Mount Sinai School of Medicine, Tisch Cancer Center, New York, NY 10029, United States.
Virology. 2012 Nov 25;433(2):356-66. doi: 10.1016/j.virol.2012.08.015. Epub 2012 Sep 13.
Adeno-associated virus (AAV) has been reported to integrate in a site-specific manner into chromosome 19 (a site designated AAVS1), a phenomenon that could be exploited for ex vivo targeted gene therapy. Recent studies employing LM-PCR to determine AAV integration loci; however, have, contrary to previous results with less reliable methods, concluded that the proclivity for AAV integration at AAVS1 is minimal. We tested this conclusion employing LM-PCR protocols designed to avoid bias. Hep G2 cells were infected with rAAV2-GFP and coinfected with wt AAV2 to supply Rep in trans. Sorted cells were cloned and cultured. In 26 clones that retained fluorescence, DNA was extracted and AAV-genomic junctions amplified by two LM-PCR methods. Sequencing was performed without bacterial cloning. Of these 26 clones it was possible to assign a genomic integration site to 14, of which 9 were in the AAVS1 region. In three additional clones, rAAV integration junction were to an integrated wt AAV genome while two were to an rAAV genome. We also show that integration of the AAV-GFP genome can be achieved without cointegration of the AAV genome. Based on the pattern of integrants we propose, for potential use in ex vivo targeted gene therapy, a simplified PCR method to identify clones that have rAAV genomes integrated into AAVS1.
腺相关病毒（AAV）已被报道以特定的方式整合到染色体 19 上（称为 AAVS1 的位点），这种现象可用于体外靶向基因治疗。最近的研究采用 LM-PCR 来确定 AAV 整合位点；然而，与以前使用不太可靠的方法得出的结果相反，他们得出的结论是 AAV 在 AAVS1 上整合的倾向最小。我们采用旨在避免偏差的 LM-PCR 方案来测试这一结论。将 rAAV2-GFP 感染 Hep G2 细胞，并与 wt AAV2 共感染以提供转座的 Rep。对分选的细胞进行克隆和培养。在保留荧光的 26 个克隆中，提取 DNA 并通过两种 LM-PCR 方法扩增 AAV 基因组接头。测序在没有细菌克隆的情况下进行。在这 26 个克隆中，有 14 个能够确定基因组整合位点，其中 9 个位于 AAVS1 区域。在另外三个克隆中，rAAV 整合接头与整合的 wt AAV 基因组有关，而两个与 rAAV 基因组有关。我们还表明，在没有 AAV 基因组共整合的情况下，可以实现 AAV-GFP 基因组的整合。基于整合子的模式，我们提出了一种简化的 PCR 方法，用于鉴定 rAAV 基因组整合到 AAVS1 的克隆，可用于体外靶向基因治疗。
