Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells.
Lack of site-specific integration of the recombinant adeno-associated virus 2 genomes in human cells.
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重组腺相关病毒 2 型基因组在人类细胞中缺乏位点特异性整合。
DOI：
10.1089/hum.1997.8.3-275
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Srivastava,A
中科院分区：
文献类型：
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作者：
Ponnazhagan,S;Erikson,D;Kearns,WG;Zhou,SZ;Nahreini,P;Wang,XS;Srivastava,A
基于腺相关病毒2(AAV)的载体系统被认为是潜在的用于人类基因治疗，因为野生型(Wt)AAV基因组似乎以位置特异性的方式整合到人类染色体DNA中。我们系统地研究了缺少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4个含四环素抗性基因(TCR)和疱疹病毒胸苷激酶(TK)启动子驱动的新霉素抗性基因(VTcNeo)、氨苄西林抗性基因(Apr)和TK-neR基因(vAp.Neo)、AAV复制基因(Rep)和TK-neR基因(vRep.neo)、AAV衣壳基因(Capsid)和TK-neR基因(vCap.Neo)的重组AAV基因组。用Aneo特异性DNA探针对人鼻咽癌细胞系(KB)的克隆分离株进行Southern杂交分析，各重组AAV基因组的整合模式明显不同。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.)共价连接。人类染色体DNA片段，推测为wt AAV基因组整合的特定位置。此外，用Aneo特异的聚合酶链式反应(PCR)扩增出276个碱基的DNA片段，但在这些克隆和Rt.Jn上没有扩增产物。在相同条件下的引物对。荧光原位杂交(FISH)分析进一步表明，重组AAV没有整合到人的19号染色体中，即使在腺病毒存在的情况下，通过挽救重组AAV基因组来确定重组AAV基因的存在也是如此。这些数据表明，重组AAV基因组整合在与wt AAV基因组特征不同的位置。这些研究可能对基于AAV的载体系统的开发有意义，因为它在人类基因治疗中的潜在应用。
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; vTcNeo), 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 aneo-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 aneo-specific primer-pair by polymerase chain reaction (PCR), no amplified DNA product was obtained usign theneo- and the Rt.Jn. primer-pair under identical conditions. Fluorescencein situhybridization (FISH) analyses further revealed the lack of integration of the recombinant AAV into human chromosome 19, even in the presence of a functionalrepgene 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.