1 Institute of Cell Biology, ZBAF, University of Witten/Herdecke , Witten, Germany .
2 Laboratory for AAV Vector Development, Center for Molecular Medicine Cologne (CMMC), University of Cologne , Cologne, Germany .
Hum Gene Ther. 2017 Dec;28(12):1169-1179. doi: 10.1089/hum.2017.025. Epub 2017 Jun 29.
Adeno-associated virus (AAV) vectors are one of the most frequently applied gene transfer systems in research and human clinical trials. Since AAV vectors do not possess an integrase activity, application is restricted to terminally differentiated tissues if transgene expression is required long term. To overcome this limitation and to generate AAV vectors that persist episomally in dividing cells, AAV vector genomes were equipped with a scaffold/matrix attachment region (S/MAR). After a mild antibiotic selection, cells transduced with AAV-S/MAR established colonies that maintained long-term transgene expression (>50 population doublings) from replicating AAV vector episomes in the absence of further selection. Unexpectedly, with a lesser but still significant efficiency, the control vector (AAV-ΔS/MAR), a standard single-stranded AAV vector, also established stable transgene-expressing colonies, most of which were maintained as replicating episomes rather than integrated vector genomes. Thus, based on the result in HeLa cells, it is concluded that AAV vector genomes per se possess the ability to establish episomal maintenance in proliferating cells, a feature that can be enhanced by incorporation of a foreign genomic element such as an S/MAR element.
腺相关病毒 (AAV) 载体是研究和人体临床试验中最常应用的基因转移系统之一。由于 AAV 载体不具有整合酶活性，如果需要长期转基因表达，则其应用仅限于终末分化组织。为了克服这一限制并产生能够在分裂细胞中持续存在于染色体外的 AAV 载体，AAV 载体基因组被配备了支架/基质附着区 (S/MAR)。经过轻度抗生素选择，用 AAV-S/MAR 转导的细胞建立了集落，这些集落能够从复制的 AAV 载体染色体外体中持续表达转基因 (>50 个细胞倍增)，而无需进一步选择。出乎意料的是，效率虽然较低但仍然显著，对照载体 (AAV-ΔS/MAR)，一种标准的单链 AAV 载体，也建立了稳定表达转基因的集落，其中大多数是以复制的染色体外体而不是整合的载体基因组的形式维持的。因此，基于 HeLa 细胞的结果，可以得出结论，AAV 载体基因组本身具有在增殖细胞中建立染色体外体维持的能力，通过掺入外源基因组元件(如 S/MAR 元件)可以增强这种能力。
