LACK OF EXPRESSION FROM A RETROVIRAL VECTOR AFTER TRANSDUCTION OF MURINE HEMATOPOIETIC STEM-CELLS IS ASSOCIATED WITH METHYLATION IN-VIVO
LACK OF EXPRESSION FROM A RETROVIRAL VECTOR AFTER TRANSDUCTION OF MURINE HEMATOPOIETIC STEM-CELLS IS ASSOCIATED WITH METHYLATION IN-VIVO
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DOI：
10.1073/pnas.91.7.2567
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发表时间：
1994-03-29
影响因子：
11.1
通讯作者：
KOHN, DB
中科院分区：
文献类型：
--
作者：
CHALLITA, PM;KOHN, DB
在小鼠基因转移/骨髓移植(BMT)模型中，我们描述了以Moloney小鼠白血病病毒(MoMuLV)为基础的逆转录病毒载体对人葡萄糖脑苷酶基因转移和表达的研究。以多能造血干细胞(HSCs)为集落形成单位，连续移植后生成脾(CFU-S)。MoMuLV长末端重复序列在原发(1度)CFU-S和重组骨髓移植受者的组织中检测到高水平的转录表达。然而，我们观察到90%的继发性(2度)CFU-S和100%的第三级(3)CFU-S的前病毒MoMuLV-LTR转录失活。我们使用甲基化敏感的限制性内切酶Sma I，通过Southern印迹分析比较了载体表达强的1度CFU-S的前病毒和转录活性不活跃的2度和3度CFU-S的前病毒的甲基化状态。研究表明，2度和3度CFU-S的前病毒LTR中Sma I位点的甲基化水平是转录活性的1度CFU-S的3到4倍。这些观察结果可能对未来逆转录病毒介导的基因转移到HSCs的临床应用具有重要意义，在HSCs中，需要持续的基因表达才能达到持久的治疗效果。
We describe studies of gene transfer and expression of the human glucocerebrosidase cDNA by a Moloney murine leukemia virus (MoMuLV)-based retroviral vector in a murine gene transfer/bone marrow transplant (BMT) model. Pluripotent hematopoietic stem cells (HSCs) were assayed as the colony-forming units, spleen (CFU-S) generated after serial transplantation. Transcriptional expression from the MoMuLV long-terminal repeat (LTR) was detected at a high level in the primary (1-degrees) CFU-S and tissues of reconstituted BMT recipients. However, we observed transcriptional inactivity of the proviral MoMuLV-LTR in >90% of the secondary (2-degrees) CFU-S and in 100% of the tertiary (3) CFU-S examined. We have compared the methylation status of the provirus in the 1-degrees CFU-S, which show strong vector expression, to that of the transcriptionally inactive provirus in the 2-degrees and 3-degrees CFU-S by Southern blot analysis using the methylation-sensitive restriction enzyme Sma I. The studies demonstrated a 3- to 4-fold increase in methylation of the Sma I site in the proviral LTR of 2-degrees and 3-degrees CFU-S compared to the transcriptionally active 1-degrees CFU-S. These observations may have important implications for future clinical applications of retroviral-mediated gene transfer into HSCs, where persistent gene expression would be needed for an enduring therapeutic effect.