Douglas J L, Lin W Y, Panis M L, Veres G
SyStemix, Palo Alto, CA 94304, USA.
Hum Gene Ther. 2001 Mar 1;12(4):401-13. doi: 10.1089/10430340150504028.
The methods available to efficiently transduce human CD34(+) hematopoietic stem cells (HSCs) derived from mobilized peripheral blood, such that they fully retain their engraftment potential and maintain high levels of transgene expression in vivo, have been unsatisfactory. The current murine retrovirus-based gene transfer systems require dividing cells for efficient transduction, and therefore the target HSCs must be activated ex vivo by cytokines to cycle, which may limit their engrafting ability. Lentivirus-based gene transfer systems do not require cell division and, thus, may allow for efficient gene transfer to human HSCs in the absence of any ex vivo cytokine stimulation. We constructed human immunodeficiency virus (HIV)-based vectors and compared them in vitro and in vivo with MuLV-based vectors in their ability to transduce unstimulated human CD34(+) HSCs isolated from mobilized peripheral blood. Both sets of vectors contained the marker gene that expresses the enhanced green fluorescent protein (EGFP) for evaluating transduction efficiency and were pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or the amphotropic murine leukemia virus envelope (A-MULV Env). The VSV-G-pseudotyped HIV-based vectors containing an internal mouse phosphoglycerate kinase promoter (PGK) were able to transduce up to 48% of the unstimulated CD34(+) cells as measured by EGFP expression. When these cells were injected into the human fetal thymus implants of irradiated SCID-hu Thy/Liv mice, up to 18% expressed EGFP after 8 weeks in vivo. In contrast, the MULV-based vectors were effective at transducing HSCs only in the presence of cytokines. Our results demonstrate that the improved HIV-based gene transfer system can effectively transduce unstimulated human CD34(+) HSCs, which can then differentiate into thymocytes and provide long-term transgene expression in vivo.
目前，有效转导源自动员外周血的人类CD34(+)造血干细胞（HSC）的方法并不理想，这些方法要能使细胞充分保留其植入潜力并在体内维持高水平的转基因表达。当前基于鼠逆转录病毒的基因转移系统需要分裂细胞才能进行有效转导，因此目标HSC必须在体外通过细胞因子激活才能进入细胞周期，这可能会限制它们的植入能力。基于慢病毒的基因转移系统不需要细胞分裂，因此在没有任何体外细胞因子刺激的情况下，可能允许将基因有效转移到人类HSC。我们构建了基于人类免疫缺陷病毒（HIV）的载体，并在体外和体内将它们与基于MuLV的载体转导从未刺激的动员外周血中分离出的人类CD34(+) HSC的能力进行了比较。两组载体都包含表达增强型绿色荧光蛋白（EGFP）的标记基因，用于评估转导效率，并用水泡性口炎病毒糖蛋白（VSV-G）或嗜异性鼠白血病病毒包膜（A-MULV Env）进行假型化。含有内部小鼠磷酸甘油酸激酶启动子（PGK）的VSV-G假型化HIV载体能够转导高达48%的未刺激CD34(+)细胞（通过EGFP表达测量）。当将这些细胞注入经辐照的SCID-hu Thy/Liv小鼠的人类胎儿胸腺植入物中时，在体内8周后高达18%的细胞表达EGFP。相比之下，基于MULV的载体仅在细胞因子存在的情况下才能有效转导HSC。我们的结果表明，改进的基于HIV的基因转移系统可以有效转导未刺激的人类CD34(+) HSC，这些细胞随后可以分化为胸腺细胞并在体内提供长期的转基因表达。
