Prospects and implications of using chromatin insulators in gene therapy and transgenesis

Abstract

Gene therapy has emerged from the idea of inserting a wild‐type copy of a gene in order to restore the proper expression and function of a damaged gene. Initial efforts have focused on finding the proper vector and delivery method to introduce a corrected gene to the affected tissue or cell type. Even though these first attempts are clearly promising, seveal problems remain unsolved. A major problem is the influence of chromatin structure on transgene expression. To overcome chromatin‐dependent repressive transgenic states, researchers have begun to use chromatin regulatory elements to drive transgene expression. Insulators or chromatin boundaries are able to protect a transgene against chromatin position effects at their genomic integration sites, and they are able to maintain transgene expression for long periods of time. Therefore, these elements may be very useful tools in gene therapy applications for ensuring high‐level and stable expression of transgenes. BioEssays 26:796–807, 2004. © 2004 Wiley Periodicals, Inc.

Protection against telomeric position effects by the chicken cHS4 β-globin insulator

“…Recent evidence supports an active role of insulators in the optimal topology conformation of chromosomal domains in the nucleus (10)(11)(12). In particular, the chicken cHS4 ␤-globin insulator can protect a transgene against CPE via the recruitment of histone acetyltransferases and methyltransferases by USF1 (5,13). This process further supports the idea that the cHS4 insulator is capable of blocking heterochromatin propagation.…”

“…Position effect variegation is defined as the variegated pattern of expression from cell to cell when a gene is translocated into the proximity of dominant heterochromatin (3,4), whereas CPE are alterations of transgene expression associated with a distinct insertion into the epigenome milieu (5). In both cases, transgene expression is affected by changes in chromatin conformation, such as those associated with histone deacetylation, H3K9me3, and DNA methylation, that can extend over considerable genomic distances (3,4).…”

“…In both cases, transgene expression is affected by changes in chromatin conformation, such as those associated with histone deacetylation, H3K9me3, and DNA methylation, that can extend over considerable genomic distances (3,4). Many studies have focused on trying to understand such phenomena, but little has been done to study the capacity of vertebrate chromatin insulators to protect against TPE (5)(6)(7)(8)(9)(10).…”

Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

“…CTRL1 consists of 46 copies of two separate, but overlapping, CTCF-binding motifs whereas CTRL2 contains 9 copies of only one of these motifs (19), perhaps explaining the greater effect of CTRL1 than CTRL2 deletion on transgene expression in the LAT locus. Recent evidence indicates that the function of insulators is dependent on additional sequences aside from CTCFbinding motifs, and thus a more complete analysis of these viral elements will be required to determine whether they have specific limitations (46). LAP2 (LATP2) has been reported to contain chromatin-binding elements that bend DNA, and such activity could enhance the function of nearby foreign promoters (47).…”

Novel approaches to in vitro transgenesis

“…Insulator elements are natural genetic sequences that establish independent domains of transcriptional activity within the eukaryotic genome and have distinct roles in establishing gene expression; acting as barrier elements to prevent the spread of heterochromatin and blocking enhancer-promoter interactions (Fig. 3A, lower panel; Kuhn & Geyer 2003, Recillas-Targa et al 2004. Thus, while insulators would not protect against insertional mutagenesis that may lead to aberrant gene expression of non-transgenes (Fig.…”