Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.
Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.
复制标题
细胞重组途径和病毒末端重复发夹结构足以实现腺相关病毒在体内和体外的整合。
DOI：
10.1128/jvi.71.12.9231-9247.1997
复制
发表时间：
1997
影响因子：
5.4
通讯作者：
Samulski,RJ
中科院分区：
文献类型：
--
作者：
Yang,CC;Xiao,X;Zhu,X;Ansardi,DC;Epstein,ND;Frey,MR;Matera,AG;Samulski,RJ
人细小病毒腺相关病毒（AAV）在其靶向病毒整合至染色体19（ch-19）上的特定位点的能力方面是独特的。重组AAV（rAAV）载体保留了整合能力，但显然失去了这种靶向能力。在这份报告中，我们描述了野生型（WT），rAAV和体外系统的末端重复介导的整合，以更好地了解这些差异。通过多种技术，包括PCR、Southern杂交和荧光原位杂交分析，对WT或rAAV潜伏的细胞系进行表征。对40多个AAV-rAAV整合接头进行克隆、测序，然后进行比较和分析。在永生化和正常二倍体人细胞中，wt AAV靶向整合至ch-19。整合的前病毒结构由头-尾串联阵列组成，其中大部分连接序列涉及AAV反向末端重复序列（ITR）。未直接观察到完整的病毒ITR。在一些实例中，发现AAV p5启动子序列在病毒-细胞连接处融合。来自PCR产物的斑点印迹分析的数据与野生型整合位点处的基因组和/或病毒DNA序列的倒位的发生一致。与wt前病毒连接不同，rAAV前病毒连接映射到非ch-19序列的子集。Southern分析支持来自两个独立细胞系的前病毒整合在CH-2上的同一位点。此外，前病毒末端重复序列存在于翻转和翻转方向，与微同源性明显的交界处。在除ITR外的所有情况下，载体完整整合。rAAV连接序列数据与通过整合基因座处的缺失和/或重排-易位发生的基因组重排一致。最后，分离并表征了在体外系统中在几种AAV底物和ch-19靶位点之间形成的连接。线性AAV底物通常利用病毒DNA底物的末端作为整合点，而在Rep蛋白存在或不存在下衍生自AAV末端重复发夹结构的产物类似于体内产生的AAV-ch-19连接。描述wt AAV、rAAV和体外整合接头的这些结果表明，病毒整合事件本身是由末端重复发夹结构通过非病毒细胞重组途径介导的，体内对ch-19具有特异性，需要额外的病毒组分。这些研究将对rAAV载体在人类基因治疗中的应用产生重要影响。
The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.