In the dominantly inherited genetic disorders where the underlying pathomechanism is dominant-negative protein interference, such as the keratin disorders, an attractive therapy strategy is to selectively inhibit expression of the mutant allele, allowing the normal allele to function.  In families with a rare recessive type of the skin blistering disorder epidermolysis bullosa simplex, we have previously shown that heterozygous carriers of a null mutation in the keratin K14 gene have perfectly normal skin. This provides proof-of-concept in humans for our therapy strategy of silencing the dominant-negative mutant allele and allowing the single normal allele to function.  The majority of keratin mutations are single point mutations leading to amino acid substitutions.  Using a medium throughput assay system, we systematically screen all possible siRNA molecules against wild-type and mutant reporter gene constructs to identify those positions where the mutant allele is potently and selectively inhibited.  These inhibitors are then taken through secondary screening using biochemical and functional assays to arrive at lead molecules for clinical development.  Currently, we are exploring non-invasive methods to deliver siRNA into the epidermis and into the cornea, using organotypic culture and animal model systems, including live-animal imaging.  Our multidisciplinary siRNA delivery programme, funded by an MRC Programme Grant and an MRC Milstein Award, includes self-delivery chemical modifications of the siRNA molecule, topical formulation chemistry and biomedical physics applications, such as ultrasound and laser-assisted drug delivery. 

References:

• Smith FJD et al., (2008) Development of therapeutic siRNAs for pachyonychia congenita.  J Invest Dermatol 128:50-58 (PubMed ID: 17762855)
• Hickerson RP et al., (2008).  Single nucleotide-specific siRNA targeting of mutant keratin 6a responsible for the dominant-negative skin disorder pachyonychia congenita.  J Invest Dermatol 128:594-605 (PubMed ID: 17914454)
• Leachman SA et al., (2010) First-in-human Mutation-targeted siRNA Phase Ib Trial of an Inherited Skin Disorder. Molecular Therapy 8:442-446 (PubMed ID: 19935778)
• McLean WHI and Moore CBT (2011) Keratin disorders: from gene to therapy. Hum Mol Genet 20:R189-197 [Epub ahead of print Sep 10] (PubMed ID: 21890491)
• Atkinson SD, McGilligan VE, Liao H, Szeverenyi I, Smith FJD, Moore CBT, McLean WHI (2011) Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex. J Invest Dermatol 131:2079-2086 [Epub ahead of print Jun 30] (PubMed ID: 21716320)