Development of a transposon-based approach for identifying novel transgene insertion sites within the replicating adenovirus
Development of a transposon-based approach for identifying novel transgene insertion sites within the replicating adenovirus
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DOI：
10.1016/j.ymthe.2005.03.019
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发表时间：
2005-07-01
影响因子：
12.4
通讯作者：
Hermiston, TW
中科院分区：
文献类型：
--
作者：
Kretschmer, PJ;Jin, F;Hermiston, TW
从溶瘤腺病毒(Ad)进行治疗性基因传递是提高基于Ad的病毒治疗癌症的效力的一种方法。为了确定与复制病毒相容的治疗性转基因插入部位，需要一种广泛扫描病毒基因组的方法。为了解决这个问题，我们修改了一种基于转座子(Tn7)的体外转座系统，以利用其无偏见的扫描能力来识别与病毒复制兼容的插入位点。使用这个系统和一个含有E3缺失的ADS的质粒，我们确定了在Ad基因组中基于启动子的表达盒插入的几个独特的位置。基于转座子的表达框由转座子所特有的PMEL限制性内切酶结合，使得表达框替换容易进行。另外两个含有不同启动子和报告基因的表达盒被替换到两个新鉴定的转基因插入位点。结果表明，表达盒替换的易感性和方向性既取决于插入位置，也取决于替换表达盒的启动子和基因。这些研究建立了基于转座子的系统，作为扫描Ad基因组和识别与病毒复制兼容的插入位点的有效方法，并代表了开发武装的癌症治疗病毒的强大工具。
Therapeutic gene delivery from an oncolytic adenovirus (Ad) is one approach to enhancing the potency of Ad-based virotherapies for cancer. To identify therapeutic transgene insertion sites compatible with the replicating virus, a methodology that broadly scans the viral genome is needed. To address this we modified a transposon (Tn7)-based in vitro transposition system to take advantage of its nonprejudiced scanning ability to identify insertion sites compatible with viral replication. Using this system with a plasmid containing an E3-deleted Ads, we identified several unique sites for promoter-based expression cassette insertions within the Ad genome. The transposon-based expression cassette is bounded by Pmel restriction endonuclease sites unique to the transposon, making expression cassette substitutions easy to perform. Additional expression cassettes containing different promoters and reporter genes were substituted into two of the newly identified transgene insertion sites. The results suggest that the ease and orientation of expression cassette substitution depend on both the insertion site location and the promoter and gene of the replacement expression cassette. These studies establish the transposon-based system as an efficient approach to scanning the Ad genome and identifying insertion sites compatible with viral replication and represents a powerful tool for the development of armed therapeutic viruses for cancer.