Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration
Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration
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DOI：
10.1016/j.ymthe.2004.03.008
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发表时间：
2004-06-01
影响因子：
12.4
通讯作者：
Kiem, HP
中科院分区：
文献类型：
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作者：
Kurre, P;Anandakumar, P;Kiem, HP
HIV-1来源的慢病毒载体为将基因输送到造血干细胞提供了独特的生物学特性，并且在感染的高度多样性(m.o.i)下使用时，允许在最小的靶细胞刺激后进行有效的基因转移。然而，这种策略已经被证明可以促进多拷贝前病毒整合，潜在地增加插入突变的风险。为了最大限度地减少细胞操作，我们以未分离的骨髓为靶点，并在M.O.i上演示了这种转导。的结果是载体修饰的外周血白细胞高达12%，并成功地用载体标记的细胞重新繁殖二次受体。实时定量聚合酶链式反应显示平均每个GFP标记的细胞有1.8个前病毒整合子。相比之下，一组动物被移植了在m.o.i转导的细胞。在其他条件不变的情况下，10个细胞中有45%出现标记，平均每个上调表达细胞有7个拷贝。两人都是犯罪嫌疑人。GFP荧光强度稳定的组表现出持续的前病毒表达。综上所述，我们已经确定了慢病毒基因转移的条件，包括最小的体外靶细胞操作，并表明m.o.i。是慢病毒转导的小鼠长期再生细胞中前病毒拷贝数的关键决定因素。因此，当需要单拷贝整合时，基因转移效率可能会受到限制，并且可能需要其他策略，如体内选择，以提高基因修饰细胞的频率。
HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of I resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per UP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.