Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors.
We have assessed the suitability of retroviral vectors for gene therapy of recessive dystrophic epidermolysis bullosa (RDEB) in dogs expressing a mutated collagen type VII. Isolation and analysis of the 9 kb dog collagen type VII cDNA identified the causative genetic mutation G1906S and disclosed the interspecies conservation of collagen type VII. Highly efficient transfer of the wild-type collagen type VII cDNA to both dog RDEB and human primary RDEB collagen type VII-null keratinocytes using recombinant vectors derived from LZRS-Ires-zeo and MSCV retroviruses achieved sustained and permanent expression of the transgene product. The expression and post-translational modification profile of the recombinant collagen type VII was comparable to that of the wild-type counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Hypermotility was fully reverted in human RDEB keratinocytes, and strongly reduced in the dog RDEB cells. This observation suggests that not only infection efficiency but also high expression levels are required to ensure therapeutic efficacy in the presence of mutated gene products. Our results set the basis for preclinical gene therapy assays in the first immune-competent large animal model for an inherited skin disease and broaden the spectrum of preclinical and clinical applications of retroviral vectors in the transfer of large recombinant genes in epithelial cells.
Baldeschi
Christine
C
INSERM U385, Faculty of Medicine, Avenue de Valombrose, 06107 Nice cedex 2, France.
Gache
Yannick
Y
Rattenholl
Anke
A
Bouillé
Pascale
P
Danos
Olivier
O
Ortonne
Jean-Paul
JP
Bruckner-Tuderman
Leena
L
Meneguzzi
Guerrino
G
D000818:Animals; D002478:Cells, Cultured; D024143:Collagen Type VII / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D004283:Dog Diseases / Q000235:genetics*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000662:veterinary*; D005455:Fluorescent Antibody Technique; D005808:Genes, Recessive / Q000235:genetics*; D015316:Genetic Therapy; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D015603:Keratinocytes; D017354:Point Mutation / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
D024143:Collagen Type VII; D017931:DNA Primers; D018076:DNA, Complementary