Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors

Zychlinski, Daniela; Schambach, Axel; Modlich, Ute; Maetzig, Tobias; Meyer, Johann; Grassman, Elke; Mishra, Anjali; Baum, Christopher F.

doi:10.1038/sj.mt.6300411

Cited by 90 publications

(148 citation statements)

References 33 publications

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“…A retroviral transactivation assay could show that the internal SFFV but not PGK or EF1a promoter was able to enhance expression from a distantly located mCMV promoter. 50 In line with these results, we could show that the SFFV promoter was also capable of efficiently activating the mCMV in a bidirectional setting. This also applied to the PGK and the CD3 promoters supporting the hypothesis that the activation of a neighboring promoter depends on enhancer strength and distance.…”

Section: Discussionsupporting

confidence: 79%

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

et al. 2009

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Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were B10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O 6 -methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.

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Section: Discussionsupporting

confidence: 79%

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

et al. 2009

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“…9 Such a cellular internal promoter is not only more physiological but also should minimize the risk of insertional mutagenesis as described with the use of cellular promoters. 22 In the specific context of the WAS LV, we show that the WPRE is required to obtain an adequate expression of the WAS transgene, although it does not seem to be required for the production of high viral titer. We also show that either of the mutant tested can be used to replace the native WPRE to express equivalent levels of transgene.…”

Section: Discussionmentioning

confidence: 84%

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

et al. 2009

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The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott-Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

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“…Extensive studies have shown that the enhancer sequences are the major cause of cell transformation, making the design of viral vectors for life-long therapy approaches even more challenging. [8][9][10] Insulators mark the boundaries of chromatin domains and limit the range of action of enhancers and silencers. 11 They are characterized by at least one of the following properties: Enhancer blocking and/or boundary.…”

Section: Introductionmentioning

confidence: 99%

“…18,23,24 A shorter sub-portion of the cHS4, namely the 250-bp insulator core element, is more suitable with viral vectors' biology, but it failed to reproduce the activity of the full-length element. 9,20,25,26 The enhancer-blocking function of the cHS4 has been attributed to the CCCTC-binding factor (CTCF), an 11-zinc-finger DNA-binding protein highly conserved in vertebrates. CTCF was implicated in diverse regulatory functions, including transcriptional activation/ repression, insulation and imprinting.…”

Section: Introductionmentioning

confidence: 99%

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

Gaussin

Modlich

Bauche³

et al. 2011

Gene Ther

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Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancerblocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

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Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors

Cited by 90 publications

References 33 publications

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors

Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors

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