15 March 2005

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2386 East Heritage Way, Suite B, Salt Lake City, Utah 84109 USA Phone +1-877-628-7300 • Email—[email protected] www.pachyonychia.org DOI: 10.1111/j.1600-0625.2012.01534.x Review Article www.blackwellpublishing.com/EXD

Progress towards genetic and pharmacological therapies for keratin genodermatoses: current perspective and future promise

Jean Christopher Chamcheu, Gary S. Wood, Imtiaz A. Siddiqui, Deeba N. Syed, Vaqar M. Adhami, Joyce M. Teng and Hasan Mukhtar Department of Dermatology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA Correspondence: Prof. Hasan Mukhtar, Helfaer Professor of Cancer Research, Director and Vice Chair for Research, Department of Dermatology, University of Wisconsin, Medical Sciences Center #B-25, 1300 University Avenue, Madison, WI-53706, USA, Tel.: (608) 263-3927; Fax: (608) 263- 5223; e-mail: [email protected]

Abstract: Hereditary keratin disorders of the skin and its appendages discovery targeting pharmacological compounds with the ability to comprise a large group of clinically heterogeneous disfiguring reinforce the compromised cytoskeleton may lead to promising blistering and ichthyotic diseases, primarily characterized by the new therapeutic strategies for treating hereditary keratinopathies. loss of tissue integrity, blistering and hyperkeratosis in severely In this review, we will summarize and discuss recent advances in affected tissues. Pathogenic mutations in keratins cause these the preclinical and clinical modelling and development of gene, afflictions. Typically, these mutations in concert with characteristic natural product, pharmacological and protein-based therapies for features have formed the basis for improved disease diagnosis, these disorders, highlighting the feasibility of new approaches for prognosis and most recently therapy development. Examples translational clinical therapy. include epidermolysis bullosa simplex, keratinopathic ichthyosis, Abbreviations: PC, pachyonychia congenita; EBS, epidermolysis bullosa pachyonychia congenita and several other tissue-specific hereditary simplex; SEI, superficial epidermolytic ichthyosis; KPI, keratinopathic keratinopathies. Understanding the molecular and genetic events ichthyosis; EI, epidermolytic ichthyosis; MAPK, mitogen-activated protein underlying skin dysfunction has initiated alternative treatment kinase,; Hsp/c, heat shock protein/cognate; SF, sulforaphane. approaches that may provide novel therapeutic opportunities for affected patients. Animal and in vitro disease modelling studies Key words: animal models of disease – genetic skin diseases – in vitro have shed more light on molecular pathogenesis, further defining disease models – keratin gene mutation – keratin genodermatoses – pharmacologic and molecular therapies – tissue-engineered human skin the role of keratins in disease processes and promoting the models translational development of new gene and pharmacological therapeutic strategies. Given that the molecular basis for these Accepted for publication 10 May 2012 monogenic disorders is well established, gene therapy and drug

Introduction epidermolytic keratinopathies are characterized by intra-epidermal The cytoskeleton of keratinocytes is basically composed of a tissue fragility, while PC is characterized by painful palmoplantar keratin intermediate filament (KIF) network, which provides (predominantly plantar) keratoderma, hypertrophic nail dystro- major cytoskeleton resilience and mechanical integrity. These phy, and often mucosal leukokeratosis (reviewed in 3). PC-related polymeric KIFs are highly susceptible to pathogenic mutations, keratins; K6a, K6b, K16 and K17 are normally expressed in the and mutations in several keratin genes lead to diverse heritable nail, hair follicle, and epidermis of palmoplantar skin, but not skin fragility disorders. Ever since the early 1990s when there was otherwise in the interfollicular epidermis, unless induced during the first indication that mutations in KIFs (K5 and K14) caused wound healing or hyperproliferative skin diseases such as psoriasis. genetic skin fragility disorders, combined efforts by researchers Other types of keratins, such as K75, K81, K83, K85 and K86, are worldwide have profoundly enhanced our knowledge of the molec- expressed only in the hair shaft and follicles, and are responsible ular basis of a wide range of distinct genodermatoses (Table S1). for keratodermas of the hair and nails (see www.interfil.org; 1–3). These culminated in the availability of prenatal and postnatal The severity of the clinical phenotypes of these major genetic testing, counselling, improved disease diagnosis, reclassifi- keratinopathic genodermatoses can vary quite widely both among cation and most recently promising therapy development individuals and within families, with some demonstrated variation (reviewed in 1–3). Three typical examples of these disorders to be in the degrees of involvement of tissue-specific associated mutant emphasized in this review include the following: (i) the basal keratins (reviewed in 3, 5, 6). epidermolysis bullosa simplex (EBS; K5 and K14 mutations), (ii) The majorities of disease-related keratin mutations are associ- the suprabasal keratinopathic ichthyoses (KPI; formerly called ated with heterozygous missense mutations or small insertion or bullous congenital ichthyosiform erythroderma) such as superficial deletion mutations and are mostly inherited as autosomal-domi- epidermolytic ichthyosis (SEI; formerly called ichthyosis bullosa of nant traits. However, autosomal recessive and mosaic cases have Siemens or IBS; K2 mutations) and epidermolytic ichthyosis (EI; been reported (reviewed in 3). The recessive mutations range from formerly called epidermolytic hyperkeratosis; K1 and K10 muta- splice site dominant-negative mutations, most often predicted to tions) (4) and (iii) pachyonychia congenita (PC; K6a, K6b, K16 lead to nonsense-mediated mRNA decay (NMD), and loss of and K17 mutations) (Fig. 1a–c). Both the basal and suprabasal mutated allele expression (7–9). Analogous to K5 stop codon/

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(a) (b) (c1) (c2) ing, promise to extended the proof-of concept efficacy of gene- based therapy recently demonstrated in reversing the plantar kera- toderma of PC (21). Several of these novel approaches are dis- cussed below. (c3) Animal and in vitro disease modelling as tools for therapy development Genetically modified mice, animal, in vitro tissue culture and bioengineering models have greatly enhanced knowledge of the (d) (e) molecular biology of several genodermatoses. These models faithfully recapitulate the features of human diseases at the clinico-pathological, ultrastructural and molecular genetics levels and represent valuable tools towards the rapid development and preclinical testing of novel gene and pharmacological therapies (reviewed in 22). Animal models for cutaneous keratin disorders Similar to humans, several naturally occuring disease-causing mutations that lead to blistering have been identified in animals, Figure 1. Clinical pictures of typical hereditary cutaneous keratin disorders and such as dogs, horses and sheep. These models have enhanced our cytoskeletal manifestation of the effects of mutations in keratin intermediate understanding of disease pathogenesis (reviewed in 23–25). In filament (KIF) genes. (a) epidermolysis bullosa simplex, mostly caused by dominant- negative mutations in either K5 or K14, expressed in epidermal basal keratinocytes. addition, genetically engineered mouse models have been gener- (b) epidermolytic ichthyosis, caused by mutations in either of the suprabasal ated for the major forms of keratinopathic genodermatoses and epidermal keratins K1 or K10, resulting in widespread hyperkeratosis of the skin. (c) PC, presenting a hererogeneous manifestation, mostly characterized by painful have significantly contributed to our current understanding of the and debilitating focal keratoderma on the sole of patients feet (c1) or oral pathomechanisms of their different forms and also provided leukokeratosis (c2), or hypertrophic nail dystrophy (c3), which are caused by insights into the complex secondary effects mediated by signalling mutations in any of the genes encoding for K6a, K6b, K6C, K16 or K17. Immunofluorescence micrographs of heat-stressed triton-extracted KIF cytoskeleton pathways and other systems that modify disease phenotypes showing normal keratin network in control keratinocytes (d) and mutant keratin (reviewed in 22, 23, 26). Despite the fact that murine models for aggregates in a severe keratin-5 mutant keratinocyte cell line EB11. human diseases often recapitulate the human phenotype, limita- tions are there that they are time-consuming, labour intensive, less truncation mutations in EBS (10, 11), heterozygous nonsense cost-effective, have lack of corresponding human genes in their mutations predicted to lead to the expression of a truncated genome and are often associated with embryonic lethality, which dominant-negative tissue-specific keratin protein that escapes in concert have led to the quest for alternative strategies (23). NMD have been reported as well in these disorders (9, 12). In vitro models of cutaneous keratin disorders Premature termination codon mutations have been reported in Ever since the first cultivation of keratinocytes by Rheinwald and dominant keratinopathies, including K5 and K14 mutations in Green (27, 28) and its application in dermatological research, Dowling–Degos (13, 14) and Naegeli–Franceschetti–Jadassohn several advances have optimized tissue culture systems suitable for syndromes (15), and K16 in PC (5). The reasons behind the gene and pharmaco-toxicological studies. Isogenic, primary and variability in disease/clinical phenotype are not known, but are immortalized cell lines derived from patients with keratin postulated to likely be a combination of genetic, epigenetic and genodermatoses and controls as well as transgenic animals have environmental factors that are associated with patient lifestyle and been invaluable in devising therapies. Such models revealed skin care in the different disorders. clumped KIF in severely keratin-defective cells (Fig. 1d,e) and New knowledge about the genetic basis of several monogenetic have helped uncover disease pathomechanisms, variations in their cutaneous keratin disorders has had a significant impact on their gene expression profiles and promote therapy development (29) diagnosis and therapy (16). Generally, EBS-, EI- and PC-associated and (reviewed in 3). Interestingly, in vitro cell culture and tissue- mutation hotspot codons have been identified, which is a useful engineered human skin disease models that mimic in vivo disease set point for the future development of gene-targeted and phenotypes (e.g. induction of keratin-clumping and cytolysis) have drugtargeted therapies. Most therapeutic goals have been challeng- been successfully established through testing the impact of cellular ing to achieve, in part because of the great complexity of genotype insults, including heat, osmotic and mechanical stresses, on –phenotype relationships, than anticipated previously (17–19). keratin-defective patient-derived cells (reviewed in 3). The skin is an ideal target for both gene and pharmacological These equivalent techniques have now been success- therapy approaches, with the potential for easy accessibility, and fully extended to bio-engineer normal and keratin-defective skin cost-effective monitoring of treatment outcomes. The delivery of (30–34), with the recent development and application of a agents such as small interfering RNA (siRNA), Spliceosome-Medi- humanized murine skin model (21, 31). ated RNA Trans-Splicing technology (SMarT), ribozymes and Humanized murine skin model for preclinical modelling antisense DNA for selective silencing of gene expression and drugs and therapy that enhance tissue integrity and positively modify diseases, Long-term studies of orthotopic and non-orthotopic skin grafting represent novel approaches to improve therapeutic outcomes (20, being problematic owing to graft degeneration (35) have recently 21). Recent technological innovations in animal and in vitro been revolutionalized using an optimized surgical strategy termed disease models, high-throughput drug screening and gene silenc- ‘skin-humanized mouse model’ which utilizes the orthotopic

ª 2012 John Wiley & Sons A/S 482 Experimental Dermatology, 2012, 21, 481–489 Progress towards genetic and pharmacological therapies for keratin genodermatoses bioengineered skin grafting strategy (36, 37). This model allows Current translational therapeutic approaches the generation of a large number of engrafted mice within a Several in vivo and in vitro model systems have provided useful relatively short time frame and consists of systematic deconstruc- molecular details, which now represent focal points for small tion–reconstruction starting with cells isolated from affected molecule discovery. These include the following: (i) modulation of patients’ skin, propagated and assembled into organotypic skin the inflammatory response, (ii) gene modifier effects on keratin equivalents, followed by grafting onto immunodeficient mice. This mutations, (iii) the role of molecular chaperones, ubiquitin–pro- strategy has been utilized to faithfully regenerate normal disease- teasome and MAP kinase systems in disease modification and (iv) free (38) and diseased humanized skin in vivo using patient- induction of compensatory molecules for protein supplementation derived or gene-modified human keratinocytes (37, 39), such as therapies. PC (31) and Junctional-EB cells (40, 41), followed by grafting to Modulation of inflammation immunodeficient mice. Pathogenic mutations in EBS and related disorders are known to Roles of molecular chaperones, ubiquitin–proteasome and cause not only tissue cell fragility, but also local inflammation via MAP kinases in the pathogenesis of keratin genodermatoses the stress responses, as exemplified by a significant upregulation Details of disease modelling and exacerbating conditions are intri- of proinflammatory cytokines in K5 knockout murine skin (57). cate steps towards refining therapy development. A common Physiological concentrations of doxycycline, one of several small feature of hereditary keratinopathic skin and eye disorders is the molecule-based strategies for EBS, increased the survival of occurrence of cytoplasmic keratin aggregates (clumps) in severe K5-deficient mice and reduced inflammation as demonstrated by forms or when induced by trauma. These abnormal KIFs a decrease in the inflammatory cytokine IL-1b levels (26, 57). aggregates are cytotoxic and further compromise cytoskeletal Moreover, doxycycline downregulated the activity of matrix integrity. In several human disorders, chaperones and the metalloproteinase 13 and reduced bulla formation, suggesting EBS ubiquitin–proteasome system have been noted to modulate disease can be ameliorated by reducing local inflammation (26, 57). severity, providing novel therapeutic targets. Furthermore, Small molecule-based modification of disease phenotypes keratinopathic genodermatoses have been proposed to be protein Intriguingly novel approaches have been developed based on small misfolding disorders (34, 42) in which keratin aggregates are remi- molecule drugs with the propensity to activate compensatory niscent of those in other protein misfolding disorders (43, 44). In keratins such as K6, K16 and K17 without injury, oxidative stress fact, keratin aggregates have been shown to interact with activated or UV light and functionally supplement defective keratins. This mitogen-activated protein (MAP) kinases, molecular chaperones approach has been exemplified by two recent strategies for EBS and components of the ubiquitin–proteasome system, as in and PC. protein folding disorders (33, 45–51). In EBS (with K14 mutation), the isothiocyanate sulforaphane Native and activated keratin states are achieved in part by the (SF) was shown to induce K16 and K17 that are complementary specific activities of Hsp70 (HspA1A) and Hsp90(HspC1), to K14. The effects in K14-mutant mouse models of EBS resulted mediated by chaperone cofactors Hip (Hsp70-interacting protein) in reprogramming of keratin synthesis, ameliorated skin blistering and Hop (Hsp70–Hsp90-organizing protein) (Fig. 2; 52). As in and thus restored murine skin integrity (58). SF is a small mole- other protein folding disorders, Hsp70 and/or Hsp90 interact with cule naturally occurring in precursor form in broccoli sprouts and the cytosolic C-terminal region of HIP (CHIP) to conserve cellular other cruciferous vegetables (59), with known cancer chemopre- resources by refolding marked ‘damaged’ proteins (Fig. 2; pathway ventive/therapeutic potential and the ability to enhance 1), and/or link it to the ubiquitin machinery which colocalizes Nrf2-dependent transcription (60). SF-mediated K16 induction with aggregated keratin and Hsp70 in the cytosol. CHIP can also was shown to depend partly on activation of the Nrf2 transcrip- divert mutant keratins for degradation via the ubiquitin–proteaso- tion factor, whereas that of K17 is related to glutathione levels in mal pathway (53), as the turnover of keratin is also regulated by mouse epidermis; both presumably involving MAP kinases (61). the ubiquitin–proteasome system (Fig. 2; pathway 2). In the Interestingly, bioactive SF in broccoli sprout extract have cleared epidermolytic keratin disorders EBS and EI, proteasomal inhibi- phase I clinical trials (62–64) and are now considered safe for use tion resulted in the upregulation of stress-induced Hsp70 and in human skin (65). In PC, K6a is the most commonly mutated Hsp90 in cell cultures and in murine model (33, 46, 54). gene, suggesting that small molecules that can specifically downre- Moreover, overexpression of the ubiquitin ligase CHIP signifi- gulate K6a expression could be therapeutic. In a small molecule cantly accelerated the degradation of mutant keratin in an Hsc70 library screening, cholesterol-lowering statins downregulated the (HspA8) or Hsc/p70-dependent mechanism, positing that Hsc/p70 expression of K6a and a subset of other keratins (66), through the chaperone machinery prime mutant keratins to the ubiquitin–pro- isoprenylation pathway, which is downstream of HMG-coA teasome system for degradation (46). So, activated MAP kinases reductase (enzyme inhibited by statins in the cholesterol biosyn- colocalize with mutant keratin aggregates and Hsp70 in stressed thesis pathway). As discussed previously, K6a expression is also cells compared with control cells, further suggesting a critical role less effectively and less specifically inhibited by retinoids, com- in disease pathogenesis (33, 50). A p38-inhibitor augmented the pared with statins; which is the basis of current consideration of proportion of keratin aggregate-positive cells in the severe EBS the combination of low-dose retinoid and statins as a therapeutic (EB11) cell line, corresponding to the requirement of phosphory- approach by the patient with PC advocacy organization PC lation of keratin aggregates prior to their degradation (46). Several Project (www.pachyonychia.org). Such strategy might be beneficial lines of evidence have implicated the involvement of MAP kinases for other epidermolytic keratinizing diseases such as EI. Other in modelling keratin genodermatoses including the correlation of drug discovery strategies are based on discoveries about biological stress-induced cell death with ERK and JNK activation (55, 56). changes in keratinopathies and could include screens for drugs

ª 2012 John Wiley & Sons A/S Experimental Dermatology, 2012, 21, 481–489 483 Chamcheu et al.

Figure 2. Proposed model showing interplay of molecular chaperones and ubiquitin–proteasome degradation pathways for mutant misfolded keratins and possible targets for therapeutic interventions. The interaction between molecular chaperones and the ubiquitin–proteasome system is a critical step in the response to cytotoxic effects of damaged keratins. Attempts to either conserve cellular resources by refolding and/or preventing toxicity by degrading the aggregated keratins are suggested modulation of either systems (pathways 1 or 2) may provide a unique therapeutic target for keratin diseases. Following nascent inactive mutant keratin polypeptide release from the ribosome, the misfolded keratin is initially recognized by Hsp40 and delivered to Hsp70. Subsequently, depending on the nature of the mutation, a multi-chaperone complex assembles containing the Hsp70 co-chaperone Hip and Hop. A number of co-chaperone networking determines the folding and degradation activities of Hsp70. At the final stage of the chaperone pathway, Hsp90 can associate with diverse binding proteins (such as heat shock protein binding protein 1HspBP1, HIP, HOP, CHIP [via its tetratricopeptide repeat (TPR), etc.] to mediate conformational changes of the misfolded keratin required to activate and refold back. Alternatively when the misfolded keratin protein is marked by phosphorylation, during cell stress and the cytotoxic effects of mutations, the activation pathway is blocked and phosphorylated keratin aggregates are targeted to the proteasome for degradation. This process involves the cochaperone CHIP and the ubiquitin ligases E3 (STUB1) forming the BAG-1 or (STUB1)–Hsp70–CHIP complex (pathway 2) which can also be ensured by increased nuclear translocation of the transcription factor pHSF-1 upon induction. BAG-1/STUB1 associate with the ATPase domain of Hsp70 and mediate interaction of Hsp70 with the proteasome via its ubiquitin-like domain (ubl), whereas CHIP binds to the carboxyl terminus and acts as chaperone-associated ubiquitin ligase to mediate the attachment of a polyubiquitin chain to the chaperone substrate (Hsp70-misfolded keratin aggregates). The misfolded keratin aggregates are targeted and cleared by the ubiquitin–proteasome degradation pathway (pathway 2). Before the tagged aggregated keratins are targeted, they are covalently modified with ubiquitin (Ub) through a series of steps that involve Ub activation (E1), conjugation (E2) and ligation (E3) and require ATP hydrolyses. This ubiquitin then forms substrate for further rounds of ubiquitination, forming the polyubiquitin chain linked by lysines and forms part of the recognition signal for the Ub-tagged aggregated keratins to be shuttled for degradation via ATP-dependent hydrolysis into the 26S proteasome by a set of protein domains such as the ubl domain of BAG-1. In the BAG-1 complex keratins are degraded and reduced to peptides, which are then released into the cytosol and further broken down by peptidases to free amino acids, while the ubiquitin chain are de-ubiquitinated and recycled to free ubiquitin. Most remarkably, BAG-1/STUB1 and CHIP associate with Hsp70 to induce the proteasome degradation of a Hsp70-bound keratin aggregates (pathway 2). When BAG-1 is displaced by binding of HspBP1 to the ATPase domain of Hsp70, the ubiquitin ligase activity of CHIP is attenuated in the formed complex, enabling CHIP to modulate Hsp70 activity without inducing degradation. The ATPase domain can also be occupied by Hip, which stimulates the chaperone activity of Hsp70 and participates in the Hsp70/Hsp90-mediated biochemical pathway regulation. At the same time, Hop displaces CHIP from the carboxyl terminus of Hsp70 and recruits Hsp90 to the chaperone complex. Intervention 1 and 2 are possible therapeutic strategies to reduce the levels of mutant keratin aggregates and cell fragility. 1, By interference-related approaches at the synthesis phase (e.g. by RNAi and SmarT). 2, By development of various small chemical molecules that can modulate molecular chaperones in favour of keratin refolding or degradation by the proteasomal pathways. Finally, the interruption or removal of mutant keratin aggregates in patients with hereditary keratinopathies may allow for the formation of a normally appearing KIF cytoskeleton with intact keratin. that can: (i) reverse the enhanced propensity of keratin-defective compensatory keratins. Further, high-throughput screening of keratinocytes towards apoptosis, as demonstrated by the reversal chemical libraries may uncover new drugs that address these of mitochondrial defects and cardiac dysfunction in desmin-null possibilities for intervention. mice upon cardiac overexpression of the anti-apoptotic Bcl-2 (67); In an effort to reinforce the compromised cytoskeleton (ii) selectively downregulate K6a expression in human resilience, alternative strategies now explore the potential to keratinocytes as demonstrated by the macrolide sirolimus or modulate intrinsic biochemical pathways such as the molecular rapamycin and statins (68, 69); (iii) upregulate partner keratins to chaperone, the ubiquitin–proteasome and MAP kinase machiner- reinforce the compromised cytoskeletal network; and (iv) slightly ies. By employing diverse cell stress models (reviewed in 3), recent downregulate normal wild-type keratins but strongly inducing studies offer a novel chemical chaperone [trimethylamine N-Oxide

ª 2012 John Wiley & Sons A/S 484 Experimental Dermatology, 2012, 21, 481–489 Progress towards genetic and pharmacological therapies for keratin genodermatoses

(TMAO) and 4-phenylbutyrate (4-PBA)]-based therapy approach tions, plantar sweating, prolonged walking and other traumatic for keratin genodermatoses such as EBS and EI (32–34, 42, 50). physical activity (98). Intradermal injection of BTX is a more These small molecules are known to modify other neurodegenera- effective remedy for focal hyperhidrosis because it blocks tive protein misfolding disorders (70–74) and have similarly been peripheral cholinergic nerve terminals (synapses), inhibits the shown to attenuate keratin aggregation and modify keratinopathic release of acetylcholine and blocks autonomic cholinergic genodermatoses in vitro (34, 42). Moreover, 4-PBA also (i) junctions (neuromuscular junction) of the postganglionic reduces the mislocalization or aggregation, and stabilizes proteins sympathetic fibres to the eccrine sweat glands, further decreasing associated with other human diseases (75–79); (ii) remodels hyperhidrosis (99, 100). Thus, patients with PC experienced dry- chromatin (80, 81); (iii) modulates heat shock protein, especially ness and remarkable reduction in pain associated with walking Hsp70 family members and MAP kinases (50); and (iv) degrades when treated with intracutaneous plantar injections of BTX-A mutant keratin aggregates via ubiquitin–proteasome (33, 46) in (101). Later, plantar injection of BTX was also suggested as effica- addition to its other gene modifying effects (70, 82). Intriguingly, cious and safe treatment to reduce painful blistering and callosities 4-PBA is an approved drug in clinical use for the treatment of in EBS (101, 102). Recently, a retrospective study including more urea cycle disorders (83), sickle cell disease and thalassaemia (74, than 14 patients with EBS and PC reported remarkable 84). Furthermore, our recent data suggest that 4-PBA and reduction in plantar pain and blistering upon local injection of TMAO-induced increases in Hsp70, which co-localized with K1 BTX, without any adverse effects except that mild anticholinergic (EI) or phosphorylated K5 (p-K5 in EBS), could possibly prime side effects were observed in two patients (103). A large popula- and facilitate the turnover of chaperone-mediated degradation of tion study is needed to ascertain the prospects of this line of mutated keratins as suggested in Fig. 2. In conformity with this investigation. concept, similar studies have delineated specific effects of chemical Retinoids and their analogues in the treatment of keratin chaperones on protein trafficking in conjunction with altered genodermatoses Hsp70 expression in other disease models (85, 86). In fact, TMAO Retinoids are known regulators of keratinocytes biology (reviewed did not only affect HSPs, but was also shown to activate and in 104). Some topical or oral synthetic retinoids are beneficial in modulate members of the MAP Kinase signalling pathways managing certain hereditary cutaneous keratinopathies including in EBS, suggesting a critical protective effect that involves EI, PC and PPK. In hyperkeratotic keratinopathies, alternative MAP Kinases (50). Colocalization of keratin aggregates with treatment options include emollients containing glycerine, lactic phosphorylated p38 (45), suggested the association of acid, urea, and other topical and systemic retinoids. While TMAO-induced keratin aggregates reduction and the activation of some patients respond well to synthetic retinoids and calcipotriol both Hsp70 and phospho-p38 (see Fig. 2). In this light, (a vitamin D3-analogue; 105, 106), orally administered etretinate pharmacological or other interventions aimed to accelerate the has been more successful in reducing thickening, but less effective turnover of aggregates may be therapeutically beneficial in at decreasing erythroderma. Importantly, several keratin gene hereditary keratin disorders (87). promoters such as the KRT6a gene contain retinoic acid response Small molecules development for clinical therapies elements (66), implying retinoids can downregulate the expression Other small molecule-based topical and or systemic therapies of these genes. A proof-of-concept study has demonstrated the include corticosteroids, cyproheptadine and antibiotics (88–90). upregulation of K4 with concomitant decrease in K2 levels in reti- Similar to doxycycline discussed earlier, there are anecdotal noid-treated EI epidermis in vivo (106). Although retinoids are reports of successful treatment with another anti-inflammatory broad spectrum in their action, evidence suggests that retinoid agent, tetracycline (91, 92), which may prevent tumor necrosis therapy could be more beneficial to EI patients with K10 rather factor alpha release or modulate the activity of metalloproteinases, than those with K1 mutations (106). Recently, using a 3D epider- to reduce bulla formation. However, a randomized trial did not mal model, retinoic acid receptor-a (RAR) agonists were reported reveal any difference between antibiotic and placebo-treated to be potent modulators of keratin 1, 2, 4 and 13 expressions groups (93). Aminoglycosides family antimicrobials may lead to (107), suggesting their use for patients with K1 and K2 mutations, translation of proteins in individuals with premature termination the latter seen in patients with superficial KPI (SEI; formerly codon mutations by transcript read through. Patients with PC treated called ichthyosis bullosa of Siemens). Their efficacy for EI therapy with rapamycin have resulted in symptomatic improvement of might be achieved via a mechanism that involves the upregulation painful plantar calluses (reduced pains and callus), suggesting oral of compensatory K4 and/or K13 proteins which may functionally or perhaps topical rapamycin or its derivatives as a therapeutic replace the mutated keratin molecules (107). It is important to option (69). investigate the effects of retinoids with affinities for different RARs Botulinum toxin type A (BTX-A) therapy on different tissue-specific keratins that might correlate with the Although topical antisweating agents, such as aluminium chloride effect on keratin expression reported in reconstituted epidermal and glutaraldehyde (94, 95) initially seemed promising for the equivalents (107). In fact, exogenous retinoid acid was shown to hyperhidrosis of localized patients with EBS (94, 95), later studies covalently bind K16 and K10 in mouse skin (108), raising the showed no benefit (96, 97). The commercially available BTX-A, is possibility that the binding of retinoid to keratins may be involved currently being evaluated as a potential therapy option in inher- in the retinoid-induced consolidation of keratin cytoskeleton ited epidermolytic keratin genodermatoses such as in EBS and PC, resilience in the epidermis; this possibility can be tested by because patients with prominent blisters and plantar callosities treating murine skin with potent RAR agonists that have an often have plantar hyperhidrosis (see Fig. 1c). Moreover, disease affinity for the different -isoforms. Although the pathomecha- manifestations are often exacerbated during hot and humid condi- nisms differ, their phenotypic expressions might share a common

ª 2012 John Wiley & Sons A/S Experimental Dermatology, 2012, 21, 481–489 485 Chamcheu et al. signalling pathway. Retinoids are known to inhibit epidermal centrations of siRNA directed against a K6a mutation were injected differentiation program causing skin irritation, fragility and into the plantar skin of a patient with PC, showed an excellent tenderness, and most patients do not tolerate long-term retinoid result with a significant decrease in plantar keratoderma (21). therapy. This has led to the need for newer, more specific drugs An alternative approach relies on exploring the functional that mimic the effect of retinoids with minimal or no side effects redundancy of keratins in tissues affected by keratin mutation or teratogenicity as exemplified by retinoic acid metabolism (121), including EBS, EI and PC. Mouse knockout studies on blocking agents (RAMBAs). KRT6A null mice, have shown that KRT6B compensate for Retinoic acid metabolism blocking agents decreased KRT6A, suggesting that complete silencing of the RAMBAs are a new group of retinoid-like drugs that block defective keratin, could be an alternative approach that has more intracellular breakdown of endogeneous RA, thus mimicking the global therapeutic application than the development of siRNAs effects of exogeneous retinoic acid (RA). The mechanism of that target individual point mutations (122, 123). Based on this action is largely through inhibiting CYP26, the enzyme responsi- concept, gene-specific siRNA has been developed and will shortly ble for the degradation of RA (109). Upon inhibition by RAM- be in clinical trials for treating the most common form of PC BAs, the intracellular all-trans-RA concentration rises to (related to KRT6A mutations; 114). therapeutic values, but primarily in the skin where all-trans-RA is SMarT technology metabolized by the CYP-dependent 4-hydroxylase. Therefore, Spliceosome-mediated RNA trans-splicing (SMaRT) is another RAMBAs are able to provide retinoid-like effects in skin with less silencing technique and uses the endogenous spliceosome machin- systemic side effects and post-treatment teratogenicity (110) and ery to effectively excise mutant exons, knocking out the mutant have proven clinically promising in a limited number of patients protein in affected cells. Wally et al. (124) reported gene expres- with keratinization disorders (109, 111). One such agent, liaroz- sion repair by SMarT, resulting in replacement of a mutation of ole, which inhibits CYP-dependent hydroxylation of all-trans-RA, the plectin gene in EBS, a strategy that has also been used to suc- has been granted orphan drug status for congenital ichthyosis. In cessfully correct collagen XVII (125) mutations. Recently, SMarT patients with ichthyosis, clinical trial of liarozole versus acitretin was used to specifically replace the first seven exons of the K14 (synthetic retinoid) showed mostly mild to moderate RA-related gene in an EBS-DM patient cell line (20). Thus, this approach adverse effects that tended to occur less frequently in the liaroz- appears a promising tool for gene therapy of several autosomal- ole-treated group, suggesting that liarozole is equally effective for dominant blistering genodermatoses because it clears the mutation ichthyosis as acitretin but showed a trend towards a more favour- while increases normal allele expression. Efforts are underway to able tolerability profile (109). increase the efficiency of SmarT therapy for clinical application Corrective gene therapy approaches (126). Owing to the dominant-negative effect most keratin gene Protein supplementation and cell-based therapy approaches mutations exert in these diseases, a conventional gene therapy A decade ago, scientists considered upregulating the expression of approach would likely be ineffective. Although increasing the ratio another intermediate filament (IF) protein to compensate for of normal keratin protein to mutant protein ameliorates the kera- keratin gene mutations. In cultured keratinocytes with dominant- tin network appearance and decreases cell fragility (19), most, negative K5 and K14 mutations, transfection with desmin, an IF strategies have focused on selective inhibition of mutant alleles normally expressed in muscle cells, restored the wild-type response (112–114). Technologies, such as short inhibitory RNAs (siRNAs) to stress (127, 128). The ectopic expression of desmin in and spliceosome-mediated RNA trans-splicing (SMarT), are the transgenic mouse epidermis resulted in the formation of a parallel most promising approaches that offer the best chances and enable and normal desmin filament network, although it did not rescue inhibition of expression of the mutant alleles without affecting the K5 null mutant phenotype (129). wild-type gene expression (17, 20, 112, 113, 115–118). Furthermore, mouse models of EBS and EI demonstrated that a SiRNA technology 50% reduction in mutant allele expression prevents blistering and Small or short inhibitory RNA (siRNA) technology was effectively normalizes skin function (19, 130). Despite the possibility that the used to downregulate mutant K6a (116) and K14 (119) allele therapeutic threshold could be greater in humans, these findings expressions in cultured PC and EBS cells respectively. The mutant- suggested the potential benefit of suppression of a mutant allele specific siRNAs led to the formation of normal keratin filaments, without increasing normal gene expression. Recessive EI disease reflecting the selective inhibition of mutant K6a in a cell culture provides evidence that knockdown to 50% of normal levels does model for PC (113). Owing to its accessibility to therapeutic not cause disease (e.g. no haploinsufficiency) as in the normal skin agents, the skin represents an attractive target for nucleic phenotype in heterozygous carriers of keratin gene defects (131, acid-based therapies (120). Using an organotypic model, Hickerson 132). The normal phenotype in KRT6A null mice, however, et al. (30) showed enhanced delivery of siRNA to inhibit gene suggests that KRT6B compensates for KRT6A’s absence, likewise, expression. Moreover, missense point mutations in the affected the observation of milder disease phenotype upon overexpression alleles of keratinopathies were silenced with siRNA, allowing of wild-type K15 in a kindred with recessive EBS because of expression of functional wild-type allele (112, 113, 116). The ablation of K14 (133). Most recently, it has been demonstrated development and optimization of this mutation-specific siRNA using K14-null mutant cells that the stress responses associated therapy approach has now progressed to an efficacious small-scale with keratin disorders can be ameliorated when cells were rescued human clinical trial, resulting in the first-in-human siRNA by the addition of wild-type keratin protein (134). Little is known treatment of a skin disorder (21). Although the strategy is not about the effect of replacing wild-type protein in cells with currently in clinical practice, this study, in which increasing con- dominant-negative KRT mutations.

ª 2012 John Wiley & Sons A/S 486 Experimental Dermatology, 2012, 21, 481–489 Progress towards genetic and pharmacological therapies for keratin genodermatoses

Induced pluripotent stem cells (iPSCs) and Revertant gene correction agents poorly penetrate cellular membranes have Mosaicism therapies low bioavailability and are formulated in undesirable solvents. The The emergence of regenerative medicine technological innovation use of nanocarriers permits the preparation of low water soluble where cells, such as dermal fibroblasts, are converted to induced medications as solid or liquid formulations which should be useful pluripotent stem cells (iPSCs), with potential to later differentiate for treating keratin genodermatoses. So far no natural, or synthetic to any cell type in the body has opened new avenues of therapeu- agents excepted have been nano-formulated for genodermatoses. tic approaches (reviewed in 23). This strategy is potentially able to However, siRNA gene correction agents are currently being nano- develop patient-specific multiremedy strategies for genodermatoses. formulated for genodermatoses. It is hoped that the development It mainly rely on ectopic expression of a limited number of tran- and implementation of nanotechnology will greatly enhance and scription factors that can reprogramme somatic cells into a tran- advance the treatment of genodermatoses and are being tested in sient embryonic stem cell state. These transient cells express cell and animal models. embryonic stem cell markers and possess both unlimited prolifera- Detailed overview of this growing field of nanotechnology/ tive capacity and the ability to differentiate into the ectoderm, nanomedicine and their therapeutic applications in dermatology is mesoderm and endoderm germ layers. beyond the scope of this review. Considerable effort has been The patient-specific iPSCs (PS-iPSCs) have now been generated generated in determining therapeutic efficacy and potential from several human diseases including heritable skin disorders negative side effects. It is thought that in healthy skin, nanoparti- (reviewed in 23), It has been demonstrated that both normal and cles delivery pose minimal health risk because they mostly utilize patient-specific (PS) iPSCs can be differentiated directly into biodegradable particles (142, 143). Even as the stability and low functional keratinocytes, with specific surface markers being used toxicity of some nanoparticles have been exploited in several to purify iPSC-derived keratinocytes and allowing for enrichment setting, the current knowledge of their toxicity effects is compara- of keratinocyte lineage cells (135). Further availability of reliable tively inadequately documented (144–147). However, available and mutation free non-viral integration of transcription factors data suggest nanoparticles can bypass the protective barriers, being will provide relevance prior to clinical application (23). Therefore, distributed and accumulate in several organs, with documented there is great promise that iPSCs may provide unprecedented toxicity in several organ systems excluding the skin (144, 148). sources of cells for heritable skin keratinopathies as well. The mechanistic adjuvant and other effects for skin barrier-defec- Revertant Mosaicism (RM) or ‘natural gene therapy’, is a tive diseases are yet to be addressed as there is limited data on naturally occurring phenomenon whereby a subpopulation of nanoparticle penetration, transport and their interactions with somatic cells spontaneous reverts to the wild-type phenotype via diseased skin. Much emphasis on quantitative studies in relation crossover or conversion which alleviates the requirement of to dose-exposure to penetration and therapeutic efficacy, as well further gene correction(136, 137). RM is now well known to not as consistency in detection sensitivity of techniques used is being a rare event and has been observed in several patients, needed. A plethora of unanswered questions emphasizes the including several types of EB (137). In heritable keratinopathies as technical challenges needed for translation of nanotechnology to revertant skin is visible and easily accessible, RM has been clinical dermatology, thus pointing to the great opportunity for reported in cases of Ichthyosis with confetti because of autosomal- further investigative studies in this arena. dominant KRT10 mutation (138); EBS because of autosomal Conclusions and future prospects recessive KRT14 mutation (139) and in severe EBS-DM This review reflects the research progress made in molecular (EBS-Dowling-Meara) with autosomal-dominant KRT14 mutation genetics and pharmacological therapy for treating inherited (140). This therefore holds promise as it may serve as a natural, cutaneous keratin disorders. Despite vigorous worldwide research patient-specific source of gene-corrected cells for its usage of efforts that have uncovered the pathophysiology of keratin revertant keratinocytes for revertant cell therapy or iPSC to genodermatoses, many treatment questions remain unanswered. provide autologous regenerative source. Progress in successful therapy development has been challenging Nanotechnology for treating keratin genodermatoses and prospects are improving, but patient management remains Several hurdles exist in developing novel agents including essentially symptomatic and palliative (97). These remedies mainly determining their optimal therapeutic combinations and effective involve prevention of secondary bacterial infections, management delivery of agents to target tissue at concentrations that achieve of blisters and wounds, and minimization of trauma and pain efficacy without toxicity. with techniques mostly relying on the individual clinician’s Nanotechnology is a relatively new and rapidly evolving field personal experience. Novel strategies include gene therapy, protein thatmaybeapplicableforthetreatmentofkeratingenodermatoses, replacement, iPSCs and pharmacological therapies, some of which as it may enhance the delivery, bioavailability and also limit any have already entered the clinical arena of dermatology. The unwanted toxicity of therapeutic agents. The structure and tunable combination of animal, in vitro and bioengineered humanized skin surface functionality of nanoparticulate systems allows them to murine approaches are relevant pathophysiological models encapsulate/conjugate single or multiple agents either in the core that are helping scientists to address new therapeutic strategies or on the surface, rendering them ideal carriers for various drugs. (38, 149), including options that attempted to neutralize the Recently, we employed nanotechnology in the field of cancer to cytotoxic effects of keratin aggregates by focusing on ameliorating improve the outcome of chemoprevention also known as ‘nano- tissue-specific damage, while maintaining normal skin function. chemoprevention’ (141). Nanotechnology may also be exploitable Pharmacological agents, for example, 4-PBA approved for clinical as an efficient delivery system for delivering siRNA and small mol- use in other human diseases, and statins, approved cholesterol- ecules for the treatment of keratin genodermatoses. Most drugs or lowering drugs are promising candidate drugs from chemical

ª 2012 John Wiley & Sons A/S Experimental Dermatology, 2012, 21, 481–489 487 Chamcheu et al. libraries for keratin disorders. Moreover, gene therapy using RNA Acknowledgements interfering agents, such as siRNA, SMarT, ribozymes, and Part of this review was presented in a lecture on May 4 at the 8th annual antisense DNA, may improve outcomes either alone or in Symposium of International Pachyonychia Congenita Consortium (IPCC) combination with drug therapy. Despite the exciting proof-of-con- during the 2011, 76th Annual Meeting of the Society for Investigative Der- cept studies showing the potential value of siRNA technology for matology, Phoenix, AR, by JCC. The first author’s original work in this keratin gene disorders, major technical hurdle persist such as the area was supported by grants from the Swedish Research Council (K2008- development of safe, effective pain-free techniques for routine 0731x), the Welander and Finsen Foundations and Uppsala University to delivery into the epidermis. The current goal is to explore other Professors Anders Vahlquist & Hans Torma. Work in HM‘s laboratory was potential delivery systems including chemical modification of the supported by US PHS Grants RO1 AR059742, T32AR055893 and ACS siRNAs, modulation of the chemistry of topical formulation, grant 120038-MRSG-11-019-01-CNE. JCC performed review of literature, microneedle arrays and nanoparticles technology approaches. writing and preparation of the manuscript. Clinical pictures on Fig. 1 were The field of nanomedicine is pursuing new strategies to deliver provided by JMT and Pachyonychia Congenita Project (www.pachyonychi- oligonucleotides topically in nanoparticle emulsions or micelles a.org). GSW, IAS, DNS, VMA JTM and HM contributed in the drafting, that can traverse the epidermal barrier and improve pharmacoki- critical analysis, revising and approval of the final version of the review netics and reduce associated side effects. Major advances in stem manuscript for publication. We thank Dr. Frances JD Smith (Nine well cell technology, particularly iPSCs, hoped that should allow Hospital, University of Dundee, UK) for her valuable comments and advice lifelong expression of introduced genes. Future approaches will regarding this manuscript and to Dr. Amy S Paller (Departments of combine an understanding of the molecular basis of Dermatology and Pediatrics, Feinberg School of Medicine, Northwestern genodermatoses with advanced technologies such as iPSCs, siRNA University, Chicago, IL) for critical reading and offering suggestions during and nanomedicine. Ultimately, personalized medicine through the course of preparation of the manuscript. specific correction of an individual’s keratin gene defects is the Conflict of interest optimal way to correct the problematic skin fragility and The authors declare no conflicts of interest with respect to the preparation uncomfortable thickening, thus, improving the quality of life of and publication of this article. patients with keratin genodermatoses.

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