Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder

Deena M. Leslie Pedrioli (Lead / Corresponding author), Dun Jack Fu, Emilio Gonzalez-Gonzalez, Christopher H. Contag, Roger L. Kaspar, Frances J. D. Smith, W. H. Irwin McLean

    Research output: Contribution to journalArticle

    33 Citations (Scopus)

    Abstract

    Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.
    Original languageEnglish
    Pages (from-to)1627-1635
    Number of pages9
    JournalJournal of Investigative Dermatology
    Volume132
    Issue number6
    DOIs
    Publication statusPublished - Jun 2012

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    Keratoderma, Palmoplantar, Epidermolytic
    Keratin-9
    RNA Interference
    Genes
    Alleles
    RNA
    Small Interfering RNA
    Assays
    Skin
    Mutation
    Keratins
    Luciferases
    Redundancy
    Therapeutics
    Mutant Proteins
    Reporter Genes
    Skin Diseases
    Proteins

    Cite this

    Leslie Pedrioli, D. M., Fu, D. J., Gonzalez-Gonzalez, E., Contag, C. H., Kaspar, R. L., Smith, F. J. D., & McLean, W. H. I. (2012). Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder. Journal of Investigative Dermatology, 132(6), 1627-1635. https://doi.org/10.1038/jid.2012.28
    Leslie Pedrioli, Deena M. ; Fu, Dun Jack ; Gonzalez-Gonzalez, Emilio ; Contag, Christopher H. ; Kaspar, Roger L. ; Smith, Frances J. D. ; McLean, W. H. Irwin. / Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder. In: Journal of Investigative Dermatology. 2012 ; Vol. 132, No. 6. pp. 1627-1635.
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    abstract = "Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85{\%} simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.",
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    Leslie Pedrioli, DM, Fu, DJ, Gonzalez-Gonzalez, E, Contag, CH, Kaspar, RL, Smith, FJD & McLean, WHI 2012, 'Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder', Journal of Investigative Dermatology, vol. 132, no. 6, pp. 1627-1635. https://doi.org/10.1038/jid.2012.28

    Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder. / Leslie Pedrioli, Deena M. (Lead / Corresponding author); Fu, Dun Jack; Gonzalez-Gonzalez, Emilio; Contag, Christopher H.; Kaspar, Roger L.; Smith, Frances J. D.; McLean, W. H. Irwin.

    In: Journal of Investigative Dermatology, Vol. 132, No. 6, 06.2012, p. 1627-1635.

    Research output: Contribution to journalArticle

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    AU - Kaspar, Roger L.

    AU - Smith, Frances J. D.

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    AB - Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.

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    Leslie Pedrioli DM, Fu DJ, Gonzalez-Gonzalez E, Contag CH, Kaspar RL, Smith FJD et al. Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder. Journal of Investigative Dermatology. 2012 Jun;132(6):1627-1635. https://doi.org/10.1038/jid.2012.28