Year: 2006
Type: article
Abstract: The ability to achieve reproducible, persistent, and therapeutic levels of gene expression in vivo by non-viral gene transfer approaches has been problematic because of the relatively transient nature of transgene expression in vivo. The mechanisms underlying gene silencing in mammalian cells are complex and remain unclear. We have previously demonstrated that removal of the transgene expression cassette from the bacterial backbone led to increased persistence of transgenes in vivo. Changes in chromatin remodeling/ conformation (in particular, the nucleosome position patterning and modifications in aminoacidic residues on histone tails) are also thought to be involved in transcriptional inactivation. We hypothesized that the mechanisms that result in changes in the chromatin remodeling/ structure could be related to the covalent linkage of bacterial DNA to the expression cassette and thus, to gene silencing. To gain insight into this, we have performed a battery of chromatin immunoprecipitations (ChIP) from DNA isolated from the livers of mice transfected with small DNA minicircles devoid of bacterial backbone (which showed marked persistence of transgene expression) or parental closed circular plasmids (which are silenced over time) expressing human alpha-1-antitrypsin (hAAT) using antibodies against different histone modifications. In all, we have tested 26 different antibodies in these assays. Our studies to date show that silencing of the transgene from the parental vector was accompanied by an increase in heterochromatin-associated histone modifications and a decrease in modifications typically associated with euchromatin. Conversely, the pattern of histone modifications on the mini-circle DNA was consistent with a euchromatin histone pattern. These data indicate that (i) episomal vectors undergo chromatinization in vivo and (ii) successful expression of the transgene is possibly dependent on the type of chromatin structure that forms on the exogenous DNA. Understanding the mechanisms by which changes in chromatin remodeling/structure are involved in transcriptional gene silencing is fundamental to unravel the mechanism of transcriptional regulation in mammalian systems. Ultimately this will assist in the development of new tools that specifically reactivate silenced transgenes delivered via gene transfer vectors.
Source: Molecular Therapy
Authors: Efren Riu, Zhi-Ying Chen, Mark A. Kay
Institution: Stanford University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
OpenAlex ID: W2069112281