A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.
A stable murine-based RD114 retroviral packaging line efficiently transduces human hematopoietic cells.
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基于鼠的稳定 RD114 逆转录病毒包装线可有效转导人类造血细胞。
DOI：
10.1016/j.ymthe.2003.07.002
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Bank,Arthur
中科院分区：
文献类型：
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作者：
Ward,Maureen;Sattler,Rose;Grossman,IRobert;BellJr,AnthonyJ;Skerrett,Donna;Baxi,Laxmi;Bank,Arthur
使用复杂的肿瘤逆转录病毒载体将治疗性基因高效转移到人类造血干细胞(HSCs)存在几个障碍。到目前为止，人类临床试验已经在稳定的逆转录病毒包装线中使用了基于Moloney白血病病毒的两性和基于长臂猿白血病病毒的信封。然而，用这些包膜假型的逆转录病毒滴度很低，这是因为无法通过离心法有效地浓缩病毒上清液而不破坏病毒，而且由于病毒靶向受体在人HSC上的低水平表达，转导效率低。来自猫内源性病毒的RD114外膜已被证明使用瞬时包装系统转导人类CD34+细胞，并通过离心法浓缩到高滴度。稳定的包装系统比瞬时系统具有潜在的优势，因为可以获得更多和更具重复性的病毒生产。因此，我们构建并测试了一株稳定表达RD114的包装细胞系，能够高水平转导人CD34+细胞。通过超速离心法，来自该细胞系的病毒颗粒被浓缩到100倍(高达107个病毒颗粒/毫升)。脐带血和镰状细胞CD34+细胞的造血祖细胞在一次暴露于该细胞株产生的浓缩RD114假型病毒后，可被含有NeoR的载体有效地转导。高达78%的转导脐血CD34+细胞和51%的镰状细胞CD34+细胞表达NeoR基因。我们还展示了利用这种新的稳定包装系统将人β-珠蛋白基因从镰状细胞患者的CD34+细胞转移到祖细胞中。结果表明，该包装线最终可能在珠蛋白基因治疗的人体临床试验中有用。
Several barriers exist to high-efficiency transfer of therapeutic genes into human hematopoietic stem cells (HSCs) using complex oncoretroviral vectors. Human clinical trials to date have used Moloney leukemia virus-based amphotropic and gibbon ape leukemia virus-based envelopes in stable retroviral packaging lines. However, retroviruses pseudotyped with these envelopes have low titers due to the inability to concentrate viral supernatants efficiently by centrifugation without damaging the virus and low transduction efficiencies because of low-level expression of viral target receptors on human HSC. The RD114 envelope from the feline endogenous virus has been shown to transduce human CD34+cells using transient packaging systems and to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient systems because greater and more reproducible viral productions can be attained. We have, therefore, constructed and tested a stable RD114-expressing packaging line capable of high-level transduction of human CD34+cells. Viral particles from this cell line were concentrated up to 100-fold (up to107viral particles/ml) by ultracentrifugation. Human hematopoietic progenitors from cord blood and sickle cell CD34+cells were efficiently transduced with a NeoR-containing vector after a single exposure to concentrated RD114-pseudotyped virus produced from this cell line. Up to 78% of progenitors from transduced cord blood CD34+cells and 51% of progenitors from sickle cell CD34+cells expressed the NeoRgene. We also show transfer of a human β-globin gene into progenitor cells from CD34+cells from sickle cell patients with this new RD114 stable packaging system. The results indicate that this packaging line may eventually be useful in human clinical trials of globin gene therapy.