Woods N B, Fahlman C, Mikkola H, Hamaguchi I, Olsson K, Zufferey R, Jacobsen S E, Trono D, Karlsson S
Department for Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine, Lund University, Lund, Sweden.
Blood. 2000 Dec 1;96(12):3725-33.
The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)
利用一种源自人类免疫缺陷病毒1（HIV-1）的载体研究了慢病毒载体将基因转移至人类造血干细胞的能力，该载体在磷酸甘油酸激酶（PGK）启动子下游表达绿色荧光蛋白（GFP），并以水泡性口炎病毒（VSV）的G蛋白进行假型化。将载体颗粒与人类脐血CD34(+)细胞过夜孵育后实现了高效转导。源自脐血CD34(+)细胞的单个集落形成单位粒细胞-巨噬细胞（CFU-GM）集落中，16%至28%通过聚合酶链反应（PCR）检测GFP基因呈阳性。通过连续移植到非肥胖糖尿病/严重联合免疫缺陷（NOD/SCID）小鼠体内评估脐血CD34(+)群体中重症联合免疫缺陷重建细胞（SRC）的转导效率。当将40万个人类脐血CD34(+)细胞移植到初代受体中时，所有初代和二代受体都含有并表达转基因。通过PCR测定，源自这些初代和二代受体骨髓的CFU-GM集落平均超过50%含有载体。以有限稀释法移植转导细胞产生了可能源自单个SRC的GFP(+)淋巴细胞和髓系祖细胞。采用反向PCR分析扩增源自SRC的集落中的载体-染色体连接片段，并证实载体已整合。这些结果表明慢病毒载体能够高效转导非常原始的人类造血祖细胞和干细胞。（《血液》。2000年；96:3725 - 3733）
