A functional screen for regulatory elements that improve retroviral vector gene expression

Abstract

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.