Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors

Abstract

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.

Current approaches and perspectives in human keratinocyte-based gene therapies

“…16 Using this animal model, it was demonstrated, in vitro, that conventional retroviral vectors efficiently transduce the dog collagen VII cDNA into the dystrophic EB keratinocytes and achieve permanent expression of the transgene with correction of the defects caused by recessive dystrophic EB. 17 Although a safety preassessment of a human transgenic skin may be feasible in the future (see below), a preclinical gene therapy study in large immunocompetent animals remains critical to define the precise specifications for future clinical trials. Thus, a preclinical study on RDEB golden retriever dogs will soon be conducted in France by the group led by Dr G Meneguzzi.…”

Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells

“…Transgenic cells have been created using retroviral, lentiviral, transposon, ⌽C31-based integrase vectors, or mini-genes. [5][6][7][8][9][10][11][12][13] The lesions in RDEB, however, are distributed over multiple and large areas of the body, both on external (skin) and internal (gastrointestinal) sites, which makes local treatments impractical and unable to target the global pathology associated with RDEB. Thus, as these measures provide only local and temporal relief from the RDEB pathology, we wanted to design a strategy with the potential for systemic and durable correction of col7 deficiency.…”

miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

“…This modification should offer new perspectives to the study of the tightly regulated miRNA turnover as reported for miR-122 or miR-141 (Katoh et al 2009;Kim et al 2011). Likewise, given the reported payload of pMSCV (Baldeschi et al 2003), we expect that 39 UTRs as long as 5 kb can be cloned in the miR-Sens vector and tested in target cells without loss of sensitivity. So far, we have prepared more than 40 39 UTRs with sizes ranging up to 3 kb without significant loss of titer.…”