Natural Gene Therapy

Natural Gene Therapy occurs in all patients with the generalized variant of non-Herlitz junctional epidermolysis bullosa caused by COL17A1 mutations

Field of research:
Dermatology

Introduction:
Epidermolysis Bullosa (EB) is a group of genetic skin diseases, characterized by blister formation due to a defect in adhesion of the basal keratinocytes to the underlying dermis. Mutations in the COL17A1 gene, encoding the protein type XVII collagen, result in non-Herlitz junctional EB (JEB-nH). Natural gene therapy, also referred to as revertant mosaicism, is the somatic reversion of an inherited mutation, thereby partially or completely restoring the phenotype. In JEB-nH this is manifested as clinically healthy revertant patches surrounded by blistering affected skin. Here we investigate the underlying reversion mechanisms in the revertant patches of four JEB-nH patients.

Material & methods:
Biopsies are taken from suspected revertant and affected skin. With immunofluorescence microscopy expression of type XVII collagen is investigated. DNA and RNA are isolated and amplified with nested PCR surrounding the mutation c.2237delG.

Results:
DNA analysis from keratinocytes with type XVII collagen expression showed 5 different second-site mutations in 7 distinct biopsy specimens, all correcting the inherited c.2237delG mutation: a transition in the same codon (c.2238C>T), a substitution in the 5’-donor splice site of intron 30 (c.2263+2T>C) and a deletion in exon 30 and intron 30 (c.2259_2263+9del). Moreover, an indel mutation (c.2228-101_2263+70delins15) and a large deletion of 2165 base pairs that included exons 30 and 31 (c.2227+153_2336-318del) were identified.

Conclusion:
All correcting COL17A1 mutations had an effect on mRNA splicing and led to skipping of the mutated exon 30, in one case even to skipping of exons 30 and 31. The type XVII collagen protein is shorter, 12 resp. 36 amino acids, but still functional, given the clinical appearance. Our results show no preference for a single repair mechanism for the inherited mutation c.2237delG.
In addition, photo-material of all 10 generalized JEB-nH patients due to mutations in COL17A1 indicates that each patient has one or more revertant skin areas. Revertant mosaicism was initially thought to be rare. Our results show however, that natural gene therapy is common and occurs in all patients with JEB-nH. This fact makes it worthwhile to study revertant cell therapy in which the patient’s own naturally corrected cells are used.