Plavec I, Voytovich A, Moss K, Webster D, Hanley M B, Escaich S, Ho K E, Böhnlein E, DiGiusto D L
SyStemix, Palo Alto, CA 94304, USA.
Gene Ther. 1996 Aug;3(8):717-24.
The expression of antiviral genes in human hematopoietic stem or progenitor cells has been proposed as a strategy for gene therapy of AIDS. To be successful, this strategy requires safe and efficient transfer of the therapeutic gene into hematopoietic cells and gene expression has to be maintained in HIV susceptible cells following differentiation. We have used retroviral vectors to transfer the gene for a transdominant inhibitor of HIV replication (RevM10) into CD34+ stem/progenitor cells isolated from human umbilical cord blood (UCB). Following transduction, cells were allowed to differentiate either in vitro in clonogenic assays and long-term stromal cell cultures or in human thymus implanted in immunodeficient scid/scid mice in vivo (SCID-hu). Following differentiation and expansion, multiple lineages of cells were shown to carry the transgene. A higher percentage of gene-marked progenitor cells (10-30% in most cases) were detected in methylcellulose colony assays and in long-term stromal cell cultures (1-5%). In contrast, gene-marked T cells derived from transduced CD34+ cells in a SCID-hu model were detected at an even lower frequency (0.01-1%). RevM10 RNA expression was detected in CD34+ cells immediately after transduction and was maintained after in vitro differentiation of those cells into CD14+ myeloid cells. In T cells, the RevM10-specific RNA was detectable by RT-PCR and also by semiquantitative RNase protection. These findings demonstrate that LTR-driven gene expression is sustained in relevant cells derived from retrovirus-transduced hematopoietic progenitor cells after extensive differentiation in vitro and in vivo and suggest that stringent in vivo, rather than in vitro assays, may be a better preclinical system to improve gene marking and expression in hematopoietic cells.
将抗病毒基因导入人类造血干细胞或祖细胞已被提议作为艾滋病基因治疗的一种策略。要成功实施该策略，需要将治疗性基因安全、高效地导入造血细胞，并且在分化后的HIV易感细胞中维持基因表达。我们使用逆转录病毒载体将HIV复制的反式显性抑制剂（RevM10）基因导入从人脐带血（UCB）中分离出的CD34+ 干细胞/祖细胞。转导后，细胞在体外进行克隆形成试验和长期基质细胞培养，或在体内植入免疫缺陷的scid/scid小鼠的人胸腺（SCID-hu）中进行分化。分化和扩增后，多个细胞谱系显示携带转基因。在甲基纤维素集落试验和长期基质细胞培养中检测到更高比例的基因标记祖细胞（大多数情况下为10%-30%）（1%-5%）。相比之下，在SCID-hu模型中，源自转导CD34+ 细胞的基因标记T细胞的检测频率更低（0.01%-1%）。转导后立即在CD34+ 细胞中检测到RevM10 RNA表达，并且在这些细胞体外分化为CD14+ 髓样细胞后仍得以维持。在T细胞中，通过RT-PCR以及半定量核糖核酸酶保护法均可检测到RevM10特异性RNA。这些发现表明，在体外和体内广泛分化后，LTR驱动的基因表达在源自逆转录病毒转导的造血祖细胞的相关细胞中得以持续，这表明严格的体内而非体外试验可能是改善造血细胞基因标记和表达的更好的临床前系统。
