Generation of recombinant skin in vitro by adeno-associated virus type 2 vector transduction

Nalini Agrawal, Hong You, Yong Liu, Maurizio Chiriva-Internati, John Bremner, Tarun Garg, Fabio Grizzi, C. Krishna Prasad, Jawahar L. Mehta, Paul L. Hermonat

Research output: Contribution to journalArticle

Abstract

It has long been recognized that skin may be a particularly good target for pharmacologic gene therapy and as a platform for the secretion of systemically distributed molecules. Adeno-associated virus type 2 (AAV) is a useful vector for skin gene therapy because skin is the natural host tissue for AAV, in which it functions as an autonomous parvovirus. We demonstrate here that recombinant (r) AAV vectors carrying the granulocyte-macrophage colony-stimulating factor (GM-CSF), human papillomavirus E6, or green fluorescent protein (GFP) transgene could transduce primary human keratinocytes in ex vivo culture. We further demonstrate that these transduced cells could be used to form a transgene-positive recombinant skin (r-skin), using the organotypic epithelial raft culture system. Transduction of keratinocytes was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR) for RNA expression, enzyme-linked immunosorbent assay (ELISA) for product secretion, intracellular staining for protein expression, vector-chromosomal junction PCR and Southern blot analysis of proviral sequences, in situ immunohistochemistry analysis of protein expression, and ultraviolet light fluorescence for GFP expression. AAV/GM-CSF/Neo-infected keratinocyte/raft skins secreted GM-CSF at levels as high as 25 ng/cm2 of skin and maintained expression to 60 days postinfection. These data support the utility and efficiency of AAV-based gene delivery to produce genetically altered keratinocytes and r-skin.

Original languageEnglish
Pages (from-to)1707-1715
Number of pages9
JournalTissue Engineering
Volume10
Issue number11-12
Publication statusPublished - Nov 2004

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Biotechnology

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