Models and treatments of Dominant Dystrophic Epidermolysis Bullosa

© 2021 Blake Robert Cordingley SmithEpidermolysis bullosa (EB) is a group of genetic blistering disorders caused by mutations in the structural proteins of the skin. EB is characterised anatomically by skin fragility, blistering and scarring, and frequently results in pain, itch and – in severe cases – death. The dystrophic subtype of EB is caused by mutations in the human COL7A1 gene which encodes for collagen VII, and can be inherited in either a dominant or recessive manner. Dominant dystrophic epidermolysis bullosa (DDEB) is commonly caused by glycine substitutions within the collagenous domain of collagen VII. Improving understanding of the pathophysiology of DDEB should enable the development of more effective treatments. To this end, having suitable mouse models for DDEB would be helpful, but none have yet been reported. This thesis describes the generation of two novel mouse lines, each of which carries a modified Col7a1 allele (p.G2037R and p.G2028R) that was introduced using CRISPR and corresponds to the most common COL7A1 mutations found in DDEB patients (p.G2043R and p.G2034R). Dominant inheritance of either of these alleles results in a phenotype that closely resembles that seen in DDEB patients. Specifically, mice carrying these alleles show recurrent blistering that is first observed transiently around the mouth and paws in the early neonatal period and then again around the digits from 5-10 weeks of age. Histologically, the mice show micro-blistering and reduced collagen VII immunostaining. Biochemically, collagen VII from these mice displays reduced thermal stability, which we also observed to be the case for DDEB patients carrying the analogous mutations. Unlike previous rodent models of EB which frequently show severe disease and early lethality, these mouse models – which to our knowledge are the first for DDEB – show no impairment in survival and have a relatively mild phenotype, and thus represent a practically and ethically tractable means for better understanding and treating EB. As proof-of-concept of the utility of these DDEB mice, animals carrying the p.G2037R allele were assessed in standardised pain behavioural tests and observed to be hypersensitive to both mechanical and thermal stimuli using the von Frey and Hargreaves’ assays respectively, mimicking what has been observed previously in EB patients. A range of different pharmacological agents – some of which are already used clinically to treat pain in EB patients and others of which are more experimental – were then objectively compared across a range of relevant doses and their analgesic effectiveness in DDEB mice was found to vary. While most drugs provided only a partial reduction in pain hypersensitivity, the non-steroidal anti-inflammatory agent, meloxicam, as well as a novel analgesic agent in early pre-clinical development were both found restore sensitivity to that of wild type mice, thus demonstrating that the DDEB mice may be useful preclinical tools for future drug development in EB