title: Generation of recombinant skin in vitro by adeno-associated virus type 2 vector transduction.
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/cm(2) 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.
authors.affiliation: Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Nalini
authors.initials: N
authors.lastname: Agrawal
authors.forename: Hong
authors.initials: H
authors.lastname: You
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Maurizio
authors.initials: M
authors.lastname: Chiriva-Internati
authors.forename: John
authors.initials: J
authors.lastname: Bremner
authors.forename: Tarun
authors.initials: T
authors.lastname: Garg
authors.forename: Fabio
authors.initials: F
authors.lastname: Grizzi
authors.forename: C
authors.initials: C
authors.lastname: Krishna Prasad
authors.forename: Jawahar L
authors.initials: JL
authors.lastname: Mehta
authors.forename: Paul L
authors.initials: PL
authors.lastname: Hermonat
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000008:administration & dosage / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017768:Skin, Artificial; D023822:Tissue Engineering / Q000379:methods; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor