The project aims to improve the molecular diagnosis of
dystrophic epidermolysis bullosa (DEB) and to develop gene therapy for severe recessive
DEB using the "shortened" version of the gene encoding type VII collagen.
DEB comprises one of the most severe groups of inherited skin diseases affecting
children and young adults. Patients affected by DEB suffer with loss of adhesion between
the epidermis (outer layer of the skin) and the dermis (inner layer). This results in
severe blistering of the skin and mucosa after mild mucosa and is evident from birth. The
condition is caused by abnormalities in the type VII collagen gene (COL7A1), which encodes
anchoring fibrils. These form attachment structures with a key role in the adhesion of the
epidermis to the dermis. The majority of patients do not produce type VII collagen protein
because they have inherited a deficient COL7A1 gene from each of their parents. The
identification of COL7A1 as the disease gene in 1991 has allowed searches for mutations in
patients and their family members to be carried out. Identification of the COL7A1 defect
in a given family has important implications for genetic counselling, e.g. the detection
of carriers, assessment of the mode of inheritance and early prenatal diagnosis in
affected families. It is also the first step towards genetic correction of the defect,
since it enables the presence of a normal copy of the gene, following gene transfer, to be
distinguished from the mutated copy.
However, COL7A1 is a complex gene comprising 118 exons, the exhaustive screening of
which remains a challenging task for each patient. The group has observed that the
mutation(s) screening remained negative in a significant number of patients after complete
screening of the COL7A1 gene. This may be due to the sensitivity of the mutation detection
method that was used (80%) and/or the to the nature and position of the mutation within
the gene. To overcome this problem, it is proposed in the first part of this project to
screen the shortened version of the gene (COL7A1cDNA), which contains only the genetic
information coding for the type VII collagen protein. In addition to being 3.7 times
smaller (still 9200 letters for the genetic code) than the COL7A1 gene (32,000 letters),
the COL7A1cDNA will greatly facilitate the detection of mutations altering the
"splicing" of the COL7A1 gene. This approach will require growth of epidermal
cells (keratinocytes) from a 5 mm skin biopsy taken under local anaesthetic from each
patient. This new approach will provide a powerful mutation screening strategy, which is
anticipated to result in the rapid identification of the majority of the defects in DEB
patients.
In the second part of the project, the group will develop genetic correction of the
condition using the "shortened" version of the COL7A1 gene. Indeed, one copy of
the normal COL7A1 gene of cDNA is sufficient to correct the condition, as indicated by the
observation that parents who produce only 50% of the normal amount of the type VII
collagen protein do not have EB. Initially, the entire COL7A1 cDNA molecule will be
isolated from normal skin. This will be labour intensive work considering the size of the
"shortened" version of COL7A1. The integrity of this molecule assembled from
smaller fragments will be checked and the group will then verify that it encodes the
normal type VII collagen. Once it has been shown that it produces normal type VII collagen
in epidermal cells, different systems will be used to deliver this normal copy of the
COL7A1 cDNA to patients’ keratinocytes in culture. Strategies will be developed
aiming to incorporate the normal copy of the COL7A1 cDNA into chromosomes of
patients’ cells. Integration of this molecule into the patients’ cells genome
should allow type VII collagen to be permanently re-expressed and normal anchoring fibrils
to be formed. This will lead to the development of a completely new approach to treat the
condition using patient’s epidermal skin grafts grown in culture and producing normal
type VII collagen.
