Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery
Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery
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DOI：
10.1016/j.ymthe.2005.06.003
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发表时间：
2005-10-01
影响因子：
12.4
通讯作者：
Salvetti, A
中科院分区：
文献类型：
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作者：
Chadeuf, G;Ciron, C;Salvetti, A
重组腺相关病毒载体（rAAV）已成功用于动物模型和患者的长期基因表达。然而，虽然rAAV的治疗潜力似乎是有希望的，但安全性问题，包括载体储备中发现的污染物，必须进一步评估。我们以前报道过，AAV-2 p5启动子内存在的顺式作用复制元件负责rAAV生产过程中观察到的rep-cap序列的重复化。在该研究中，我们还注意到，可以发现质粒衍生的原核序列（如氨苄青霉素抗性基因）被包装到AAV衣壳中。在本报告中，首先，我们通过分析使用不同程序生产的rAAV原液证实并扩展了后一种观察结果。其次，我们证明了这些质粒衍生的序列被转移，并在体内持续rAAV注射到不同的组织后。第三，我们的数据显示，这些包装的质粒分子中至少有一些与AAV ITR连接，并且以可以通过细菌转化拯救的形式存在于体内。这项研究强调了需要更严格的表征rAAV股票，并提供了有用的信息，开发的rAAV生产方法，能够规避或限制这种不需要的颗粒的产生。
Recombinant adeno-associated virus vectors (rAAV) have been successfully used for long-term gene expression in animal models and in patients. However, while the therapeutic potential of rAAV appears promising, safety issues, including contaminants found in vector stocks, must be further evaluated. We previously reported that a cis-acting replication element present within the AAV-2 p5 promoter was responsible for the encapsidation of rep-cap sequences observed during rAAV production. In that study, we also noticed that plasmid-derived prokaryotic sequences (such as the ampicillin resistance gene) could be found packaged into AAV capsids. In this report, first we confirmed and extended the latter observation by analyzing rAAV stocks produced using different procedures. Second, we demonstrated that these plasmid-derived sequences were transferred and persisted in vivo after rAAV injection into different tissues. Third, our data showed that at least some of these packaged plasmid molecules were linked to the AAV ITRs and were present in vivo in a form that could be rescued through bacterial transformation. This study highlights the need for more stringent characterization of rAAV stocks and provides useful information on the development of rAAV production methods that are able to circumvent or limit the generation of such undesirable particles.