Abstract
The chromatin insulator cHS4 can reduce silencing chromosomal position effects and genotoxicity associated with integrating viral vectors. However, the fully active version of this element can also reduce vector titers and is only partially effective. In order to identify alternatives to cHS4, we developed a functional lentiviral vector-based reporter screen for enhancer-blocking insulators. Using this system, we screened candidate sequences that were initially identified by chromatin profiling for binding by CTCF and for DNase hypersensitivity. All 12 analyzed candidates blocked enhancer-promoter activity. The enhancer-blocking activity of the top two candidates was confirmed in two complementary plasmid-based assays. Studies in a gammaretroviral reporter vector indicated these two candidates have little to no effect on vector titers, and do not diminish vector expression in primary mouse bone marrow cultures. Subsequent assessment in a mouse in vivo tumor formation model demonstrated that both candidates reduced the rate of gammaretroviral vector-mediated genotoxicity as effectively as the cHS4 insulator. In summary, we have developed a novel lentiviral vector-based method of screening candidate elements for insulator activity, and have used this method to identify two new insulator elements capable of improving the safety of retroviral vectors without diminishing vector titers or expression. These findings expand the limited arsenal of insulators functionally validated to reduce the rate of retroviral vector-mediated genotoxicity.
Highlights
Chromatin insulators have been shown to improve both the expression and safety of integrating viral vectors [1]
Most studies of insulators in gammaretroviral and lentiviral vectors have focused on the prototypical chromatin insulator cHS4, which was derived from DNase Hypersensitive Site (DHS) 4 of the chicken β-globin locus control region [2]
In the most widely-used functional assay for enhancerblocking chromatin insulators, a candidate element is placed between an enhancer and a promoter linked to a neomycin phosphotransferase (Neo) drugresistance gene (Figure 1A), and the level of transgene expression is determined by measuring the rate of drugresistant colony formation following plasmid transfection of a mammalian cell line [3]
Summary
Chromatin insulators have been shown to improve both the expression and safety of integrating viral vectors [1]. Most studies of insulators in gammaretroviral and lentiviral vectors have focused on the prototypical chromatin insulator cHS4, which was derived from DNase Hypersensitive Site (DHS) 4 of the chicken β-globin locus control region [2] This element acts as a barrier insulator capable of reducing the silencing of viral vectors by chromosomal position effects [3,4,5], and an enhancer-blocking insulator capable of reducing the activation of cellular proto-oncogenes by vector enhancers [6,7,8]. A smaller 250 bp core from the cHS4 element with suboptimal enhancer-blocking activity failed to prevent malignant transformation in a clinical trial, but is thought to have played a role in the transformation event [19]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
