Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors

Gene Therapy volume 6, pages 432–441 (1999)Cite this article

Abstract

Gene transfer into the skin is a promising approach to treat inherited or acquired dermatological diseases and systemic monogenic deficiencies. For this purpose, the efficient and sustained gene delivery into keratinocytes is of critical importance. Recombinant adeno-associated virus (rAAV) vectors hold the potential to achieve a long-term gene transfer into various human organs. In order to evaluate this potential for skin gene therapy, human keratinocytes were transduced in vitro with rAAV vectors encoding the reporter genes β-galactosidase (rAAV/LacZ) or green fluorescent protein (rAAV/GFP). Using rAAV/LacZ at a multiplicity of infection (MOI) of five transducing particles per cell, up to 70% of human keratinocytes were trans- duced within 48 h. This effect was independent of individual skin donors and different body areas serving as the source for keratinocyte isolation. rAAV had no significant influence on cell viability, but induced a growth arrest in transduced keratinocytes. This growth arrest was overcome by replating cells in fresh media. rAAV/GFP-transduced keratinocytes could be passaged several times, expressed GFP for up to 50 days, and passed the transgene to their daughter cells, suggesting that keratinocyte precursor cells were also transduced. Taken together, the results suggest that rAAV is a promising gene transfer vehicle for skin gene therapy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 2ab
Figure 1
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Rheinwald JG, Green H . Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells Cell 1975 6: 331–344

    Article  CAS  PubMed  Google Scholar 

  2. Wang X, Zinkel S, Polonsky K, Fucks E . Transgenic studies with a keratin promotor-driven growth hormone transgene Proc Natl Acad Sci USA 1997 94: 219–226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Navsaria HA, Myers SR, Leigh IM, McKay IA . Culturing skin in vitro for wound therapy TIBTECH 1995 13: 91–100

    Article  CAS  Google Scholar 

  4. Braun-Falco M, Hallek M . Hautgentherapie-Perspektiven des Gentransfers in Keratinozyten Hautarzt 1998 49: 536–544

    Article  CAS  PubMed  Google Scholar 

  5. Fenjves ES . Approaches to gene transfer in keratinocytes J Invest Dermatol 1994 103: 70S–75S

    Article  CAS  PubMed  Google Scholar 

  6. Greenhalgh DA, Rothnagel JA, Roop DR . Epidermis: an attractive target tissue for gene therapy J Invest Dermatol 1994 103: 63S–69S

    Article  CAS  PubMed  Google Scholar 

  7. Krueger GG et al. Genetically modified skin to treat disease: potential and limitations J Invest Dermatol 1994 103: 76S–84S

    Article  CAS  PubMed  Google Scholar 

  8. Vogel JC, Walker PS, Hengge UR . Gene therapy for skin diseases Adv Dermatol 1996 11: 383–398

    CAS  PubMed  Google Scholar 

  9. Andree C et al. In vivo transfer and expression of a human epidermal growth factor gene accelerates wound repair Proc Natl Acad Sci USA 1994 91: 12188–12192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eming SA et al. Genetically modified human epidermis overexpressing PDGF-A directs the development of a cellular and vascular connective tissue stroma when transplanted to athymic mice-implications for the use of genetically modified keratinocytes to modulate dermal regeneration J Invest Dermatol 1995 105: 756–763

    Article  CAS  PubMed  Google Scholar 

  11. Sun L et al. Transfection with aFGF cDNA improves wound healing J Invest Dermatol 1997 108: 313–318

    Article  CAS  PubMed  Google Scholar 

  12. Carreau M et al. Functional retroviral vector for gene therapy of xeroderma pigmentosum group D patients Hum Gene Ther 1995 6: 1307–1315

    Article  CAS  PubMed  Google Scholar 

  13. Zeng L et al. Retrovirus-mediated gene transfer corrects DNA repair defect of xeroderma pigmentosum cells of complementation groups A, B and C Gene Therapy 1997 4: 1077–1084

    Article  CAS  PubMed  Google Scholar 

  14. Jensen TG et al. Correction of steroid sulfatase deficiency by gene transfer into basal cells of tissue-cultured epidermis from patients with recessive X-linked ichthyosis Exp Cell Res 1993 209: 392–397

    Article  CAS  PubMed  Google Scholar 

  15. Choate KA, Medalie DA, Morgan JR, Khavari PA . Corrective gene transfer in the human skin disorder lamellar ichthyosis Nature Med 1996 2: 1263–1267

    Article  CAS  PubMed  Google Scholar 

  16. Choate KA et al. Transglutaminase1 delivery to lamellar ichthyosis keratinocytes Hum Gene Ther 1996 7: 2247–2253

    Article  CAS  PubMed  Google Scholar 

  17. Fenjves ES, Schwartz PM, Blaese RM, Taichman LB . Keratinocyte gene therapy for adenosine deaminase deficiency: a model approach for inherited metabolic disorders Hum Gene Ther 1997 8: 911–917

    Article  CAS  PubMed  Google Scholar 

  18. Gerrard AJ, Hudson DL, Brownlee GG, Watt FM . Towards gene therapy for haemophilia B using primary human keratinocytes Nat Genet 1993 3: 180–183

    Article  CAS  PubMed  Google Scholar 

  19. DeLuca M, Pellegrini G . The importance of epidermal stem cells in keratinocyte-mediated gene therapy Gene Therapy 1997 4: 381–383

    Article  CAS  Google Scholar 

  20. Fenjves ES, Yao S-N, Kurachi K, Taichman LB . Loss of expression of a retrovirus-transduced gene in human keratinocytes J Invest Dermatol 1996 106: 576–578

    Article  CAS  PubMed  Google Scholar 

  21. Mathor MB et al. Clonal analysis of stably transduced human epidermal stem cells in culture Proc Natl Acad Sci USA 1996 93: 10371–10376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Hallek M et al. Recombinant adeno-associated virus vectors Curr Res Mol Ther 1998 1: 417–430

    CAS  Google Scholar 

  23. Kremer EJ, Perricaudet M . Adenovirus and adeno-associated virus mediated gene transfer Br Med Bull 1995 51: 31–44

    Article  CAS  PubMed  Google Scholar 

  24. Flotte TR, Carter BJ . Adeno-associated virus vectors for gene therapy Gene Therapy 1995 2: 357–362

    CAS  PubMed  Google Scholar 

  25. Kotin RM . Prospects for the use of adeno-associated virus as a vector for human gene therapy Hum Gene Ther 1994 5: 793–801

    Article  CAS  PubMed  Google Scholar 

  26. Samulski RJ . Adeno-associated virus: integration at a specific chromosomal locus Curr Opin Genet Dev 1993 3: 74–80

    Article  CAS  PubMed  Google Scholar 

  27. Muzyczka N . Use of adeno-associated virus as a general transduction vector for mammalian cells Curr Top Microbiol Immunol 1992 158: 97–127

    CAS  PubMed  Google Scholar 

  28. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus J Virol 1998 72: 2224–2232

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Chiorini JA et al. High-efficiency transfer of the T cell co-stimulatory molecule B7–2 to lymphoid cells using high-titer recombinant adeno-associated virus vector Hum Gene Ther 1995 6: 1531–1541

    CAS  PubMed  Google Scholar 

  30. Flotte TR et al. An improved system for packaging recombinant adeno-associated virus vectors capable of in vivo transduction Gene Therapy 1995 2: 29–37

    CAS  PubMed  Google Scholar 

  31. Podsakoff G, Wong KKJ, Chatterjee S . Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors J Virol 1994 68: 5656–5666

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Alexander IE, Russel DW, Miller D . DNA-damaging agents greatly increase the transduction of nondividing cells by adeno-associated virus vectors J Virol 1994 68: 8282–8287

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Xiao X, Li J, Samulski RJ . Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector J Virol 1996 70: 8098–8108

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Monahan PE et al. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia Gene Therapy 1998 5: 40–49

    Article  CAS  PubMed  Google Scholar 

  35. Snyder RO et al. Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice Hum Gene Ther 1997 8: 1891–1900

    Article  CAS  PubMed  Google Scholar 

  36. Afione SA et al. In vivo model of adeno-associated virus vector persistence and rescue J Virol 1996 70: 3235–3241

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Conrad CK et al. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung Gene Therapy 1996 3: 658–668

    CAS  PubMed  Google Scholar 

  38. Kaplitt MG et al. Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain Nat Genet 1994 8: 148–154

    Article  CAS  PubMed  Google Scholar 

  39. Flotte TR et al. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector Proc Natl Acad Sci USA 1993 90: 10613–10617

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kaplitt MG et al. Long-term gene transfer in porcine myocardium after coronary infusion of an adeno-associated virus vector Ann Thorac Surg 1996 62: 1669–1676

    Article  CAS  PubMed  Google Scholar 

  41. Fisher KJ et al. Recombinant adeno-associated virus for muscle directed gene therapy Nature Med 1997 3: 306–312

    Article  CAS  PubMed  Google Scholar 

  42. Manning WC et al. Transient immunosuppression allows transgene expression following readministration of adeno-associated virus vectors Hum Gene Ther 1998 9: 477–485

    Article  CAS  PubMed  Google Scholar 

  43. Descamps V, Blumenfeld N, Beuzard Y, Perricaudet M . Keratinocytes as a target for gene therapy Arch Dermatol 1996 132: 1207–1211

    Article  CAS  PubMed  Google Scholar 

  44. Klein-Bauerschmitt P, zur Hausen H, Schlehofer JR . Induction of differentiation-associated changes in established human cells by infection with adeno-associated virus type 2 J Virol 1992 66: 4191–4200

    Google Scholar 

  45. Maass G et al. Recombinant adeno-associated virus vectors for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells Hum Gene Ther 1998 9: 1049–1059

    Article  CAS  PubMed  Google Scholar 

  46. Lane EB, Alexander CM . Use of keratin antibodies in tumour diagnosis Semin Cancer Biol 1990 1: 165–179

    CAS  PubMed  Google Scholar 

  47. Van Muijen GNP, Warnaar SO, Ponec M . Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, newborn, and adult epidermis Exp Cell Res 1987 171: 331–345

    Article  CAS  PubMed  Google Scholar 

  48. Ponnazhagan S et al. Adeno-associated virus type 2-mediated transduction in primary human bone marrow-derived CD34+ hematopoietic progenitor cells: donor variation and correlation of transgene expression with cellular differentiation J Virol 1997 71: 8262–8267

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Nead MA, McCance DJ . Poly-L-ornithine-mediated transfection of human keratinocytes J Invest Dermatol 1995 105: 668–671

    Article  CAS  PubMed  Google Scholar 

  50. Deng H, Lin Q, Khavari PA . Sustainable cutaneous gene delivery Nat Biotech 1997 15: 1388–1391

    Article  CAS  Google Scholar 

  51. Russell DW, Alexander IE, Miller AD . DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors Proc Natl Acad Sci USA 1995 92: 5719–5723

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Verma IM . ‘Green light’ for gene transfer Nat Biotech 1996 14: 576

    Article  CAS  Google Scholar 

  53. Kube DM, Ponnazhagan S, Srivastava A . Encapsidation of adeno-associated virus type 2 rep proteins in wild-type and recombinant progeny virions: Rep-mediated growth inhibition of primary human cells J Virol 1997 71: 7361–7371

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Jiang C-K, Connolly D, Blumenberg M . Comparison of methods for transfection of human epidermal keratinocytes J Invest Dermatol 1991 97: 969–973

    Article  CAS  PubMed  Google Scholar 

  55. Staedel C et al. High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes J Invest Dermatol 1994 102: 768–772

    Article  CAS  PubMed  Google Scholar 

  56. Sawamura D et al. In vivo transfer of a foreign gene to keratinocytes using the hemagglutinating virus of Japan-liposome method J Invest Dermatol 1997 108: 195–199

    Article  CAS  PubMed  Google Scholar 

  57. Lu B et al. Topical application of viral vectors for epidermal gene transfer J Invest Dermatol 1997 108: 803–808

    Article  CAS  PubMed  Google Scholar 

  58. Surosky RT et al. Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome J Virol 1997 71: 7951–7959

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Ferrari FK, Samulski T, Shenk T, Samulski RJ . Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors J Virol 1996 70: 3227–3234

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Fisher KJ et al. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis J Virol 1996 70: 520–532

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Doll RF et al. Comparison of promoter strengths on gene delivery into mammalian brain cells using AAV vectors Gene Therapy 1996 3: 437–447

    CAS  PubMed  Google Scholar 

  62. Dong J-Y, Fan P-D, Frizzell RA . Quantitative analysis of the packaging capacity of recombinant adeno-associated virus Hum Gene Ther 1996 7: 2101–2112

    Article  CAS  PubMed  Google Scholar 

  63. Sanes JR, Rubenstein JLR, Nicolas J-F . Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos EMBO J 1986 5: 3133–3142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Leigh IM, Watt FM . Keratinocyte Methods Cambridge University Press: Cambridge 1994

  65. Jones PH, Watt FM . Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression Cell 1993 73: 713–724

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratorium für Molekulare Biologie, Genzentrum, Ludwig-Maximilians-Universität, München, Germany

    M Braun-Falco, A Doenecke & M Hallek

  2. Dermatologische Klinik der Universität Köln, Germany

    H Smola

Authors
  1. M Braun-Falco
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Doenecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H Smola
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Hallek
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Braun-Falco, M., Doenecke, A., Smola, H. et al. Efficient gene transfer into human keratinocytes with recombinant adeno-associated virus vectors. Gene Ther 6, 432–441 (1999). https://doi.org/10.1038/sj.gt.3300815

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3300815

Keywords

This article is cited by

Search

Advanced search

Quick links