Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells.
Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells.
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发表时间：
1994
期刊：
影响因子：
20.3
通讯作者：
S. Goodman;X. Xiao;R. Donahue;A. Moulton;J. Miller;C. Walsh;N. Young;R. Samulski;A. Nienhuis
中科院分区：
文献类型：
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作者：
S. Goodman;X. Xiao;R. Donahue;A. Moulton;J. Miller;C. Walsh;N. Young;R. Samulski;A. Nienhuis
仅含野生型病毒反向末端重复序列(ITR)的重组腺相关病毒(RAAV)能够稳定整合到宿主细胞基因组中，并在培养细胞中表达插入的基因。我们现在已经定义了rAAV将基因导入初级造血祖细胞的能力。在强病毒启动子的控制下，构建了含有β-半乳糖苷酶(β-GAL)编码序列(包括核定位信号)的载体。通过将载体质粒与第二个含有AAV蛋白编码序列的质粒共转染到腺病毒感染的人胚胎肾细胞中，制备了具有感染性的载体颗粒。这些载体在人K562红白血病细胞和底特律6细胞中转移和表达了β-半乳糖基因。阳性免疫选择产生了一组富含CD34+细胞的细胞，这些细胞与rAAVβ-Gal载体转导。在60%到70%的感染细胞中有核定位酶的表达。用半定量聚合酶链式反应(PCR)方法显示，在克隆培养2周后形成的祖细胞来源的克隆具有病毒相关DNA，每个细胞的拷贝数估计为1-2。利用野生型病毒将AAV整合到造血祖细胞中，因为其基因组可能整合在19号染色体上的首选位置。我们的数据表明，rAAV将在原始造血祖细胞中高频率地转移和表达基因，并支持这种治疗性基因转移载体系统的发展。
Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta-galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral-associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.