Gervaix A, Schwarz L, Law P, Ho A D, Looney D, Lane T, Wong-Staal F
Department of Medicine, University of California at San Diego, La Jolla 92093-0665, USA.
Hum Gene Ther. 1997 Dec 10;8(18):2229-38. doi: 10.1089/hum.1997.8.18-2229.
Gene therapy is a promising treatment modality for acquired immunodeficiency syndrome (AIDS). Autologous transplantation with genetically altered pluripotent hematopoietic stem cells encoding anti-human immunodeficiency virus (HIV) genes could in theory completely and permanently reconstitute all blood lineages and immune functions with cells resistant to HIV. Recent studies showed that CD34+ stem cell can be mobilized in HIV-infected individuals after granulocyte colony-stimulating factor (G-CSF) administration without major side effects or increase of viral load. In this study, peripheral blood CD34+ cells of five HIV-infected individuals were mobilized with G-CSF and after leukapheresis and enrichment, subjected to retroviral transduction with genes encoding anti-HIV ribozyme-decoy fusion molecules. These cells were tested for the ability to give rise to progeny cells, for retroviral transduction efficiency, and for expression of the transgene. CD34+-derived macrophage-like cells were also challenged with HIV. Results showed that CD34+ cells from HIV-infected individuals gave rise to similar numbers of progeny colonies as cells from healthy donors. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. CD34+-derived macrophages expressed anti-HIV genes and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. Furthermore, we showed that after thawing, cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. Thus, CD34+ cells from HIV-infected patients can be stored for further genetic manipulations with improved vectors or anti-HIV genes as they become available.
基因治疗是获得性免疫缺陷综合征（艾滋病）一种很有前景的治疗方式。用编码抗人类免疫缺陷病毒（HIV）基因的基因改造多能造血干细胞进行自体移植，理论上可以用对HIV有抗性的细胞完全且永久地重建所有血细胞谱系和免疫功能。最近的研究表明，在给予粒细胞集落刺激因子（G-CSF）后，HIV感染个体中的CD34+干细胞可以被动员起来，且无重大副作用或病毒载量增加。在本研究中，5名HIV感染个体的外周血CD34+细胞用G-CSF进行动员，在白细胞分离和富集后，用编码抗HIV核酶-诱饵融合分子的基因进行逆转录病毒转导。检测这些细胞产生子代细胞的能力、逆转录病毒转导效率和转基因表达。CD34+来源的巨噬样细胞也用HIV进行攻击。结果显示，HIV感染个体的CD34+细胞产生的子代集落数量与健康供体的细胞相似。通过对单个集落中转基因进行DNA聚合酶链反应（PCR）评估，这些细胞的转导效率在68.8%至100%之间。与未转导的细胞相比，CD34+来源的巨噬细胞表达抗HIV基因，并对HIV复制表现出显著且持续的抑制作用。此外，我们还表明，解冻后，这些个体的冷冻保存CD34+细胞的存活、增殖和转导参数与新鲜细胞相当。因此，HIV感染患者的CD34+细胞可以储存起来，以便在有更好的载体或抗HIV基因时进行进一步的基因操作。
