Small-Molecule Therapies for Genetic Skin Fragility
Authors: Leena Bruckner-Tuderman
Journal: Molecular Therapy (2014)

Of the known genetic skin diseases, dystrophic epidermolysis bullosa (DEB) is a prime target for therapy development. It is a monogenic, strongly disabling disorder with high unmet medical need. Lifelong generalized skin blistering as a consequence of minor mechanical friction, chronic wounds, excessive scarring, and progressive soft-tissue fibrosis are clinical hallmarks. Joint contractures and mitten deformities of the extremities cause severe disability, and the fibrosis of the skin increases tissue stiffness and favors development of aggressive squamous cell carcinoma, a feared complication of the disease.1Fine J-D Bruckner-Tuderman L Eady RAJ Bauer EA Bauer JW Has C et al.The classification of inherited epidermolysis bullosa (EB): report of the Fourth International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar For obvious reasons, many investigators have pursued curative gene-, cell-, and protein-based therapy approaches for DEB, but these have turned out to be much more challenging than anticipated and have shown very limited benefit in pilot clinical trials.2Bruckner-Tuderman L McGrath JA Robinson EC Uitto J Progress in epidermolysis bullosa research: summary of DEBRA International Research Conference 2012.J Invest Dermatol. 2013; 133: 2121-2126Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar In the recent past, it has become clear that alternative approaches are urgently needed to improve the health and quality of life of severely affected patients. In line with this, Cogan et al.3Cogan J Weinstein J Wang X Hou Y Martin S South AP et al.Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.Mol Ther. 2014; 22: 1741-1752Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar report in this issue on the use of aminoglycosides to induce readthrough of premature termination codons (PTCs) and to restore functional protein production in DEB keratinocytes and fibroblasts in vitro. DEB is caused by mutations in the COL7A1 gene, which encodes collagen VII, the major component of anchoring fibrils, which attach the epidermis to the dermis and are pivotal for the integrity of the skin. All clinical forms of DEB—mild and severe, recessive (RDEB) and dominant—are allelic disorders caused by different mutations in the COL7A1 gene. To date, more than 700 distinct mutations have been reported, and homozygous, heterozygous, and compound heterozygous mutation constellations produce a broad spectrum of phenotypes.4Has C Bruckner-Tuderman L The genetics of skin fragility.Annu Rev Genomics Hum Genet. 2014; 15: 16.1-16.24Crossref Scopus (52) Google Scholar It is estimated that 10–25% of disease-causing COL7A1 mutations represent nonsense mutations that generate a PTC. Cogan et al.3Cogan J Weinstein J Wang X Hou Y Martin S South AP et al.Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.Mol Ther. 2014; 22: 1741-1752Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar treated cultured primary fibroblasts and keratinocyte cell lines derived from RDEB skin with three different aminoglycosides (geneticin, gentamicin, and paromomycin) to suppress PTC mutations and to induce collagen VII expression in the cells. The treatment resulted in the synthesis and secretion of collagen VII in both cell types, reaching 15–30% of expected normal levels. The effects were dose-dependent and reached a peak about one week after the treatment, tapering off thereafter, but collagen VII protein could still be observed in the cultures 14 days after the treatment. Functional tests demonstrated that the abnormal migratory phenotype of collagen VII–deficient cells improved after aminoglycoside treatment, and in organotypic skin-equivalent cultures constructed with drug-treated RDEB keratinocytes and fibroblasts, collagen VII was deposited at the epidermal–dermal interface, suggesting that the neosynthesized collagen may have normal structure and functions. The suppression of PTC was also demonstrated in HEK293 cells transiently transfected with constructs containing 22 known RDEB nonsense mutations. After aminoglycoside treatment, collagen VII synthesis reached 15–30% of expected physiological levels—a positive finding. Preclinical studies in mice and genotype–phenotype correlations in humans have shown that the quantity of collagen VII and the density of anchoring fibrils in the skin determine the extent of its integrity.1Fine J-D Bruckner-Tuderman L Eady RAJ Bauer EA Bauer JW Has C et al.The classification of inherited epidermolysis bullosa (EB): report of the Fourth International Consensus Meeting on Diagnosis and Classification of EB.J Am Acad Dermatol. 2014; 70: 1103-1126Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar,5Fritsch A Loeckermann S Kern JS Braun A Bösl MR Bley T et al.A hypomorphic mouse model for dystrophic epidermolysis bullosa reveals disease mechanisms and responds to fibroblast therapy.J Clin Invest. 2008; 118: 1669-1679Crossref PubMed Scopus (169) Google Scholar We have shown that about 30–35% of physiological collagen VII levels provide reasonable skin integrity in mouse models.5Fritsch A Loeckermann S Kern JS Braun A Bösl MR Bley T et al.A hypomorphic mouse model for dystrophic epidermolysis bullosa reveals disease mechanisms and responds to fibroblast therapy.J Clin Invest. 2008; 118: 1669-1679Crossref PubMed Scopus (169) Google Scholar,6Nyström A Velati D Mittapalli VR Kern JS Fritsch A Bruckner-Tuderman L Collagen VII plays a dual role in wound healing.J Clin Invest. 2013; 123: 3498-3509Crossref PubMed Scopus (132) Google Scholar These data are important from the point of view of therapy development for completely collagen VII–deficient DEB: it is reassuring that a 100% restoration of collagen VII will not be required for improvement of the phenotype. The precise structure and functionality of collagen VII synthesized in aminoglycoside-treated RDEB cells in vitro remain elusive at present. Under physiological conditions, the collagen is expressed in and secreted by both keratinocytes and fibroblasts, and it undergoes a series of post-translational modification/maturation steps during protein folding into triple-helical collagen and supramolecular assembly into functional anchoring fibrils. Although Cogan et al.3Cogan J Weinstein J Wang X Hou Y Martin S South AP et al.Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.Mol Ther. 2014; 22: 1741-1752Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar undertook preliminary characterization of the newly synthesized collagen VII using immunofluorescence and immunoblotting assays, future studies using powerful proteomics and structural biology techniques will have to provide accurate data on detailed structure–function relationships of the aminoglycoside-induced collagen. It has been about 15 years since the first proof of concept for treating genetic disorders resulting from PTC mutations was reported for muscular dystrophy.7Barton-Davis ER Cordier L Shoturma DI Leland SE Sweeney HL Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice.J Clin Invest. 1999; 104: 375-381Crossref PubMed Scopus (496) Google Scholar Since then, the approach has been validated in vitro and in animal models, such as zebrafish or mouse, for a number of disorders including muscular dystrophies, cystic fibrosis, and xeroderma pigmentosum.8Bidou L Allamand V Rousset J-P Namy O Sense from nonsense: therapies for premature stop codon diseases.Trends Mol Med. 2012; 18: 679-688Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,9Li M Andersson-Lendahl M Sejersen T Arner A Muscle dysfunction and structural defects of dystrophin-null sapje mutant zebrafish larvae are rescued by ataluren treatment.FASEB J. 2014; 28: 1593-1599Crossref PubMed Scopus (38) Google Scholar,10Kuschal C DiGiovanna JJ Khan SG Gatti RA Kraemer KH Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons.Proc Natl Acad Sci USA. 2013; 110: 19483-19488Crossref PubMed Scopus (45) Google Scholar However, translation into human therapies has been limited by the well-known toxicity of this group of drugs or lack of efficacy.8Bidou L Allamand V Rousset J-P Namy O Sense from nonsense: therapies for premature stop codon diseases.Trends Mol Med. 2012; 18: 679-688Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar One reason for the latter may be the fact that the molecular mechanisms of translation termination and nonsense-mediated messenger RNA decay (NMD) are not yet fully understood. Although the core NMD machinery is evolutionarily highly conserved among species, and it is known that the specific nucleotide context in the environment of the PTC plays an important role, additional complexity of these processes is emerging. Different models of PTC recognition and NMD that are employed depending on the exon–intron structure of the specific genes have been presented (for review, see ref. 8Bidou L Allamand V Rousset J-P Namy O Sense from nonsense: therapies for premature stop codon diseases.Trends Mol Med. 2012; 18: 679-688Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The family of collagen genes, including COL7A1, encompasses 42 large multiexon genes that are associated with a number of genetic disorders in many tissues.11Myllyharju J Kivirikko KI Collagens, modifying enzymes and their mutations in humans, flies and worms.Trends Genet. 2004; 20: 33-43Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar So far, published data on PTC recognition and NMD associated with mutations in collagen genes support two fundamentally different mechanisms: (i) NMD dependent on the exon junction complex in the case of collagens I and VI and (ii) PTC recognition with the involvement of the 3′-untranslated region in the case of collagen X.12Fang Y Bateman JF Mercer JF Lamandé SR Nonsense-mediated mRNA decay of collagen—emerging complexity in RNA surveillance mechanisms.J Cell Sci. 2013; 126: 2551-2560Crossref PubMed Scopus (26) Google Scholar No information exists on these mechanisms in relation to COL7A1 nonsense mutations. Therefore, the applicability of the current PTC recognition and NMD models for the collagen VII gene and, consequently, corresponding therapeutic strategies remain unclear.12Fang Y Bateman JF Mercer JF Lamandé SR Nonsense-mediated mRNA decay of collagen—emerging complexity in RNA surveillance mechanisms.J Cell Sci. 2013; 126: 2551-2560Crossref PubMed Scopus (26) Google Scholar The in vitro study by Cogan et al.3Cogan J Weinstein J Wang X Hou Y Martin S South AP et al.Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.Mol Ther. 2014; 22: 1741-1752Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar raises cautious optimism for biologically valid treatments of RDEB. The application of specific PTC readthrough compounds targeting the individual nonsense mutation constellation of the patient holds promise for noninvasive drug therapies, which would, of course, be highly desirable for this intractable disease. Such treatments might benefit a subset of patients with RDEB carrying certain nonsense mutations—perhaps up to a tenth of the affected individuals—and improve their skin integrity and quality of life. However, substantial hurdles lie ahead and must be overcome before clinical applications can become realistic. First, the specific molecular basis of NMD and PTC readthrough associated with COL7A1 nonsense mutations will have to be elucidated, and maximally specific aminoglycosides or aminoglycoside-like compounds identified for each individual patient. Second, given that PTC readthrough introduces an amino acid in lieu of the stop codon, the effects of the substitution must be assessed in light of the fact that single amino acid substitution mutations in collagen VII are also known to cause DEB, albeit milder phenotypes. Third, the toxicity of aminoglycosides poses a serious challenge. Because RDEB manifests at birth and progresses steadily, treatment must be initiated at an early age. Indeed, adverse effects of a long-term treatment in children may pose insurmountable problems, if no alternative molecules facilitating PTC readthrough will be available. The option of topical application of aminoglycosides may seem attractive at first glance but may still exhibit toxicity and not suffice to repair internal mucosal lesions and extracutaneous organ involvement in RDEB. Therefore, alternative systemic agents should be sought. Several aminoglycoside derivatives or nonaminoglycoside compounds that facilitate PTC readthrough without the toxicity of aminoglycosides have been identified, and current screening procedures are likely to discover new compounds.8Bidou L Allamand V Rousset J-P Namy O Sense from nonsense: therapies for premature stop codon diseases.Trends Mol Med. 2012; 18: 679-688Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar Of these, PTC124, or ataluren, has been extensively investigated and seemed promising to stop progress of muscular dystrophies and cystic fibrosis in preclinical disease models.13Peltz SW Morsy M Welch EM Jacobson A Ataluren as an agent for therapeutic nonsense suppression.Annu Rev Med. 2013; 64: 407-425Crossref PubMed Scopus (135) Google Scholar Not all of the phase II and III clinical trials have been conclusively evaluated (http://www.ptcbio.com/clinical_trials), but it seems that the drug has a good safety profile and improves some symptoms,13Peltz SW Morsy M Welch EM Jacobson A Ataluren as an agent for therapeutic nonsense suppression.Annu Rev Med. 2013; 64: 407-425Crossref PubMed Scopus (135) Google Scholar although not all patients benefit from the treatment.14Kerem E Konstan MW De Boeck K Accurso FJ Sermet-Gaudelus I Wilschanski M et al.Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial.Lancet Respir Med. 2014; 2: 539-547Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar Presumably, as delineated above, future therapies must be applied on an individualized basis, selecting patients with optimal mutation and PTC recognition constellations that respond to a particular drug and its mode of action. Of note, in the study by Cogan et al.,3Cogan J Weinstein J Wang X Hou Y Martin S South AP et al.Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.Mol Ther. 2014; 22: 1741-1752Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar all DEB cells and expression constructs carrying COL7A1 nonsense mutations were refractory to PTC124 treatment. Therefore, it is likely that novel compounds with different specificities, or novel strategies to block NMD, will need to be found for clinically applicable drug therapies for DEB. Hopefully, continually increasing insights into the molecular mechanisms of PTC recognition and NMD,8Bidou L Allamand V Rousset J-P Namy O Sense from nonsense: therapies for premature stop codon diseases.Trends Mol Med. 2012; 18: 679-688Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,13Peltz SW Morsy M Welch EM Jacobson A Ataluren as an agent for therapeutic nonsense suppression.Annu Rev Med. 2013; 64: 407-425Crossref PubMed Scopus (135) Google Scholar in general and with collagen genes, will lead to discovery of small-molecule compounds with true therapeutic potential for DEB.