High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors
High-efficiency gene transfer into ex vivo expanded human hematopoietic progenitors and precursor cells by adenovirus vectors
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DOI：
10.1182/blood.v91.8.2781.2781_2781_2792
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发表时间：
1998-04-15
期刊：
影响因子：
20.3
通讯作者：
Rafii, S
中科院分区：
文献类型：
--
作者：
Frey, BM;Hackett, NR;Rafii, S
复制缺陷型腺病毒载体（AdVec）能够高效、低毒且易于递送地感染循环和非循环细胞，为研究控制造血不同阶段的调控基因的表达提供了理想的载体。为了检查 AdVec 在造血前体细胞和祖细胞中的感染效率，我们使用了表达绿色荧光蛋白 (AdGFP) cDNA 人源化形式的复制缺陷腺载体，从而可以使用流式细胞术和荧光显微镜评估活造血细胞中转基因表达的感染效率和动力学。对感染复数 (MOI) 为 100 AdGFP 的离体扩增造血前体细胞进行流式细胞术分析，结果显示 78% 的巨核细胞（CD41a(+) 和 CD42b(+)）细胞、82% 的树突状细胞（CD1a(+)）、41% 的红细胞前体细胞（血型糖蛋白 A(+)）、 32%的单核细胞（CD14(+)）表达GFP。在相同条件下感染的外周血白细胞去除术产品中新鲜分离的 CD34(+) 细胞中有 19%+/-1% 表达 GFP。体外扩增的AdGFP感染的CD34(+)细胞的形态学评估显示正常成熟，通过量化克隆形成效率和增殖能力来分析AdGFP感染的CD34(+)细胞的功能能力，以1至100的MOls感染CD34(+)祖细胞不会损害CD34(+)细胞的克隆形成效率。然而，MOI 大于 100 会导致粒细胞/粒细胞巨噬细胞集落形成单位 (CFU-G/GM) 的形成受到显着抑制。在 3 周的连续稀释扩增中（Delta 测定），暴露于 MOls 为 1 至 1,000 的 AdGFP 后，细胞因子驱动的 CD34(+) 细胞的增殖潜力并未受到损害。 GFP(+)群体在高MOls（500至1,000）下扩增10至15倍，表明最初感染的CD34(+)细胞中存在转基因的多个拷贝，并在随后的后代中表达。这些数据表明，AdVec能够高效、低毒地向造血祖细胞和前体细胞传递转基因，通过AdVec将GFP等标记基因引入造血细胞将为研究造血前体细胞和祖细胞的体外和体内发育、归巢和运输提供有价值的系统。此外，这些结果为 AdVec 治疗血液疾病的基因治疗策略的设计提供了见解。 (C) 1998 年，美国血液学会。
Replication-deficient adenoviral vectors (AdVec), which infect cycling and noncycling cells with high efficiency, low toxicity, and ease of delivery, provide ideal vehicles to study the expression of regulatory genes controlling different stages of hematopoiesis. To examine the infection efficiency of AdVec in hematopoietic precursor and progenitor cells, we used a replication-deficient adenovector expressing the humanized form of the cDNA for green fluorescent protein (AdGFP), permitting assessment of infection efficiency and kinetics of transgene expression in viable hematopoietic cells using flow cytometry and fluorescence microscopy. Flow-cytometric analysis of ex vivo expanded hematopoietic precursor cells infected with a multiplicity of infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic (CD41a(+) and CD42b(+)) cells, 82% of dendritic (CD1a(+)) cells, 41% of RBC precursors (glycophorin A(+)), and 32% of monocytic (CD14(+)) cells expressed GFP. Nineteen percent +/- 1% of freshly isolated CD34(+) cells from peripheral blood leukapheresis products infected under the same conditions expressed GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected CD34(+) cells showed normal maturation, The functional capacity of AdGFP-infected CD34(+) cells was analyzed by quantifying clonogeneic efficiency and proliferative capacity, Infection of CD34(+) progenitor cells with MOls of 1 to 100 did not impair clonogeneic efficiency of CD34(+) cells. However, MOI greater than 100 resulted in a significant inhibition of colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) formation, In sequential dilution expansion over 3 weeks (Delta assay), the cytokine-driven proliferative potential of CD34(+) cells was not impaired following exposure to AdGFP at MOls of 1 to 1,000, The GFP(+) population expanded 10- to 15-fold at high MOls (500 to 1,000), indicating multiple copies of the transgene in the initially infected CD34(+) cells, which were expressed in subsequent progenies. These data show that AdVec deliver transgenes with high efficiency and low toxicity to hematopoietic progenitor and precursor cells, Introduction of marker genes such as GFP into hematopoietic cells by AdVec will provide a valuable system for study of development, homing, and trafficking of hematopoietic precursor and progenitor cells in vitro and in vivo. Furthermore, these results provide insights into the design of gene therapy strategies for treatment of hematologic disorders by AdVec. (C) 1998 by The American Society of Hematology.