TY - JOUR
T1 - Generation of recombinant skin in vitro by adeno-associated virus type 2 vector transduction
AU - Agrawal, Nalini
AU - You, Hong
AU - Liu, Yong
AU - Chiriva-Internati, Maurizio
AU - Bremner, John
AU - Garg, Tarun
AU - Grizzi, Fabio
AU - Prasad, C. Krishna
AU - Mehta, Jawahar L.
AU - Hermonat, Paul L.
PY - 2004
Y1 - 2004
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=19944429826&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=19944429826&partnerID=8YFLogxK
U2 - 10.1089/ten.2004.10.1707
DO - 10.1089/ten.2004.10.1707
M3 - Article
C2 - 15684679
AN - SCOPUS:19944429826
SN - 1076-3279
VL - 10
SP - 1707
EP - 1715
JO - Tissue Engineering
JF - Tissue Engineering
IS - 11-12
ER -