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An integration-defective lentivirus-based resource for site-specific targeting of an edited safe-harbour locus in the human genome

Gene Therapy volume 21, pages 343–352 (2014)Cite this article

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Abstract

Optimized gene transfer into human cells are still challenging the promise of human stem and induced pluripotent stem cells as resources for disease models, diagnostic screens and personalized cell therapy. These potential applications require precise control of the spatio-temporal action of gene switches and the coordinated regulation of modulators, effectors and differentiation factors during pluripotency, differentiation and homeostasis. Most studies require identical transgene environments for comparable analysis; however, this cannot be achieved by standard methods for transgenesis in human cells because of unintended epigenetic modifications, genetic instability, dose-dependent effects, and disruption or activation of host genes. Although gene targeting can circumvent these problems, human cells have proved difficult to target, and there is therefore a need to develop tools for targeted transgenesis at efficiencies similar to those achieved in mice. We present a simple strategy, KASTRINA 2.0, for reliable transgenesis in human cells, based on targeted recombinase-mediated cassette exchange and the safe episomal status conferred by integrase-deficient lentivirus (IDLV). By driving limited cre recombinase expression, the IDLV yields single site-specific recombination of a selectable donor cassette (TRINA) at the ‘safe-harbour’ AAVS1 locus previously edited by zinc-finger nuclease to contain an acceptor site (KAS2.0).

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References

  1. Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M et al. T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science 1995; 270: 475–480.

    Article  CAS  Google Scholar 

  2. Hockemeyer D, Soldner F, Beard C, Gao Q, Mitalipova M, DeKelver RC et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 2009; 27: 851–857.

    Article  CAS  Google Scholar 

  3. Zou J, Maeder ML, Mali P, Pruett-Miller SM, Thibodeau-Beganny S, Chou B-K et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 2009; 5: 97–110.

    Article  CAS  Google Scholar 

  4. Chapdelaine P, Pichavant C, Rousseau J, P Acirc Ques F, Tremblay JP . Meganucleases can restore the reading frame of a mutated dystrophin. Gene Ther 2010; 17: 846–858.

    Article  CAS  Google Scholar 

  5. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 2011; 29: 143–148.

    Article  CAS  Google Scholar 

  6. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al. RNA-guided human genome engineering via Cas9. Science 2013; 339: 823–826.

    Article  CAS  Google Scholar 

  7. Turan S, Galla M, Ernst E, Qiao J, Voelkel C, Schiedlmeier B et al. Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 2011; 407: 193–221.

    Article  CAS  Google Scholar 

  8. DeKelver RC, Choi VM, Moehle EA, Paschon DE, Hockemeyer D, Meijsing SH et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res 2010; 20: 1133–1142.

    Article  CAS  Google Scholar 

  9. Sadelain M, Papapetrou EP, Bushman FD . Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer 2012; 12: 51–58.

    Article  CAS  Google Scholar 

  10. Torres R, García A, Payá M, Ramirez JC . Non-integrative lentivirus drives high-frequency cre-mediated cassette exchange in human cells. PLoS One 2011; 6: e19794.

    Article  CAS  Google Scholar 

  11. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee Y-L, Kim KA et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol 2007; 25: 1298–1306.

    Article  CAS  Google Scholar 

  12. Kolb AF . Selection-marker-free modification of the murine beta-casein gene using a lox2272 [correction of lox2722] site. Anal Biochem 2001; 290: 260–271.

    Article  CAS  Google Scholar 

  13. Qiao J, Oumard A, Wegloehner W, Bode J . Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. J Mol Biol 2009; 390: 579–594.

    Article  CAS  Google Scholar 

  14. Silver DP, Livingston DM . Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol Cell 2001; 8: 233–243.

    Article  CAS  Google Scholar 

  15. Fehse B, Kustikova OS, Bubenheim M, Baum C . Pois(s)on-it’s a question of dose. Gene Ther 2004; 11: 879–881.

    Article  CAS  Google Scholar 

  16. Muzumdar MD, Tasic B, Miyamichi K, Li L, Luo L . A global double-fluorescent Cre reporter mouse. genesis 2007; 45: 593–605.

    Article  CAS  Google Scholar 

  17. Smith JR, Maguire S, Davis LA, Alexander M, Yang F, Chandran S et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells 2008; 26: 496–504.

    Article  CAS  Google Scholar 

  18. van Rensburg R, Beyer I, Yao XY, Wang H, Denisenko O, Li Z-Y et al. Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther 2013; 20: 201–214.

    Article  CAS  Google Scholar 

  19. Wanisch K, Yáñez-Muñoz RJ . Integration-deficient lentiviral vectors: a slow coming of age. Mol Ther 2009; 17: 1316–1332.

    Article  CAS  Google Scholar 

  20. Brown JP, Wei W, Sedivy JM . Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science 1997; 277: 831–834.

    Article  CAS  Google Scholar 

  21. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega S et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 2009; 460: 1149–1153.

    Article  Google Scholar 

  22. Li H, Collado M, Villasante A, Strati K, Ortega S, Cañamero M et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 2009; 460: 1136–1139.

    Article  CAS  Google Scholar 

  23. Hockemeyer D, Jaenisch R . Gene targeting in human pluripotent cells. Cold Spring Harbor Symposia on Quantitative Biology 2011; 75: 201–209.

    Article  Google Scholar 

  24. González F, Boué S, Belmonte J . Methods for making induced pluripotent stem cells: reprogramming a la carte. Nat Rev Genet 2011; 12: 231–242.

    Article  Google Scholar 

  25. Neff NF, Quake SR, Lu R, Weissman IL . Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding. Nat Biotechnol 2011; 29: 928–933.

    Article  Google Scholar 

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Acknowledgements

This work was supported by grant of the Ministry of Economy and Compititiveness [INNPACTO IPT-010000-2010-40] to JCR; and National Centre for Cardiovascular Research (CNIC) institutional funding from the Fundacion Pro-CNIC to the Viral Vector Technical Unit.

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Authors and Affiliations

  1. Viral Vector Facility, Fundacion CNIC, Melchor Fernandez Almagro 3, Madrid, Spain

    R Torres, A Garcia, M Jimenez & J C Ramirez

  2. Molecular Cytogenetics Group, Fundacion CNIO, Melchor Fernandez Almagro 3, Madrid, Spain

    S Rodriguez

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  1. R Torres
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  2. A Garcia
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  3. M Jimenez
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  4. S Rodriguez
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Correspondence to J C Ramirez.

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The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Torres, R., Garcia, A., Jimenez, M. et al. An integration-defective lentivirus-based resource for site-specific targeting of an edited safe-harbour locus in the human genome. Gene Ther 21, 343–352 (2014). https://doi.org/10.1038/gt.2014.1

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