Reiser J, Harmison G, Kluepfel-Stahl S, Brady R O, Karlsson S, Schubert M
Molecular and Medical Genetics Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15266-71. doi: 10.1073/pnas.93.26.15266.
The use of Moloney murine leukemia virus (Mo-MLV)-based vectors to deliver therapeutic genes into target cells is limited by their inability to transduce nondividing cells. To test the capacity of HIV-based vectors to deliver genes into nondividing cells, we have generated replication-defective HIV type 1 (HIV-1) reporter vectors carrying neomycin phosphotransferase or mouse heat stable antigen, replacing the HIV-1 sequences encoding gp160. These vectors also harbor inactive vpr, vpu, and nef coding regions. Pseudotyped HIV-1 particles carrying either the ecotropic or the amphotropic Mo-MLV envelope proteins or the vesicular stomatitis virus G protein were released after single or double transfections of either human 293T or monkey COS-7 cells with titers of up to 10(7) colony-forming units per milliliter. A simple ultrafiltration procedure resulted in an additional 10- to 20-fold concentration of the pseudotyped particles. These vectors along with Mo-MLV-based vectors were used to transduce primary human skin fibroblasts and human peripheral blood CD34+ cells. The HIV-1 vector system was significantly more efficient than its Mo-MLV-based counterpart in transducing human skin fibroblasts arrested at the G0/G1 stage of the cell cycle by density-dependent inhibition of growth. Human CD34+ cells were transduced efficiently using HIV-1 pseudotype particles without prior stimulation with cytokines.
基于莫洛尼鼠白血病病毒（Mo-MLV）的载体向靶细胞递送治疗性基因的应用受到其无法转导非分裂细胞的限制。为了测试基于HIV的载体向非分裂细胞递送基因的能力，我们构建了携带新霉素磷酸转移酶或小鼠热稳定抗原的复制缺陷型1型HIV（HIV-1）报告载体，取代了编码gp160的HIV-1序列。这些载体还含有无活性的vpr、vpu和nef编码区。用人293T细胞或猴COS-7细胞进行单次或双次转染后，释放出携带亲嗜性或双嗜性Mo-MLV包膜蛋白或水泡性口炎病毒G蛋白的假型HIV-1颗粒，滴度高达每毫升10^7个集落形成单位。一个简单的超滤程序使假型颗粒的浓度额外增加了10到20倍。这些载体与基于Mo-MLV的载体一起用于转导原代人皮肤成纤维细胞和人外周血CD34+细胞。在通过密度依赖性生长抑制使细胞周期停滞在G0/G1期的人皮肤成纤维细胞中，HIV-1载体系统在转导方面明显比其基于Mo-MLV的对应物更有效。使用HIV-1假型颗粒无需事先用细胞因子刺激就能有效地转导人CD34+细胞。
