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Stable nonviral genetic correction of inherited human skin disease

Nature Medicine volume 8, pages 1166–1170 (2002)Cite this article

A Corrigendum to this article was published on 01 February 2003

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Abstract

Current gene-transfer technologies display limitations in achieving effective gene delivery. Among these limitations are difficulties in stably integrating large corrective sequences into the genomes of long-lived progenitor-cell populations. Current larger-capacity viral vectors suffer from biosafety concerns, whereas plasmid-based approaches have poor efficiency of stable gene transfer1,2. These barriers hinder genetic correction of many severe inherited human diseases, such as the blistering skin disorder recessive dystrophic epidermolysis bullosa (RDEB)3, caused by mutations in the large COL7A1 gene. To circumvent these barriers, we used the φC31 bacteriophage integrase4,5,6, which stably integrates large DNA sequences containing a specific 285-base-pair attB sequence into genomic 'pseudo-attP sites'. φC31 integrase–based gene transfer stably integrated the COL7A1 cDNA into genomes of primary epidermal progenitor cells from four unrelated RDEB patients. Skin regenerated using these cells displayed stable correction of hallmark RDEB disease features, including Type VII collagen protein expression, anchoring fibril formation and dermal-epidermal cohesion. These findings establish a practical approach to nonviral genetic correction of severe human genetic disorders requiring stable genomic integration of large DNA sequences.

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Figure 1: Stable marker gene delivery to primary human epidermal progenitors using the φC31-integrase.
Figure 2: Restoration of full-length Type VII collagen protein expression to RDEB keratinocyte progenitors.
Figure 3: Restoration of Type VII collagen protein expression in regenerated human RDEB patient skin tissue in vivo.

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  • 23 September 2002

    This was incorrect in AOP version but corrected in print. Added corrected info

References

  1. Hengge, U.R., Walker, P.S. & Vogel, J.C. Expression of naked DNA in human, pig, and mouse skin. J. Clin. Invest. 97, 2911–2916 (1996).

    Article  CAS  Google Scholar 

  2. Choate, K.A. & Khavari, P.A. Direct cutaneous gene delivery in a human genetic skin disease. Hum. Gene Ther. 8, 1671–1677 (1997).

    Google Scholar 

  3. Marinkovich, M.P., Herron, G.S., Khavari, P.A. & Bauer, E.A. Hereditary epidermolysis bullosa. in Dermatology in General Medicine, Vol. I (eds. Freedberg, I.M. et al.) 690–702 (McGraw-Hill, New York, 1999).

    Google Scholar 

  4. Kuhstoss, S. & Rao, R.N. Analysis of the integration function of the streptomycete bacteriophage φ C31. J. Mol. Biol. 222, 897–908 (1991).

    Article  CAS  Google Scholar 

  5. Thyagarajan, B., Olivares, E.C., Hollis, R.P., Ginsburg, D.S. & Calos, M.P. Site-specific genomic integration in mammalian cells mediated by phage φ C31 integrase. Mol. Cell Biol. 21, 3926–3934 (2001).

    Article  CAS  Google Scholar 

  6. Groth, A.C., Olivares, E.C., Thyagarajan, B. & Calos, M.P. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA 97, 5995–6000 (2000).

    Article  CAS  Google Scholar 

  7. Sclimenti, C.R., Thyagarajan, B. & Calos, M.P. Directed evolution of a recombinase for improved genomic integration at a native human sequence. Nucleic Acids Res. 29, 5044–5051 (2001).

    Article  CAS  Google Scholar 

  8. Christiano, A.M. et al. Structural organization of the human type VII collagen gene (COL7A1), composed of more exons than any previously characterized gene. Genomics 21, 169–179 (1994).

    Article  CAS  Google Scholar 

  9. Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84, 2302–2306 (1987).

    Article  CAS  Google Scholar 

  10. Medalie, D.A. et al. Evaluation of human skin reconstituted from composite grafts of cultured keratinocytes and human acellular dermis transplanted to athymic mice. J. Invest. Dermatol. 107, 121–127 (1996).

    Article  CAS  Google Scholar 

  11. Choate, K.A., Medalie, D.A., Morgan, J.R. & Khavari, P.A. Corrective gene transfer in the human skin disorder lamellar ichthyosis. Nature Med. 2, 1263–1267 (1996).

    Article  CAS  Google Scholar 

  12. Freiberg, R.A. et al. A model of corrective gene transfer in X-linked ichthyosis. Hum. Molec. Genet. 6, 937–933 (1997).

    Article  Google Scholar 

  13. Seitz, C.S., Giudice, G.J., Balding, S.D., Marinkovich, M.P. & Khavari, P.A. BP180 gene delivery in junctional epidermolysis bullosa. Gene Ther. 6, 42–47 (1999).

    Article  CAS  Google Scholar 

  14. Robbins, P.B. et al. In vivo restoration of laminin 5 β3 expression and function in junctional epidermolysis bullosa. Proc. Natl. Acad. Sci. USA 98, 5193–5198 (2001).

    Article  CAS  Google Scholar 

  15. Bruckner-Tuderman, L., Hopfner, B. & Hammami-Hauasli, N. Biology of anchoring fibrils: Lessons from dystrophic epidermolysis bullosa. Matrix Biol. 18, 43–54 (1999).

    Article  CAS  Google Scholar 

  16. Heinonen, S. et al. Targeted inactivation of the type VII collagen gene (Col7a1) in mice results in severe blistering phenotype: A model for recessive dystrophic epidermolysis bullosa. J. Cell Sci. 112, 3641–3648 (1999).

    CAS  PubMed  Google Scholar 

  17. Burton, E.A. & Davies, K.E. Muscular dystrophy—reason for optimism? Cell 108, 5–8 (2002).

    Article  CAS  Google Scholar 

  18. Evans, J.T. & Garcia, J.V. Lentivirus vector mobilization and spread by human immunodeficiency virus. Hum. Gene Ther. 11, 2331–2339 (2000).

    Article  CAS  Google Scholar 

  19. Kochanek, S. High-capacity adenoviral vectors for gene transfer and somatic gene therapy. Hum. Gene Ther. 10, 2451–2459 (1999).

    Article  CAS  Google Scholar 

  20. Kolodka, T.M., Garlick, J.A. & Taichman, L.B. Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes. Proc. Natl. Acad. Sci. USA 95, 4356–4561 (1998).

    Article  CAS  Google Scholar 

  21. Stockschlader, M.A., Schuening, F.G., Graham, T.C. & Storb, R. Transplantation of retrovirus-transduced canine keratinocytes expressing the β-galactosidase gene. Gene Ther. 1, 317–322 (1994).

    CAS  PubMed  Google Scholar 

  22. Pfutzner, W., Joari, M.A., Hengge, U.R. & Vogel, J.C. A raft culture model demonstrates the feasibility of topical selection for keratinocytes expressing a desired gene. J. Invest. Dermatol. 112, 552 (1999).

  23. Freiberg, R.A., Ho, S.N. & Khavari, P.A. Transcriptional control in keratinocytes and fibroblasts using synthetic ligands. J. Clin. Invest. 99, 2610–2615 (1997).

    Article  CAS  Google Scholar 

  24. Mathor, M.B. et al. Clonal analysis of stably transduced human epidermal stem cells in culture. Proc. Natl. Acad. Sci. USA 93, 10371–10376 (1996).

    Article  CAS  Google Scholar 

  25. Khavari, P.A., Peterson, C.L., Tamkun, J.W., Mendel, D.B. & Crabtree, G.R. BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription. Nature 366, 170–174 (1993).

    Article  CAS  Google Scholar 

  26. Keene, D.R., Sakai, L.Y., Lunstrum, G.P., Morris, N.P. & Burgeson, R.E. Type VII collagen forms an extended network of anchoring fibrils. J. Cell Biol. 104, 611–621 (1987).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M.P. Marinkovich for antibody reagents and helpful discussions at all stages of this work; J. Vogel for COL7A1 sequences; F. Scholl, Z. Siprashvili, A. Nguyen, N. Griffiths, P. Bernstein, P.B. Robbins and other members of the Khavari laboratory for helpful discussions; and S. Tufa for technical support. This work was supported by the USVA Office of Research and Development and by NIH AR44012 (to P.A.K.) and support from the Nu Skin Center for Dermatologic Research and the Epidermolysis Bullosa Medical Research Foundation. NOTE: In the version of the article initially published online, the Acknowledgments section inadvertently omitted funding support to one of the authors. This has been corrected in the HTML and PDF version, and will appear correctly in the forthcoming print issue.

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Authors and Affiliations

  1. VA Palo Alto Healthcare System, Palo Alto, California and the Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Susana Ortiz-Urda, Qun Lin, Min Fang & Paul A. Khavari

  2. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA

    Bhaskar Thyagarajan & Michele P. Calos

  3. Shriners Hospital for Children, Portland, Oregon, USA

    Douglas R. Keene

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  1. Susana Ortiz-Urda
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  2. Bhaskar Thyagarajan
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  4. Qun Lin
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  7. Paul A. Khavari
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Correspondence to Paul A. Khavari.

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Ortiz-Urda, S., Thyagarajan, B., Keene, D. et al. Stable nonviral genetic correction of inherited human skin disease. Nat Med 8, 1166–1170 (2002). https://doi.org/10.1038/nm766

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