Cheng L, Du C, Lavau C, Chen S, Tong J, Chen B P, Scollay R, Hawley R G, Hill B
SyStemix, Inc, Palo Alto, CA, USA.
Blood. 1998 Jul 1;92(1):83-92.
Inefficient retroviral-mediated gene transfer to human hematopoietic stem cells (HSC) and insufficient gene expression in progeny cells derived from transduced HSC are two major problems associated with HSC-based gene therapy. In this study we evaluated the ability of a murine stem cell virus (MSCV)-based retroviral vector carrying the low-affinity human nerve growth factor receptor (NGFR) gene as reporter to maintain gene expression in transduced human hematopoietic cells. CD34(+) cells lacking lineage differentiation markers (CD34(+)Lin-) isolated from human bone marrow and mobilized peripheral blood were transduced using an optimized clinically applicable protocol. Under the conditions used, greater than 75% of the CD34(+) cell population retained the Lin- phenotype after 4 days in culture and at least 30% of these expressed a high level of NGFR (NGFR+) as assessed by fluorescence-activated cell sorter analysis. When these CD34(+)Lin-NGFR+ cells sorted 2 days posttransduction were assayed in vitro in clonogenic and long-term stromal cultures, sustained reporter expression was observed in differentiated erythroid and myeloid cells derived from transduced progenitors, and in differentiated B-lineage cells after 6 weeks. Moreover, when these transduced CD34(+)Lin-NGFR+ cells were used to repopulate human bone grafts implanted in severe combined immunodeficient mice, MSCV-directed NGFR expression could be detected on 37% +/- 6% (n = 5) of the donor-type human cells recovered 9 weeks postinjection. These findings suggest potential utility of the MSCV retroviral vector in the development of effective therapies involving gene-modified HSC.
逆转录病毒介导的基因向人类造血干细胞（HSC）的低效转移以及转导的HSC衍生的子代细胞中基因表达不足是与基于HSC的基因治疗相关的两个主要问题。在本研究中，我们评估了携带低亲和力人类神经生长因子受体（NGFR）基因作为报告基因的基于小鼠干细胞病毒（MSCV）的逆转录病毒载体在转导的人类造血细胞中维持基因表达的能力。使用优化的临床适用方案转导从人骨髓和动员的外周血中分离出的缺乏谱系分化标志物的CD34(+)细胞（CD34(+)Lin-）。在所使用的条件下，培养4天后，超过75%的CD34(+)细胞群体保留了Lin-表型，并且通过荧光激活细胞分选分析评估，这些细胞中至少30%表达高水平的NGFR（NGFR+）。当在转导后2天分选的这些CD34(+)Lin-NGFR+细胞在体外进行克隆形成和长期基质培养时，在源自转导祖细胞的分化红细胞和髓细胞以及6周后的分化B谱系细胞中观察到持续报告基因表达。此外，当这些转导的CD34(+)Lin-NGFR+细胞用于重新填充植入严重联合免疫缺陷小鼠的人骨移植物时，在注射后9周回收的供体类型人类细胞的37%±6%（n = 5）上可以检测到MSCV指导的NGFR表达。这些发现表明MSCV逆转录病毒载体在涉及基因修饰HSC的有效治疗方法开发中的潜在用途。
