NIH Public Access Author Manuscript J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. NIH-PA Author Manuscript Published in final edited form as: J Dermatol Sci. 2008 September ; 51(3): 151–157. doi:10.1016/j.jdermsci.2008.04.003. Therapeutic siRNAs for dominant genetic skin diseases including pachyonychia congenita Sancy A. Leachman1#, Robyn P. Hickerson2, Peter R. Hull3, Frances J. D. Smith4, Leonard M. Milstone5, E. Birgitte Lane6, Sherri J. Bale7, Dennis R. Roop8, W. H. Irwin McLean4, and Roger L. Kaspar2# 1Department of Dermatology and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 2TransDerm Inc., Santa Cruz, CA 3Department of Dermatology, Royal University Hospital, University of Saskatchewan, Saskatchewan NIH-PA Author Manuscript 4Human Genetics Unit, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK 5Department of Dermatology, Yale University, New Haven, CT 6Institute of Medical Biology, Singapore 138665, Singapore 7GeneDx, Gaithersburg, MD 8Department of Dermatology and Regenerative Medicine and Stem Cell Biology Program, University of Colorado at Denver and Health Sciences Center, Aurora, CO Abstract NIH-PA Author Manuscript 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. #Corresponding authors: Sancy Leachman, 801 585-1810, sancy.leachman@hci.utah.edu, Roger Kaspar, 831 420-1684, roger.kaspar@transderminc.com. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Leachman et al. Page 2 Introduction NIH-PA Author Manuscript The human genome project has provided accessible and comprehensive documentation of the human genome. This has greatly facilitated the development of new gene discovery technologies. It has also spawned the field of functional genomics, which attempts to assign functional relevance to copious sequence data. One of the most successful functional genomics technologies yet developed involves the use of small interfering RNAs (siRNAs) to interrogate the function of human genes. The capacity of siRNA to specifically and potently block gene expression in vitro has led to the consideration of siRNA as a candidate therapeutic agent as well. The continuing rapid pace of discovery and development in genetics and genomics portends the advent of individualized medicine, in which 1) patient genetic information can be rapidly analyzed, 2) disease mutations can be identified and 3) mutation-specific siRNAs can be selected, synthesized, tested for safety and efficacy, and efficiently delivered as novel therapeutics. Emergence of a variety of sequence-specific therapies [1] for ultra -rare, nonlethal, dominant-negative skin disorders, such as pachyonychia congenita (PC), would have been unthinkable without the rapid and relatively inexpensive synthetic and analytic technologies that developed along with the genome project. NIH-PA Author Manuscript A major task in the years ahead is the development of treatments for all dominant-negative disorders using findings from structural, functional and genetic basic science investigation. One such disease-targeted treatment involves the use of siRNAs. SiRNAs are a new class of RNA inhibitors that act via the RNA-induced silencing complex (RISC) to specifically degrade target RNAs. Inhibition of gene expression by siRNA is mediated by hybridized RNAs, typically containing a 19 bp complementary region with two nucleotide 3’ overhangs (19 + 2 design), that are sufficiently small so as to avoid immune surveillance [2]. This mechanism is distinct from the classical antisense activity of single-stranded oligonucleotides mainly with respect to the involvement of RISC, which catalytically cleaves the target mRNA and thereby exerts activity for a period of time dictated by RISC turnover. Unlike antisense oligonucleotides, persistence of the siRNA within cells outside of the RISC is theoretically not required for continued RNAi activity. Therefore, an intermittent dosing schedule for the siRNA can be rationalized in clinical trials. Pachyonychia congenita is an ideal “proof of principle” model for siRNA therapeutics NIH-PA Author Manuscript To date, 54 functional keratin genes have been identified and mutations in 20 keratin genes have been associated with human genetic disorders [3–5]. Typically, the sites of these mutations lie in the alpha helical domains involved in protein-protein interactions. Since all known keratins act in pairs, mutation of one member of the pair also affects the function of the partner protein, resulting in disruption of higher-order intermediate filament formation or assembly kinetics [6]. Significant basic scientific data regarding the molecular etiology of keratinizing disorders have been available since the mid-1990s. Although some therapeutic strategies have been suggested from experimental work, no therapeutic agents until now have reached the point of clinical trial. Pachyonychia congenita (PC) is a well-characterized genetic disorder predominantly affecting nails and skin, which is caused by mutations in keratins KRT6A or KRT16 (PC-1, OMIM 167200) and KRT6B or KRT17 (PC-2, OMIM 167210). The physical findings most commonly include grossly thickened nails coupled with palmoplantar hyperkeratosis [7,8]. The thickened fingernails are disfiguring and hinder fine fingertip actions used in a multitude of tasks. Importantly, PC patients experience severe incapacitating pain associated with the plantar keratoderma (Fig. 1). This necessitates significant lifestyle modification including the use of wheelchairs or crutches, and regular pain medication. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 3 NIH-PA Author Manuscript The discovery of PC as a keratin disorder followed a genetic linkage analysis study of a large Scottish pedigree. This linkage study showed co-segregation of the disease with polymorphic markers within the type I keratin gene cluster at 17q12-q21 [9]. Subsequently, a heterozygous missense mutation was identified in the family in KRT17 [10]. Simultaneously, a missense mutation was detected in KRT16 in a sporadic case of PC-1 [10]. A second gene was shown to be associated with PC-1 when mutations were identified in KRT6A [11]. Some time later a mutation was detected in KRT6B in an extended family with the PC-2 phenotype [12]. To date, there are published reports of 55 causative mutations responsible for PC in 108 independently ascertained families (www.interfil.org). The gene most commonly mutated is KRT6A, and the most common site of mutation is at position 171 in the amino acid sequence (Fig. 2). NIH-PA Author Manuscript PC serves as a prototype indication for siRNA-based therapy. The molecular basis of PC is known and the defective genes responsible have been identified. In addition, many different keratins are expressed in the epidermis and there is probable functional redundancy. SiRNA technology permits selective targeting of the dominant mutant allele, potentially eliminating only the functionally disruptive version of the keratin. A further advantage is the external location and focal nature of the plantar skin lesions permitting minimally-invasive localized treatments to be performed on patients. Localized siRNA therapy for a skin disease also has a major advantage over other tissue targets since the results of the therapeutic intervention can be directly observed and if necessary sampled, a feature which recently facilitated the first-inman gene therapy grafting of a junctional EB patient with a laminin mutation [13]. Any advance in the development of siRNA-based therapeutics for PC will likely be directly applicable to treatment of other dominant genodermatoses, as well as indirectly applicable to a much larger number of human disorders that have dominant-negative etiology. Further, development of methods for delivery of siRNA-based therapeutics may have downstream benefit for treatment of unrelated genetic disorders that also affect the skin. Development of mutation and gene-specific siRNAs In PC, two approaches to the development of siRNAs have been undertaken. The first is the development of mutation specific siRNAs, which have the disadvantage of limiting the number of families who could be treated but are highly specific. The second approach was directed at developing gene specific siRNAs that simultaneously target both wildtype and mutant genes. Identification of K6a N171K mutation-specific siRNAs NIH-PA Author Manuscript Several viable approaches are available to identify optimal siRNAs for targeting mutant mRNAs in dominant negative diseases. A rational approach, taking advantage of existing algorithms that predict good target sites as well as the site most likely to yield discrimination can be employed. Alternatively, all possible siRNAs targeting the mutation site can be prepared (sequence walk) and tested, assuring that all possible effective siRNAs are identified. Both approaches have been used with success [14–16] and a combination of the two may be most effective, eliminating those sequences that are known to be ineffective or non-discriminating. Efficient and effective screening requires an assay that will quickly and accurately identify suitable candidates. One approach is to make bicistronic constructs, in which the mutant (and wildtype as control) is linked to a reporter construct such as a fluorescent protein or firefly luciferase and cloned into a plasmid expression vector ([14] and data not shown). The effectiveness of the candidate siRNAs can then be scored by fluorescence or luciferase assay following plasmid co-transfection with candidate siRNAs. Using this type of approach, we identified K6a.513a.12, an siRNA that has no effect on wildtype K6a expression in tissue J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 4 culture or animal studies, but silences the mutant form containing a single nucleotide change [14]. NIH-PA Author Manuscript The single nucleotide specificity of siRNA treatment can be readily observed by linking wildtype and mutant targeted cDNAs to tags such as fluorescent proteins. Fig. 3A shows the intermediate filaments formed following transfection of expression plasmids containing KRT6A wildtype cDNA linked to plum fluorescent protein as well as mutant KRT6A linked to yellow fluorescent protein (YFP), which results in aggregates of mutant protein. Cotransfection with differentially-tagged wildtype (plum) and mutant (YFP) K6a expression constructs allows visualization of the distinctly-tagged wildtype or mutant keratin 6a (K6a) proteins, which can be selectively inhibited with siRNAs that are specific to either the wildtype or mutant forms of KRT6A mRNA (Fig. 3B), thereby demonstrating the single nucleotide specificity of the siRNA agents. The specificity of the K6a.513a.12 siRNA is further demonstrated by inhibition of mutant KRT6A mRNA and not wildtype mRNA in immortalized keratinocytes derived from a K6a N171K patient biopsy (Fig. 4). NIH-PA Author Manuscript While effective siRNAs targeting the N171K mutation were readily identified, other mutations may be more problematic and effective siRNAs may be more difficult to identify. The rapid progress of siRNA development, however, suggests that even difficult sites may be amenable to targeting, by modification of one of both strands in the siRNA molecule. For example, modification of the antisense strand to facilitate stronger hybridization to the target may allow tuning to specific mutation sites [15]. Identification of gene-specific siRNAs The overlapping structure and function of the KRT6A and KRT6B genes, as well as studies of KRT6A knockout mice [17–19], strongly suggest that reduction or elimination of K6a expression can be compensated for by K6b or other keratins. Thus, siRNAs targeting both the mutant and wildtype forms of K6a, may be a viable approach to treating PC. We have recently identified siRNAs that specifically block expression of both wildtype and mutant forms of K6a with no effect on homologous keratin gene expression such as K6b [16]. NIH-PA Author Manuscript Comparison of the DNA sequences of the cDNAs encoding human KRT6A (Genbank RefSeq accession number NM_005554) and KRT6B (Genbank RefSeq accession number NM_005555) revealed that only a few isolated bases can distinguish these two genes in terms of their protein-encoding sequences. However, these two genes do differ significantly in their non-coding 3’UTR sequences. Using the Dharmacon siDESIGN center, four inhibitors were designed within the 3’UTR of K6a that were predicted to inhibit KRT6A expression without affecting the expression of KRT6B or other type II keratin genes due to significant sequence differences. Animal models relevant to studying dominant negative disorders The parallel mouse and human genome projects have greatly facilitated logical construction of animal models. Comparison of the human and mouse genome sequences revealed that only about 300 human genes do not have a murine ortholog [20], facilitating the rapid generation of knockout or knock- in mutant mice corresponding to most human genes. In the case of the keratin-related genodermatoses, the most useful and realistic models are mice in which dominant-negative mutations, equivalent to those commonly found in human patients, can be activated in the epidermis by topical application of a small-molecule inducer [21,22]. These revolutionary mice allow induction of epidermal fragility phenotypes in small regions of the skin to allow the study of pathogenetic mechanisms and therapy systems, without major distress to the animal or lethality. Analogous inducible PC mice are under development currently. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 5 PC clinical trial utilizing siRNA NIH-PA Author Manuscript As discussed above, PC is an ultra -rare disorder and current treatment modalities primarily center on symptomatic relief. Recent registry data suggests that the incidence of PC is likely to be on the order of a few thousand individuals worldwide (see www.pachyonychia.org and [23]). The development of the potent and exquisitely selective siRNA targeting N171K mutant KRT6A mRNA discussed above has created the opportunity to undertake the first siRNA clinical trial (initiated early 2008) for any skin disorder. This first trial investigates the safety and tolerability of intra-lesional injections of siRNA into PC patient calluses. The study will be a split-body and double-blinded investigation, injecting drug into one foot and vehicle into a matched callus on the other foot of a PC patient. The primary purpose of the study is to assess dose safety and tolerance of an increasing volume and concentration of siRNA. A secondary objective is to evaluate patients for any signs of efficacy at the injection site (and elsewhere on the skin). Multiple measures of efficacy will be assessed including clinical examination, subjective patient scoring systems of pain and quality of life, and a state-of-theart real time RT-PCR assay that quantitatively distinguishes wildtype and mutant keratin mRNAs in callus shavings (Hickerson, Leachman et al., in preparation). NIH-PA Author Manuscript Future perspectives NIH-PA Author Manuscript The transition of siRNA agents into routine clinical use is on the horizon. To date, only a handful of siRNA therapeutics (including those developed for macular degeneration and respiratory syncytial virus) have entered clinical trials and none has yet obtained FDA approval [24–26]. Here we report on the progress of a new siRNA entering clinical trials in PC patients with the KRT6A N171K mutation, with a gene-specific KRT6A siRNA study possibly to follow. This is the first-in-man siRNA therapeutic trial for a skin indication and the first siRNA to target a gene mutation. In the case of PC, the rapid trajectory of the siRNA inhibitors into clinical trials has only been made possible by the strength of the basic scientific knowledge and technologies resulting from the human genome project (and related efforts). The clinical trial with this agent will represent an important proof-of-principle human experiment, rigorously designed with quantifiable endpoints to test whether this siRNA therapeutic is not only safe, but also holds promise in the treatment of this disorder. The potential for specificity in the design of these siRNA agents offers unprecedented potential in the field of tailored and individualized medicine. If this siRNA proves effective in the treatment of PC, it may herald the onset of tailored siRNA therapeutics for any number of dominant skin diseases as well as other disorders. If efficacy is proven, siRNA agents may be a new class of drug with the potential to cause a paradigm shift in the treatment of dominant negative genetic disorders. Acknowledgements The Pachyonychia Congenita Project (PC Project, see www.pachyonychia.org) has been critically instrumental in effectively organizing an international group of PC patients, patient advocates, and the International PC Consortium (IPCC), a group of physicians and scientists who have agreed to work together to develop PC therapeutics. The authors thank PC Project, and in particular the director, Mary Schwartz, for unwavering support of this project and for providing the patient photograph. We are grateful to PC patients for their strong support and also thank IPCC members for stimulating discussion and insights. Finally, we thank Roger Tsien (UC San Diego) for providing the plum fluorescent protein construct. References 1. Lewin AS, Glazer PM, Milstone LM. Gene therapy for autosomal dominant disorders of keratin. J Investig Dermatol Symp Proc 2005;10:47–61. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. 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Development of therapeutic siRNAs for pachyonychia congenita. J Invest Dermatol 2008;128:50– 58. [PubMed: 17762855] 17. Wojcik SM, Bundman DS, Roop DR. Delayed wound healing in keratin 6a knockout mice. Mol Cell Biol 2000;20:5248–5255. [PubMed: 10866680] 18. Wojcik SM, Longley MA, Roop DR. Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b. J Cell Biol 2001;154:619– 630. [PubMed: 11489919] 19. Wong P, Colucci-Guyon E, Takahashi K, Gu C, Babinet C, Coulombe PA. Introducing a null mutation in the mouse K6alpha and K6beta genes reveals their essential structural role in the oral mucosa. J Cell Biol 2000;150:921–928. [PubMed: 10953016] 20. Waterston RH, Lindblad-Toh K, Birney E, et al. Initial sequencing and comparative analysis of the mouse genome. Nature 2002;420:520–562. [PubMed: 12466850] 21. Cao T, Longley MA, Wang XJ, Roop DR. An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy. J Cell Biol 2001;152:651–656. [PubMed: 11157990] 22. Arin MJ, Longley MA, Wang XJ, Roop DR. Focal activation of a mutant allele defines the role of stem cells in mosaic skin disorders. J Cell Biol 2001;152:645–649. [PubMed: 11157989] 23. Kaspar RL. Challenges in Developing Therapies for Rare Diseases including Pachyonychia Congenita. The Journal of Investigative Dermatology Symposium Proceedings 2005;10:62–66. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 7 NIH-PA Author Manuscript 24. de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J. Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007;6:443–453. [PubMed: 17541417] 25. Kim DH, Rossi JJ. Strategies for silencing human disease using RNA interference. Nat Rev Genet 2007;8:173–184. [PubMed: 17304245] 26. Dykxhoorn DM, Lieberman J. Knocking down disease with siRNAs. Cell 2006;126:231–235. [PubMed: 16873051] Biographies NIH-PA Author Manuscript Sancy A. Leachman, MD, PhD, is a tenured Associate Professor in the Department of Dermatology at the University of Utah School of Medicine, chairperson of the International Pachyonychia Congenita Consortium, and Medical Director of PC-Project, a non-profit public charity founded to treat pachyonychia congentia. She studies genetic skin disorders with an emphasis on hereditary melanoma and pachyonychia congenita. Her research focuses on the application of basic science knowledge and state-of-the-art technology to treat these genetic skin disorders. In addition to her work on pachyonychia congenita, Dr. Leachman is Director of the Melanoma and Cutaneous Oncology Program at Huntsman Cancer Institute and directs the Tom C. Mathews Jr. Familial Melanoma Research Program, which is dedicated to investigation of the familial melanoma syndrome. Before joining Huntsman Cancer Institute, Leachman was a resident and post-doctoral fellow in dermatology at Yale University School of Medicine, where she worked on a DNA-based vaccination technology to prevent and treat papillomavirus-induced squamous cell carcinoma. She earned her MD and PhD from the University of Texas Southwestern Medical School, and was awarded the prestigious Doris Duke Clinical Scientist Development Award in 2000. NIH-PA Author Manuscript Roger L. Kaspar, PhD, is the CEO and scientific founder of TransDerm, a company focused on developing novel therapeutics, including inhibitors based on RNA interference (RNAi) technology, for skin disorders. Dr. Kaspar received his doctorate from the University of Washington (Seattle) in biochemistry (David Morris) and performed post-doctoral work at M.I.T. (Lee Gehrke), Stanford University (Helen Blau), and Chiba University (Tomohito Kakegawa). After serving on the faculty at Brigham Young University (Utah) in the Department of Chemistry and Biochemistry, he left academia to work at SomaGenics, prior to founding TransDerm. Drawing on his expertise is in the area of post-transcriptional gene regulation, his current efforts are focused on designing highly potent and selective therapeutic siRNAs that target disease-causing genes in skin disorders, including pachyonychia congenita, and investigating methods to efficiently deliver agents such as siRNAs to appropriate skin cells. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 8 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1. NIH-PA Author Manuscript 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. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 9 NIH-PA Author Manuscript 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. NIH-PA Author Manuscript NIH-PA Author Manuscript J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 10 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 3. NIH-PA Author Manuscript 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. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1. Leachman et al. Page 11 NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 4. NIH-PA Author Manuscript 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. J Dermatol Sci. Author manuscript; available in PMC 2009 September 1.