放置一个弗林蛋白酶切割位点 (RKRRKR)，以允许将嵌合蛋白切割成两个片段。此外，这些构建体包含纤维连接蛋白分泌信号序列，导致基因产物从转导细胞中持续分泌。体外研究表明，转染 HEK293 细胞后，无论转基因盒中基因的顺序如何，都会发生适当的切割和持续分泌。在体内，将 rAAV 载体注入梨状皮层会导致 GFP 表达和甘丙肽显著减弱海人酸诱导的癫痫发作活动。因此，目前的结果确立了一种蛋白水解方法在单个 AAV 载体转基因盒中表达和分泌多种基因产物的实用性。
