Gardner J P, Zhu H, Colosi P C, Kurtzman G J, Scadden D T
Division of Experimental Hematology, AIDS Research Center, MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA.
Blood. 1997 Dec 15;90(12):4854-64.
Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2- progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited.
重组腺相关病毒（rAAV）已被提议作为造血干细胞的基因转移载体，由于其高滴度以及在靶细胞整合方面相对不依赖细胞周期，因而比其他基于病毒的系统具有优势。我们使用体外T淋巴细胞生成系统，在诱导分化为T细胞的原代人CD34⁺CD2⁻祖细胞的背景下，评估了一种含有在巨细胞病毒（CMV）启动子控制下的LacZ报告基因的rAAV载体。来自成人骨髓或脐带血的靶细胞被高效转导，在暴露于rAAV-CMV-LacZ后，71%至79%的CD2⁺细胞表达LacZ标记基因mRNA并产生LacZ编码蛋白。通过对病毒暴露细胞免疫表型亚群的连续定量分析，评估了转基因表达对T细胞分化的影响，与对照相比未发现改变。评估了转基因表达的持久性，发现到第35天时其衰减，动力学取决于感染复数。此外，通过DNA聚合酶链反应，在CD4或CD8亚选的CD3⁺细胞中未检测到载体DNA。这些数据表明，rAAV载体可能在经历T细胞谱系定向分化的原始细胞中导致强大的转基因表达，而不会产生毒性或改变T细胞分化模式。然而，表达是短暂的，转基因不太可能整合。重组AAV载体对于T细胞个体发生过程中事件的基因操作可能是有价值的基因转移工具，但它们在获得性免疫缺陷综合征等疾病的基因治疗策略中的潜力有限。
