Bokhoven Marieke, Stephen Sam L, Knight Sean, Gevers Evelien F, Robinson Iain C, Takeuchi Yasuhiro, Collins Mary K
Infection and Immunity, University College London, Windeyer Building, 46 Cleveland Street, London W1T 2AH, United Kingdom.
J Virol. 2009 Jan;83(1):283-94. doi: 10.1128/JVI.01865-08. Epub 2008 Oct 22.
Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).
γ逆转录病毒载体和慢病毒载体是基因治疗中很有前景的工具，但它们可能具有致癌性。更安全载体的开发依赖于插入诱变的定量测定方法。在此，我们报告一种快速、廉价且可重复的测定方法，该方法使用小鼠细胞系来测量白细胞介素-3（IL-3）非依赖性突变体的频率。慢病毒载体和γ逆转录病毒载体导致插入诱变的频率相似；然而，它们使用不同的机制。基于人类免疫缺陷病毒（HIV）的载体仅通过插入生长激素受体（Ghr）基因座产生突变体。在该基因座中，HIV增强子/启动子在没有HIV Tat蛋白的情况下具有活性，并且HIV/Ghr剪接转录本表达GHR，细胞对生长激素（GH）产生反应。在基于自我失活HIV的载体中删除增强子/启动子可防止这种插入诱变机制。相比之下，γ逆转录病毒载体插入到其他基因座，包括IL-3以及在逆转录病毒标记癌症基因数据库（RTCGD）中被鉴定为常见插入位点的基因。
