Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, WA, USA.
Blood Cells Mol Dis. 2010 Dec 15;45(4):343-50. doi: 10.1016/j.bcmd.2010.08.005. Epub 2010 Sep 16.
Recombinant retroviruses constitute the most common class of gene delivery vectors used in hematopoietic cell-based gene therapy. However, the use of these vectors can be limited by inadequate levels of transgene expression, often mediated by expression variegation and vector silencing due to chromosomal position effects. Toward the goal of addressing this problem, we sought to identify cis-regulatory elements from the human genome that can improve the level and stability of retroviral vector gene expression. Libraries of size-selected fragments from the human genome were cloned into the "double-copy" position of the gammaretroviral reporter vector MGPN2, and the resulting vectors underwent several rounds of transduction and selection for high-level vector GFP expression. From this screen we identified both enhancer-like elements and vector mutations associated with increased vector expression. One element, H-11, exhibited enhancer activity in a mouse bone marrow progenitor colony assay, a human promoter trap assay, and a long-term mouse bone marrow transplant assay. This element seems to be an orientation-dependent, tissue-independent enhancer.
重组逆转录病毒构成了用于基于造血细胞的基因治疗的最常见的基因传递载体类别。然而，由于染色体位置效应引起的转基因表达的不均一性和载体沉默，这些载体的使用可能会受到限制。为了实现这一目标，我们试图从人类基因组中寻找顺式调控元件，以提高逆转录病毒载体基因表达的水平和稳定性。从人类基因组中大小选择片段的文库被克隆到γ逆转录病毒报告载体 MGPN2 的“双拷贝”位置，并且所得载体经历了几轮转导和选择，以获得高水平的载体 GFP 表达。从该筛选中，我们鉴定了与增加载体表达相关的增强子样元件和载体突变。一个元件 H-11 在小鼠骨髓祖细胞集落测定、人启动子陷阱测定和长期小鼠骨髓移植测定中表现出增强子活性。该元件似乎是一种依赖于取向的、组织独立的增强子。
