Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34+ hematopoietic cells
Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34+ hematopoietic cells
复制标题
DOI：
10.1634/stemcells.19-3-236
复制
发表时间：
2001-01-01
期刊：
影响因子：
5.2
通讯作者：
Gewirtz, AM
中科院分区：
文献类型：
--
作者：
Choi, JK;Hoang, N;Gewirtz, AM
基于HIV的慢病毒载体可以抑制非分裂细胞，这在转导缓慢分裂的造血干细胞时是基于鼠白血病病毒（MLV）的载体的重要优势。然而，我们发现在人CD 34（+）造血细胞中，具有内部巨细胞病毒（CMV）启动子的基于HIV的载体表达的转基因比基于MLV的逆转录病毒载体鼠干细胞病毒（MSCV）少100至1，000倍。为了增加整合的慢病毒的表达，我们用劳斯肉瘤病毒或MSCV的启动子替换CMV启动子，并获得表达的适度增强。当去除CMV增强子/启动子并将HIV长末端重复序列（LTR）替换为新型HIV/MSCV杂合LTR时，观察到更显著的效果。该载体保留了使非分裂细胞增殖的能力，但现在表达的转基因（增强的绿色荧光蛋白）比原始基于HIV的载体高10至100倍。当在相同条件下比较时，具有杂合I，TR的HIV载体比基于MSCV的逆转录病毒载体转导更高百分比的CD34（+）细胞（19.4%对2.4%）。转导细胞的数量和转基因表达水平在5 - 8周内保持恒定，如通过长期培养起始细胞、荧光激活细胞分选和非肥胖糖尿病/严重联合免疫缺陷再增殖测定所确定的。
HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34(+) hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV), To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR, This vector retains the ability to transduce nondividing cells hut now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid I,TR transduced a higher percentage of CD34(+) cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by longterm culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.