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Site-specific integration and tailoring of cassette design for sustainable gene transfer

Nature Methods volume 8, pages 861–869 (2011)Cite this article

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Abstract

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

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Figure 1: Targeted integration and transgene expression in CCR5 and AAVS1 of human lymphoblastoid cells.
Figure 2: Upregulation of endogenous genes at the integration site depends on the exogenous promoter and target locus.
Figure 3: Cassette design for unperturbed target gene expression.
Figure 4: Epigenetic analysis of CCR5 and AAVS1 before and after transgene insertion.
Figure 5: AAVS1-targeted integration and transgene expression without perturbing endogenous gene expression in primary human T lymphocytes.
Figure 6: Transgene expression in human stem cells and their progeny after targeted integration into AAVS1.

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References

  1. Ellis, J. Silencing and variegation of gammaretrovirus and lentivirus vectors. Hum. Gene Ther. 16, 1241–1246 (2005).

    Article  CAS  Google Scholar 

  2. Naldini, L. Ex vivo gene transfer and correction for cell-based therapies. Nat. Rev. Genet. 12, 301–315 (2011).

    Article  CAS  Google Scholar 

  3. Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).

    Article  CAS  Google Scholar 

  4. Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97–110 (2009).

    Article  CAS  Google Scholar 

  5. Maeder, M.L. et al. Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol. Cell 31, 294–301 (2008).

    Article  CAS  Google Scholar 

  6. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  Google Scholar 

  7. Carroll, D. Progress and prospects: zinc-finger nucleases as gene therapy agents. Gene Ther. 15, 1463–1468 (2008).

    Article  CAS  Google Scholar 

  8. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  Google Scholar 

  9. Gondo, Y., Fukumura, R., Murata, T. & Makino, S. Next-generation gene targeting in the mouse for functional genomics. BMB Rep. 42, 315–323 (2009).

    Article  CAS  Google Scholar 

  10. Frazer, K.A., Murray, S.S., Schork, N.J. & Topol, E.J. Human genetic variation and its contribution to complex traits. Nat. Rev. Genet. 10, 241–251 (2009).

    Article  CAS  Google Scholar 

  11. de Boer, B.A., Ruijter, J.M., Voorbraak, F.P. & Moorman, A.F. More than a decade of developmental gene expression atlases: where are we now? Nucleic Acids Res. 37, 7349–7359 (2009).

    Article  CAS  Google Scholar 

  12. Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009).

    Article  CAS  Google Scholar 

  13. Aiuti, A. et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458 (2009).

    Article  CAS  Google Scholar 

  14. Samulski, R.J. et al. Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 10, 3941–3950 (1991).

    Article  CAS  Google Scholar 

  15. Liu, R. et al. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86, 367–377 (1996).

    Article  CAS  Google Scholar 

  16. Smith, J.R. et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells 26, 496–504 (2008).

    Article  CAS  Google Scholar 

  17. Henckaerts, E. et al. Site-specific integration of adeno-associated virus involves partial duplication of the target locus. Proc. Natl. Acad. Sci. USA 106, 7571–7576 (2009).

    Article  CAS  Google Scholar 

  18. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  Google Scholar 

  19. Zou, J. et al. Oxidase deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease mediated safe harbor targeting. Blood 117, 5561–5572 (2011).

    Article  CAS  Google Scholar 

  20. Matrai, J. et al. Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk. Hepatology 53, 1696–1707 (2011).

    Article  CAS  Google Scholar 

  21. Hafenrichter, D.G. et al. Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes. Blood 84, 3394–3404 (1994).

    CAS  PubMed  Google Scholar 

  22. Okuyama, T. et al. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Hum. Gene Ther. 7, 637–645 (1996).

    Article  CAS  Google Scholar 

  23. Miao, C.H. et al. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol. Ther. 1, 522–532 (2000).

    Article  CAS  Google Scholar 

  24. Ogata, T., Kozuka, T. & Kanda, T. Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J. Virol. 77, 9000–9007 (2003).

    Article  CAS  Google Scholar 

  25. Li, C. et al. A small regulatory element from chromosome 19 enhances liver-specific gene expression. Gene Ther. 16, 43–51 (2009).

    Article  Google Scholar 

  26. Zhou, V.W., Goren, A. & Bernstein, B.E. Charting histone modifications and the functional organization of mammalian genomes. Nat. Rev. Genet. 12, 7–18 (2011).

    Article  Google Scholar 

  27. Cavazzana-Calvo, M. et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature 467, 318–322 (2010).

    Article  CAS  Google Scholar 

  28. Papapetrou, E.P. et al. Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat. Biotechnol. 29, 73–78 (2011).

    Article  CAS  Google Scholar 

  29. Jacquier, A. The complex eukaryotic transcriptome: unexpected pervasive transcription and novel small RNAs. Nat. Rev. Genet. 10, 833–844 (2009).

    Article  CAS  Google Scholar 

  30. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  31. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  32. Bondanza, A. et al. Suicide gene therapy of graft-versus-host disease induced by central memory human T lymphocytes. Blood 107, 1828–1836 (2006).

    Article  CAS  Google Scholar 

  33. Kaneko, S. et al. IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes. Blood 113, 1006–1015 (2009).

    Article  CAS  Google Scholar 

  34. Riddell, S.R. & Greenberg, P.D. The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J. Immunol. Methods 128, 189–201 (1990).

    Article  CAS  Google Scholar 

  35. Vescovi, A.L. et al. Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp. Neurol. 156, 71–83 (1999).

    Article  CAS  Google Scholar 

  36. Vescovi, A.L. & Snyder, E.Y. Establishment and properties of neural stem cell clones: plasticity in vitro and in vivo. Brain Pathol. 9, 569–598 (1999).

    Article  CAS  Google Scholar 

  37. Neri, M. et al. Efficient in vitro labeling of human neural precursor cells with superparamagnetic iron oxide particles: relevance for in vivo cell tracking. Stem Cells 26, 505–516 (2008).

    Article  CAS  Google Scholar 

  38. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  39. Roy, N.S. et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259–1268 (2006).

    Article  CAS  Google Scholar 

  40. Pfaffl, M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  Google Scholar 

  41. Zhao, S. & Fernald, R.D. Comprehensive algorithm for quantitative real-time polymerase chain reaction. J. Comput. Biol. 12, 1047–1064 (2005).

    Article  CAS  Google Scholar 

  42. Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. & Vandesompele, J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 8, R19 (2007).

    Article  Google Scholar 

  43. Lee, T.I., Johnstone, S.E. & Young, R.A. Chromatin immunoprecipitation and microarray-based analysis of protein location. Nat. Protoc. 1, 729–748 (2006).

    Article  CAS  Google Scholar 

  44. Wuarin, J. & Schibler, U. Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. Mol. Cell Biol. 14, 7219–7225 (1994).

    Article  CAS  Google Scholar 

  45. Masternak, K., Peyraud, N., Krawczyk, M., Barras, E. & Reith, W. Chromatin remodeling and extragenic transcription at the MHC class II locus control region. Nat. Immunol. 4, 132–137 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Celona, A. Anselmo and F. Ungaro for help with some experiments, A. Agresti, M. Bianchi and D. Gabellini for critical discussion, C. Di Serio and A. Nonis for statistical counseling, K. Ponder (Washington University, St. Louis), C. Miao (University of Washington, Seattle) and A. Recchia (University of Modena and Reggio Emilia) for providing reagents. Research was supported by Telethon (Telethon Institute for Gene Therapy grant), 7th EU Framework Programme (grant agreement 222878, PERSIST), European Research Council Advanced grant (249845 Targeting Gene Therapy), Fondazione Cariplo (Nobel Project) to L.N., Italian Ministry of Health (Giovani Ricercatori) to V.B.; Telethon (TGT06B02) to A.G.; Italian Ministry of Research and University (Ideas), Italian Ministry of Health (Giovani Ricercatori), Associazione Italiana per la Ricerca sul Cancro and Fondazione Cariplo to C.B.

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Author notes

  1. Bruno Di Stefano

    Present address: Present address: Hematopoietic Stem Cell Biology and Differentiation Group, Department of Differentiation and Cancer Centre for Genomic Regulation, Barcelona, Spain.,

  2. Daniela Cesana, Pietro Genovese, Bruno Di Stefano, Elena Provasi and Daniele F Colombo: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Regenerative Medicine, San Raffaele Telethon Institute for Gene Therapy, Gene Therapy and Stem Cells, San Raffaele Institute, Milan, Italy

    Angelo Lombardo, Daniela Cesana, Pietro Genovese, Daniele F Colombo, Margherita Neri, Alessio Cantore, Pietro Lo Riso, Martina Damo, Oscar M Pello, Angela Gritti & Luigi Naldini

  2. Vita-Salute San Raffaele University, Milan, Italy

    Angelo Lombardo, Daniela Cesana, Pietro Genovese, Elena Provasi, Daniele F Colombo, Margherita Neri, Alessio Cantore, Pietro Lo Riso, Martina Damo & Luigi Naldini

  3. Division of Neurosciences, Stem Cell and Neurogenesis Unit, San Raffaele Institute, Milan, Italy

    Bruno Di Stefano & Vania Broccoli

  4. Division of Regenerative Medicine, Experimental Hematology Unit, Gene Therapy and Stem Cells, Program in Immunology and Bio-immunotherapy of Cancer, San Raffaele Scientific Institute, Milan, Italy

    Elena Provasi, Zulma Magnani & Chiara Bonini

  5. Sangamo BioSciences Inc., Richmond, California, USA

    Michael C Holmes & Philip D Gregory

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Contributions

A.L., D.C., P.G., B.D.S., E.P. and D.F.C. designed and performed experiments, and interpreted data. M.N., Z.M., A.C., P.L.R., M.D. and O.M.P. performed experiments and interpreted data. M.C.H. and P.D.G. provided ZFNs. A.G., V.B. and C.B. coordinated NSC, iPSC and T-cell work, respectively. A.L. and L.N. conceived the project, coordinated all work and wrote the paper.

Corresponding author

Correspondence to Luigi Naldini.

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Competing interests

M.C.H. and P.D.G. are employees of Sangamo BioSciences Inc.

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Lombardo, A., Cesana, D., Genovese, P. et al. Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods 8, 861–869 (2011). https://doi.org/10.1038/nmeth.1674

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