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Review
. 2008 Sep;51(3):151-7.
doi: 10.1016/j.jdermsci.2008.04.003. Epub 2008 May 20.

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita

Sancy A Leachman  1 , Robyn P Hickerson, Peter R Hull, Frances J D Smith, Leonard M Milstone, E Birgitte Lane, Sherri J Bale, Dennis R Roop, W H Irwin McLean, Roger L Kaspar
Affiliations
Review

Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita

Sancy A Leachman et al. J Dermatol Sci. 2008 Sep.

Abstract

The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. The establishment of a non-profit organization, PC Project, has led to an unprecedented coalescence of patients, scientists, and physicians with a unified vision of developing novel therapeutics for PC. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

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Figures

Figure 1
Figure 1
Typical painful and debilitating plantar hyperkeratosis observed in pachyonychia congenita harboring the K6a N171K mutation. Treatment with siRNA will be by injection into the calluses.
Figure 2
Figure 2
A schematic representation of the protein domain organization common to all keratins. Four coiled coil domains, 1A, 1B, 2A, 2B, are separated by non-helical linkers, L1, L12 and L2. Shaded in red are the helix boundary domains that are highly conserved in sequence between all keratins. The majority of mutations identified in PC (in K6a, K16, K6b, K17) fall within these domains. The position of the most common amino acid mutated in K6a, N171, is shown.
Figure 3
Figure 3
Ability of siRNAs to specifically target the single nucleotide KRT6A N171K mutation responsible for the dominant disorder pachyonychia congenita. A. Human PLC hepatoma cells were transfected with wildtype KRT6A fused to plum fluorescent protein or alternatively N171K mutant KRT6A fused to yellow fluorescent protein (YFP) and visualized by fluorescence microscopy[14]. B. Co-transfection of tagged mutant (YFP) and wildtype (plum) KRT6A expression plasmids with siRNA. Co-transfection with non-specific control (NSC4) siRNA had no effect on expression plasmid expression with both plum-colored filaments (wildtype K6a) or yellow/green aggregates (N171K mutant K6a) observed. Addition of mutant-specific siRNA (K6a.513a.12) blocked mutant K6a expression along with its YFP tag, resulting in only wildtype expression, which leads to intermediate filament formation (plum coloration). As a further control, cells were treated with siRNA specific to the wildtype form (K6a.513c.12), resulting in no filaments being formed and only yellow/green (from YFP) aggregates observed.
Figure 4
Figure 4
Mutant-specific siRNA potently reduces mutant K6a mRNA levels without affecting wildtype levels in immortalized keratinocytes prepared from a PC patient. PC-10_K6a_N171K cells (immortalized keratinocytes prepared from a K6a N171K PC patient skin biopsy) were treated with K6a.513a.12 (targets N171K mutant mRNA) or control irrelevant siRNA (targets EGFP) at time 0 h. At the indicated timepoints, RNA was isolated, reversed transcribed and the resulting cDNA subjected to real time qRT-PCR analysis (Hickerson, Leachman et al., manuscript in preparation) using custom gene expression assays for wildtype and mutant K6a (GAPDH gene expression assay was used as the endogenous control). Normalized mutant K6a expression divided by wildtype K6a expression is plotted for each sample.
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