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Assessment of optimal transduction of primary human skin keratinocytes by viral vectors
Author(s) -
GagnouxPalacios Laurent,
Hervouet Catherine,
Spirito Flavia,
Roques Stephanie,
Mezzina Mauro,
Danos Olivier,
Meneguzzi Guerrino
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.768
Subject(s) - transduction (biophysics) , viral vector , clonogenic assay , transgene , biology , keratinocyte , genetic enhancement , flow cytometry , cell culture , gene delivery , microbiology and biotechnology , hek 293 cells , ex vivo , recombinant dna , virology , gene , in vivo , genetics , biochemistry
Background Genetically modified keratinocytes generate transplantable self‐renewing epithelia suitable for delivery of therapeutic polypeptides. However, the variety of viral vectors and experimental conditions currently used make fragmented or contradictory the information on the transduction efficiency of the human primary keratinocytes. To compare the suitability of the most currently used viral vectors for efficient gene transfer to human keratinocytes, we have performed a comparative study using a panel of recombinant constructs. Methods For each vector, the transduction efficiency and the persistence of the transgene expression were quantified by fluorescence microscopy and flow cytometry analysis of the infected cells. Results We show that: (1) canine and human adenoviral vectors achieve a highly efficient but transient transduction of both primary and immortalized keratinocytes; (2) the adenovirus‐associated virus (AAV) vectors transduce immortalized keratinocytes, albeit with a short‐lived gene expression (<4 days), but fail to infect primary keratinocytes; and (3) under appropriate conditions, the oncoretroviral and lentiviral vectors can permanently transduce up to 100% of primary keratinocytes, but the highly clonogenic keratinocytes are more efficiently targeted by lentiviral vectors. Conclusions Therefore, AAV vectors are unsuitable to transduce primary keratinocytes, while human and canine adenoviral vectors appears to be appropriate to achieve short‐term delivery of therapeutic products. Recombinant retroviruses provide sustained expression of the transgene, but the lentiviral vectors are the most suitable for ex vivo gene therapy because of their ability to transduce clonogenic primary keratinocytes. Copyright © 2005 John Wiley & Sons, Ltd.