Assessment of optimal transduction of primary human skin keratinocytes by viral vectors.
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.
Gagnoux-Palacios
Laurent
L
INSERM U634, Faculty of Medicine, Nice, France.
Hervouet
Catherine
C
Spirito
Flavia
F
Roques
Stephanie
S
Mezzina
Mauro
M
Danos
Olivier
O
Meneguzzi
Guerrino
G
D006516:Adenoviruses, Canine / Q000235:genetics*; D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000627:therapeutic use*; D006801:Humans; D015603:Keratinocytes / Q000637:transplantation*; D016086:Lentivirus / Q000235:genetics; D009052:Leukemia Virus, Murine; D051379:Mice; D010957:Plasmids; D011995:Recombination, Genetic; D014161:Transduction, Genetic