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

Gene Therapy volume 19, pages 15–24 (2012)Cite this article

Abstract

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 enhancer-blocker 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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Acknowledgements

This work was performed with support from EC-DG research (FP6-NoE), CLINIGENE (LSHB-CT-2006-018933 to NM, OCH and CB) and the University of Lausanne (NM). UM and CB also received support from the Food and Drug Administration (project FDA-09-1056211-KP) and the German Ministry of Research and Education (BMBF project iGene, 01GU0813). The kind gift of plasmids by Dr G Felsenfeld is acknowledged. We thankJulien DeRoyer (LBAP, ENSC); Johanna Krause (Department of Experimental Hematology, Hannover); Dr Jérémy Catinot and Pieric Doriot for technical help.

AUTHOR CONTRIBUTION

AG designed and performed experiments on the identification and assay of insulators, analyzed data and wrote the paper with NN under the supervision of NM; C Bauche constructed insulators and insulated hybrid LTRs; CD and AA performed experiments on the implementation of insulators in viral vectors, and analyzed data under the supervision of OCH who initiated the program with NM; and UM and AS performed experiments to assay the cytotoxicity of insulated viral vectors, and analyzed data under the supervision of C Baum.

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Authors and Affiliations

  1. Institute of Biotechnology,

    A Gaussin, N J Niederländer & N Mermod

  2. , University of Lausanne, Lausanne, Switzerland

    A Gaussin, N J Niederländer & N Mermod

  3. Doctoral program in Biotechnology and Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    A Gaussin

  4. Department of Experimental Hematology, Hanover Medical School, Hanover, Germany

    U Modlich, A Schambach & C Baum

  5. Laboratory of Biotechnology and Applied Pharmacogenetics, UMR 8113, Ecole Normale Supérieure de Cachan, Cachan, France

    C Bauche, C Duros, A Artus & O Cohen-Haguenauer

  6. Université Paris-Diderot & Hopital Saint-Louis, Paris Cedex 10, France

    O Cohen-Haguenauer

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  1. A Gaussin
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  2. U Modlich
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Correspondence to O Cohen-Haguenauer or N Mermod.

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Gaussin, A., Modlich, U., Bauche, C. et al. CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors. Gene Ther 19, 15–24 (2012). https://doi.org/10.1038/gt.2011.70

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