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Journal of Virology, February 2002, p. 1496-1504, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1496-1504.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

Ulrich Kuhn,^, Atsushi Terunuma, Wolfgang Pfutzner, Ruth Ann Foster, and Jonathan C. Vogel^*

Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Received 24 May 2001/ Accepted 25 October 2001

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.

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* Corresponding author. Mailing address: Dermatology Branch, National Cancer Institute, NIH, Bldg. 10, Room 12N260, 10 Center Dr., MSC 1908, Bethesda, MD 20892-1908. Phone: (301) 496-9002. Fax: (301) 496-5370. E-mail: jonvogel{at}mail.nih.gov.

Present address: Laboratory of Tissue Engineering, Instituto Dermopatico dell’ Immacolata, 00040 Pomezia, Rome, Italy.

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Journal of Virology, February 2002, p. 1496-1504, Vol. 76, No. 3
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.3.1496-1504.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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