Wang Gary P, Levine Bruce L, Binder Gwendolyn K, Berry Charles C, Malani Nirav, McGarrity Gary, Tebas Pablo, June Carl H, Bushman Frederic D
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
Mol Ther. 2009 May;17(5):844-50. doi: 10.1038/mt.2009.16. Epub 2009 Mar 3.
Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.
基于慢病毒载体的基因疗法已被用于使用反义env载荷靶向人类免疫缺陷病毒（HIV）。我们分析了三名接受治疗个体的慢病毒载体整合位点。我们将体外载体转导的CD4+细胞产物中的整合位点与输注后多次回收的细胞中的位点进行了比较。使用454焦磷酸测序分析整合位点，从体外产物中获得了总共7782个独特的整合位点，从输注后回收的细胞中获得了237个独特的位点。发现两个数据集中的整合载体拷贝在活性基因内以及与活性转录单元相关的表观遗传标记附近强烈富集。相对于靶DNA上的核小体结构分析整合表明，倾向于整合在核小体表面向外的DNA大沟中。没有迹象表明输注后转导细胞的生长导致原癌基因5'端附近或肿瘤抑制基因内的整合位点富集。因此，在输注基因修饰的CD4+细胞后首次观察慢病毒载体转导细胞的纵向演变，没有提供证据表明由于载体介导的原癌基因插入激活而导致细胞异常扩增。
