AbstractEpidermolytic palmoplantar keratoderma (EPPK) is an autosomal-dominant keratin disorder. It is clinically characterised by diffuse hyperkeratosis of the palms and soles, demarcated by an erythematous border. Mutations altering the coding sequence of the keratin 9 (KRT9) gene, a palmoplantar-specific type I keratin, have been identified as the cause of EPPK. We hypothesise that effective, long-term treatment of EPPK will require silencing, or limiting the activity of, these causative mutant variants. To this end, a two-pronged strategy for disrupting mutant KRT9 allele with short interfering RNAs (siRNAs) was explored as a potential therapeutic approach. This led to the development and in vitro validation of potent mutation- and gene-specific siRNA inhibitors.
Despite its importance in EPPK, little is known about the physical requirement for KRT9 or its functional role in the palmoplantar epidermis. Here, a K9-null mouse was generated to define the functional importance of KRT9 (Krt9 in mice) in the palmoplantar epidermis, and determine the extent to which silencing is therapeutically viable. Krt9-/- mice developed epidermal fragility phenotypes localised to the epidermal footpad regions, demonstrating that Krt9 is essential for the structural integrity of palmoplantar epidermis. However, one functional Krt9 allele was sufficient for the normal development of epidermis, strongly suggesting that our mutation-specific siRNAs would be viable in a therapeutic context.
To further advance our candidate siRNA therapeutics into clinically applicable treatments, two additional novel mouse models were generated. A Krt9 luciferase reporter mouse model was developed to facilitate live animal epidermal imaging, and thus serve as a platform to identify and develop in vivo siRNA delivery methodologies. An EPPK phenotypic mouse model was also generated, which exhibited EPPK-like epidermal fragility phenotypes. Following the development of an optimal siRNA delivery system with the reporter gene mice, this phenotypic model will enable preclinical validation of its phenotype-resolution capabilities. These novel mouse models will further our understanding of KRT9 and how mutated variants give rise to EPPK, as well as, bring candidate siRNA therapeutics closer to clinical use.
|Date of Award||2014|
|Supervisor||Irwin McLean (Supervisor) & Deena Leslie Pedrioli (Supervisor)|