Ponnazhagan S, Erikson D, Kearns W G, Zhou S Z, Nahreini P, Wang X S, Srivastava A
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Hum Gene Ther. 1997 Feb 10;8(3):275-84. doi: 10.1089/hum.1997.8.3-275.
The adeno-associated virus 2 (AAV)-based vector system has been suggested for its potential use in human gene therapy because the wild-type (wt) AAV genome appears to integrate into the human chromosomal DNA in a site-specific manner. We systematically investigated the integration patterns of the recombinant AAV genomes lacking one or both the viral coding sequences. Four recombinant AAV genomes were constructed containing the genes for resistance to tetracycline (TcR) and the herpesvirus thymidine kinase (TK) promoter-driven gene for resistance to neomycin (neoR; vTc.Neo), the genes for resistance to ampicillin (ApR) and TK-neoR (vAp.Neo), the genes for AAV replication (rep) genes and TK-neoR (vRep.Neo), and the AAV capsid (cap) genes and TK-neoR (vCap.Neo). The integration pattern of each of the recombinant AAV genomes in individual clonal isolates of the human nasopharyngeal carcinoma cell line (KB) analyzed on Southern blots using a neo-specific DNA probe was distinctly different. In addition, in none of the clones examined was the proviral genome covalently linked to the previously described AAV right-junction (Rt.Jn.) human chromosomal DNA fragment, the putative specific-site of integration for the wt AAV genome. Furthermore, whereas a 276-bp DNA fragment could be readily amplified from each of these clones, using a neo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained using the neo- and the Rt.Jn. primer-pair under identical conditions. Fluorescence in situ hybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functional rep gene as determined by rescue of the recombinant AAV genome in the presence of adenovirus. These data suggest that the recombinant AAV genomes integrate at sites that are different from that characterized for the wt AAV genome. These studies may have implications in the development of the AAV-based vector system for its potential use in human gene therapy.
基于腺相关病毒2（AAV）的载体系统因其潜在的人类基因治疗用途而受到关注，因为野生型（wt）AAV基因组似乎以位点特异性方式整合到人类染色体DNA中。我们系统地研究了缺失一个或两个病毒编码序列的重组AAV基因组的整合模式。构建了四个重组AAV基因组，分别包含四环素抗性基因（TcR）和疱疹病毒胸苷激酶（TK）启动子驱动的新霉素抗性基因（neoR；vTc.Neo）、氨苄青霉素抗性基因（ApR）和TK-neoR（vAp.Neo）、AAV复制（rep）基因和TK-neoR（vRep.Neo）以及AAV衣壳（cap）基因和TK-neoR（vCap.Neo）。使用neo特异性DNA探针在Southern印迹上分析每个重组AAV基因组在人鼻咽癌细胞系（KB）的单个克隆分离物中的整合模式，结果明显不同。此外，在所检查的克隆中，没有一个前病毒基因组与先前描述的AAV右连接（Rt.Jn.）人类染色体DNA片段共价连接，而该片段被认为是wt AAV基因组的假定特异性整合位点。此外，虽然使用neo特异性引物对通过聚合酶链反应（PCR）可以从每个这些克隆中轻松扩增出一个276 bp的DNA片段，但在相同条件下使用neo和Rt.Jn.引物对未获得扩增的DNA产物。荧光原位杂交（FISH）分析进一步表明，即使在腺病毒存在下通过重组AAV基因组的拯救确定存在功能性rep基因，重组AAV也未整合到人类19号染色体中。这些数据表明，重组AAV基因组整合的位点与wt AAV基因组的特征位点不同。这些研究可能对基于AAV的载体系统在人类基因治疗中的潜在应用开发具有启示意义。
