*Address correspondence to: [email protected] #, ¶ Equalcontribution. 5. Leiden University Medical Center, Department of Human Genetics, Leiden, the Netherlands. Groningen,Netherlands. the 4. University ofGroningen, University Medica Groningen,Netherlands. the Dermatology, of Department Groningen, Center Medical University Groningen, of University 3. Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria. 2. EBHouseAustria, Research Program forMolecular Therapy ofGenodermatoses, Department of 1. Department ofDermatology, Medical Center – Affiliations: This article is protected by copyright. All rights reserved. 10.1111/exd.13141 differences between this version and the Version of Record. Please cite this article asdoi: through the copyediting, typesetting, pagination and proofreading process, which may leadto This article has been accepted for publication and undergone full peer review but has not been Olivier Bornert genodermatoses for therapies RNA-based Article type : Review Article 29-Jun-2016 : Date Accepted :16-Jun-2016 Date Revised :28-Oct-2015 Date Received Accepted ArticleAnnemieke Aartsma-Rus 1 , Patricia Peking 5¶ , AnnaM.G.Pasmooij, 2# , Jeroen Bremer 3¶ University ofFreiburg, Freiburg, Germany. , Eva M.Murauer l Center Groningen, Department ofGenetics, 3# , Ulrich Koller , Ulrich 2¶ 2 , Alexander Nyström , Peter C. van denAkker 1¶ * 3,4¶ , Introduction of patients and families affected by agenodermatosis. of this review is to set the stage for the development of new and better therapies toimprove the lives critically discuss their opportunities, limitations and the challenges that remain to besolved. The aim antisenseand oligonucleotides. Wepresent thecurrent state ofthese therapeuticapproaches and and genome editing by splicing-modulation by antisense oligonucleotides and RNA are emerging asapowerful tool to treat genodermat diseases, forwhich treatment isgenerally limited tomanagement ofsymptoms.RNA-based therapies Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of Abstract file; 5 supplementary figures. Manuscript information: interfering RNA. recessive dystrophic epidermolysis bullosa; RTM, pre-mRNA dystrophy; IVT mRNA, oligonucleotide;AON, antisense DEB,dystrophic epidermolysis DMD, bullosa; Duchenne muscular Abbreviations This article is protected by copyright. All rights reserved. Accepted ArticleKeywords: Skin, antisenseoligonucleotides,

in vitro in vitro word count 3943; references 89; 1 table; 1 figure; 1 Supplementary text transcribed mRNAs,and gene knockdown by small interfering RNA transcribed mRNA; PTC, premature termination codon; RDEB, in vitro transcribed mRNA, RNA siRNA, transcribed oses. In this review, wediscuss in detail RNA

trans trans -splicing molecule; siRNA, small -splicing, transcript-replacement trans -splicing. vitro Further, the large area that has to betreated, the avascula This article is protected by copyright. All rights reserved. Acceptedgenodermatoses and may appear tobe challenging. 17). However, the therapeutic potential of direct cell therapy (15). The technique has already beenused tosuccessfully correct skin cells promising approach (10-14). In addition, genome editing isemerging as a powerful tool for gene and diseases (8, 9). At present, transplantation of gene-corrected skin grafts seems to be the most viral vector capacity) are limitations that impede development ofgene therapy approaches for skin in vivo, viral vectors have forcertain organs (7). The limited number ofvectors efficiently targeting skin cells defective gene into affected organs. To achieve this, these approaches utilize the specific tropisms viralClassic vector-based gene replacement therapies aim correct cDNA the introduce of to copies genome editing, protein replacement, cell-based and RNA-based therapies. Therapies targeting the cause ofgenetic diseases can bedivided into five groups: gene replacement, Article anchoring structures andwell-developed cells accessibility ofthe skin, the ability toculture skin development oftherapies for genodermatoses has its specific advantages andchallenges. directThe impedes the development of common therapeutic approaches. In comparison to other diseases the that mutations inover 500 uniquegenes cause disorders with adistinct skin phenotype (4), which increased photosensitivity, inherited tumorigenesis and deepdermal trauma (1-3). Studiesindicate They display diverse clinical manifestations such as superficial epidermal andmucosal involvement, Genodermatoses – the inherited disorders of the skin –comprise agroup of heterogeneous diseases. , facilitate research. On theother hand, there are challenges such as multiple organ involvement, poor vector transmission to the epidermis and the size of some transgenes (e.g. larger than the for some diseases, the in vitro organ reconstruction is too simplistic because it simplistic lackstoo is reconstruction organ immune (6). cells, and the possibility toreconstitute the organ r epidermis, andsite-specific heterogeneity (5). in vivo

correction remains to beevaluated for in vitro (16, in This article is protected by copyright. All rights reserved. target rather than the therapeutic tool. To make clear a distinction between these two discrete Acceptedthrough are therapeutic options as well. However, for theseapproaches, the RNA isthetherapeutic RNA-editing andmodulation of translation of mutated mRNA transcripts by translational read- exogenously delivered RNA molecules. Post-transcriptional modification of RNA-transcripts by and limitations (summarized inTable 1). Wewill focus onapproaches that directly utilize applications for genodermatoses. Further, and importantly, wewill critically discuss their advantages future and current their with therapies RNA-based specific introduce will we sections following protein replacement therapies, as well as the unique challenges posedby treating skin. In the RNA-based therapies can overcome some ofthedifficulties that accompany thesecell, gene and corrected stem cells, on the other hand, might beamore efficacious approach (8,26-28). stem cells inthe epidermis andits long-term poten further cell andniche manipulation, does notseem tosubstantially increase the number ofcurative some genodermatoses (24, 25). However, bone marro sustain organ homeostasis, which is evident from long-lastingmosaic patches that occur naturally in remains elusive (22, 23). The epidermis contains a pool of self-renewing stem cells sufficient to Articlestudies presented no clear evidence of wild-type protein synthesis, the exact underlying mechanism transient symptomatic improvement also in connective tissue genodermatoses. Nevertheless, as the bone marrow transplantations (21). It has been s Several genodermatoses caused by primary immunodeficiencies can bemanaged by HLA-matched wounds or by microneedle injections (19, 20). dermal-epidermal junction (18).Epidermal delivery canbe achieved by direct application onto likely bemost effective for proteins naturally expressed in the dermal extracellular matrix orthe Systemic protein replacement therapy may beanalternative for a subset of genodermatoses and will hownthatmarrow bone transplantation induced some tial is thus uncertain. The local application of w transplantation, presently performed without

This article is protected by copyright. All rights reserved. significantly improve theexonskipping efficiency in hope is that the experience gained from developing AON-based therapy for these diseases will help to and spinal muscular atrophy (32), and in preclinical stages for several other genetic conditions. The AON-mediated splicing modulation iscurrently inclinical trials for Duchenne muscular dystrophy efficiency of type VII collagen restoration was low. partially restored type VII collagen synthesis. Although these first results were promising, the rats, back the onto of grafted human into skin-equivalents with this AON, directly injection single of Goto which is caused by genetic deficiency of type VII collagen, encoded by AON-mediated splicing modulation is being pursued fordystrophic epidermolysis bullosa (DEB), translation of the generated transcript will result in an internally deleted protein. longer recognized by the splicing machinery andis skipped from themature transcript. Subsequent mutated, in-frame exonduring pre-mRNA splicing (Fig. 1andFig. S1). The targeted exon is no to e.g. knock downgene expression or to manipulate splicing. For the latter, AONs hybridize to a Antisense oligonucleotides are small(AON) pieces of RNA. modified DNAor They canbeexploited AON-mediated exon skipping modulatingSplice therapies information (SupplementaryInformation). Supplementary the in approaches latter the of review a place to chosen therefore have we strategies,

AcceptedCOL7A1 Article et al et ., (29) showed that evoked de novo type VII collagen synthesis in patient keratinocytes. Subsequently, a in vitro transfection with a specific AON targeting the mutated exon 70 exon mutated the AONtargeting specific a with transfection COL7A1

to reach therapeutically relevant levels COL7A1 (29-31). In 2006, option for the repair of mutations on the mRNA level. SMaRT uses the cellular splicing machinery to This article is protected by copyright. All rights reserved. AcceptedIn recent years, spliceosome-mediated RNA RNA therapeutic effect in some disorders. low for a too remain may andformulation, still AONchemistry in improvements despite efficiency, woul AONs with treatment life-long known whether the therapy will be transient. Therefore, continuous treatment cycles will be needed, and it is not yet of effect the proteins, and transcripts AONs, rate of turnover to due Furthermore, worldwide. patients when a only anAONcan product, used fewfor from be and generating the revenue trials clinical exon, and even mutation. This poses obvious challenges todrug development such as performing to need approaches AON-based Therefore, function. be in-frame, andskipping shouldnot remove amino acidsequences that are essential for protein exonsneed to as targeted targets, suitable are mutations or genes not all AONsis of that A limitation Articlespecificity. cells that naturally express the protein, thereby avoiding concerns regarding organ and cell type the in synthesis protein only restore AONs Finally, (38). events adverse limited cause and toxicity low have tomanufacture, easy are AONs are that advantages Additional (37). systemically administered be can AONs that is advantage major a organs (4) multiple affect often genodermatoses that Given restored protein synthesis might yield significant phenotypic improvement (34-36). of level alow even which for diseases, recessive severe with patients for foreseen is AON treatment caused by mutant genes with a predominant in-frame exonorganization. The greatest benefit from genodermatoses other for attractive become will AONs that DEB,we envision for AON therapy of type VII collagen synthesis (26, 31, 33,34). Anticipating such advances in the development of an trans -splicing trans -splicing (SMaRT) has emerged as an attractive be critically evaluated for each targeted gene, targeted for each evaluated critically be d be tolerated and safe. Finally, the skipping the Finally, tolerated and d be safe.

PLEC on EB,forwhich RTMshave beensuccessfully developed and optimized forpreclinical studies on generation of skin grafts is conceivable. Most of the research on genodermatoses has so far focused Depending onthe disorder, either region, and consequently enhancing thereby reducing studies have revealed that the most efficient binding domains hybridize to exon/intron junctions, tool has been developed to optimize determination ofthe recombination frequency (50,51). Further, afluorescence-based RTMscreening of the repair molecules. Recent studies have shown that the binding domain is the crucial factor in the therapeutic efficiency in the earlier studies. Since then, much effort has been put into optimization hemoph VIII (39), muscular dystrophy ( and animal models for avariety ofhuman genetic diseases including Alzheimer’s disease ( generated, andwild-type protein synthesis restored. Proof-of-concept has been demonstrated in cell disease-causing mutation. After successful recombination, a hybrid full-length wild-type mRNA is internal wild-type coding region to replace the part of the endogenous pre-mRNA containing the S2). RTMThe is composed ofabinding domain, whichconfers target-specificity, and a 5’,3’ or The first preclinical proof-of-concept of the potential of epidermolysis bullosa (46-49). ( This article is protected by copyright. All rights reserved. pre-mRNA recombine an endogenous target pre-mRNA containi Accepted ArticleCFTR , )(43), hypertrophic cardiomyopathy ( KRT14 trans ilia A-knockout mice (42).Low (< 5%) recombination frequency significantly hampered , COL17A1 -splicing molecule (RTM) coding for part of the wild-type transcript (Fig.1 and Fig. cis -splicing dueto the blockage of competit and DYSF COL7A1 in situ and trans- trans (46-48, 50,55). administration (54) or correction of epidermal stem cells and TTN splicing (46, 48,51). -splicing efficiency efficiency -splicing ) MYBPC3 (40,41), hemophilia A( ng amutation with an exogenously delivered ) (44), retinitis pigmentosa (

trans ive splicing elements within the target in vitro -splicing came from studies in factor (50, 52, 53). Collectively, the FVIII )(42), cystic fibrosis RHO ) (45),and MAPT ) Transcript-replacement In vitro splicingmodulation, reaching clinically relevant trans-splicing efficiency may prove tobeahurdle. cell clonessingle have that undergone safety careful (10). profiling Just AON-mediated as with for a clinical application would be correction of stem cells followed by generation of skin grafts from further validating thesafety ofthis approach. Inmeantime, the themost promising andsafest strategy carefully addressed. Therefore, future efforts shoul or the RTMs themselves exhibiting unspecific into the clinic can be realized. Concerns related to the safety ofthe vectors needed forRTM delivery maintained (56, 57). Still, however, there are challenges that have to be overcome before translation additional advantage ofRNA broader range ofvectors to beused, which minimizes the risk ofgenetic rearrangements. An a allows turn in This cDNA). full of the instead used is cDNA the of portion a only (i.e. delivered majorA advantage of This article is protected by copyright. All rights reserved. of the keratin 14 gene ( been demonstrated for autosomal dominant disorder VII collagen-composed rudimentary anchoring fibrils wasobserved (48). Proof-of-concept has also used to generate skin equivalents that were subsequently transplanted onto mice, formation of type from apatient suffering from recessive DEB (RDEB). Further, when the corrected keratinocytes were mutation led to restoration of full-length type VII collagen synthesis in cultured primarykeratinocytes of encoding the cytolinker protein plectin (49). Subsequently, proof-of-concept was obtained for repair The first correction of anEB-associated gene by RNA

Accepted ArticleCOL7A1 transcribed mRNA-based therapy mRNA-based transcribed (48) . Replacement ofa 3.3kb 3’ portion of the KRT14 trans -splicing isthat it allows areduction insize of thetransgene to be ), which resulted in aphenotypic reversion of patient cells trans -splicing is that the endogenous control of gene expression is trans s usingexchangeSMaRT to thefirst sevenexons d focuson investigating off-target events and trans -splicinghavebeen raised andneed tobe

-splicing was achieved for the COL7A1 mRNA harboring apathogenic in vitro PLEC (47). gene This article is protected by copyright. All rights reserved. AcceptedSurfactant protein B(SFTPB) deficiency (OMIM 178640) groundbreaking pre-clinical study onamonogenetic disorder affecting another squamous epithelium. Transcript-replacement therapy is still in its infancy. Wehere illustrate its potential by a alternative option (20). Achieving cell- andtissue-specific delivery isanother challenge (68). avascular epidermis will be more difficult to so dependent uptake of mRNA occurs in dermal cells (67). Because efficient systemic targeting of the highly vascularized. Another option could be intradermal injections, since spontaneous calcium- target the skin. The dermis could be targeted by systemic administration because its deeper region is improved transfection vehicles are needed. A few different delivery approaches are conceivable to overcome (59-61, 66). Nevertheless, low transfecti and its recognition by the host,issues withdegradation and immune activation have inpart been consideration of transcript-replacement as a therapeutic tool. Through increased knowledge ofRNA (59), and inefficient uptake ofmRNA innon-immune cells (65) these studies did notlead towider as rapid degradation of mRNA by RNases in the extracellular environment (63, 64), immunogenicity Articlerecognized in the early 1990s (60, 62).However, due to inherent difficulties with theapproach, such potentialThe of injecting puremRNA torestore protein synthesis and revert disease phenotypes was the cap and/or nucleosides to produce less immunogenic andmore stable variants (61). mRNA canbe further modified to enhance transcribed drug, aDNAtemplate, based on wild-type sequence-engineered or cDNA(59,60), has to be reduced functionality and immunogenicity of recombinant proteins (59). createTo an active mRNA protein guarantees correct posttranslational modification, which helps overcome concerns about proteins (Fig.1Fig. and S3) (58). Transcript-replacement therapy introduces mRNA transcripts into cells todrive synthesis of wild-type in vitro and a5’capandpoly-A tailadded (61) (Fig. S3). In situ production of a protein in cells that naturally express this in vivo lve, topical application of mRNA couldbean protein translation. Examples are modification of on efficiency remains amajor hurdle (59) and

is an autosomal isan recessivecondition with In vitro transcribed (IVT) Sftpb been obtained from This article is protected by copyright. All rights reserved. refinement is needed to make itan attractive alternative. TALEN. This indicates thatmRNA-mediated gene editin directed repair, but with much lower efficiency than transfection with a plasmid encoding the vitro IVT mRNA-mediated genome editing has been used to correct through non-homologous end joining. breaks the repair will system DNArepair the provided, is DNA template when no correct note, Of correction of the mutated gene, and restoration of wi between the breaks during repair by homologous recombination. The outcome of this approach is may inserted be DNAtemplate mRNA, by IVT the the which encoded is bynuclease, the created breaks double stranded After a with DNA template. together cells to beprovided can nucleases the genomic sequence. To permanently correct agene, anIVT mRNA encoding suchsite-specific by determined which are sites, target specific at DNAbreaks double-stranded generate to designed be nucleases, e.g. zinc fingers, transcription activator-like nucleases (TALEN) or CRISPR-Cas9 thatcan IVT mRNA canalso be used for genome editing. Here, the IVT mRNAs encode site-specific IVT mRNA-mediated genome editing protein translation by mRNA modifications (68). protein B(68). This success wasachieved after optimizing bothdelivery ofthemRNA tothelung and neonatal lethality due to respiratory failure (69). Direct administration of Accepted Article under highpressure rescuedconditional (70). Transfection withIVT mRNA encoding patient-specific promotedTALEN homology- knockout mouse (71).

restoration of surfactant protein B synthesis in the above-mentioned conditional Sftpb knockout mice from lethality by synthesis of surfactant ld-type protein synthesis (Fig.1 andFig. S3)(59).

In vivo g is feasible in skin cells, but that further proof-of-concept of the approach has COL7A1 RDEB patient fibroblasts Sftpb mRNA tothe lung in discriminate between two sequences differing by only onenucleotide, it constitutes aparticularly (reviewed in(73)) pachyonychia congenita (74) ( most work hasbeenperformed onthedominantly inherited diseases such askeratinopathies interesting therapeutic option fordominantly inherited genodermatoses (Fig.1 and Fig. S4).Therefore, palmoplantar keratoderma (75,76), ( (RISC). They werefirst used in1995 for silencing protein synthesis long) that target mRNA fordegradation utilizing the endogenous RNAinduced silencing complex Small interfering RNAs (siRNAs) are double-stranded RNAmolecules (frequently 20-25 base pairs knockdown gene/allele Mutant templates to the target cells. in a future clinical phase will again be th challenge unexplored Ayet concern. a remain delivery DNAtemplate vector-based viral and activity than vector-mediated transfer (59, 71). Still, safety issues from both potential off-target nuclease activityis limited to theinherited intimedue instabil because the natural regulation of gene expression is not changed. Furthermore, because the nuclease in contrast to direct IVT mRNA delivery, cell- and tissue-specific targeting is not a pre-requisite, theoretically require only alimited number oftreatments toobtain long-standing effects. In addition, Although the results were promising, with regressi mutant. Intradermal injections were performed in symmetric plantar calluses onopposite feet. TD101, an siRNAspecifically designed against the mRNA encoding thekeratin 6ap.Asn171Lys split-body, vehicle-controlled, dose-escalation trial (74). The trial evaluated the safety and efficacy of ThisphaseIb trial for treatment ofpachyonychia congenita wasa single-patient, double-blinded, “siRNA” for www.clinicaltrials.gov This article is protected by copyright. All rights reserved. Accepted Article A clear advantage of in situ genome editing is that thegenome itself is corrected, which would identified onegenodermatosisidentified study among trials. 39registered KRT9 ), and on EB simplex ( e delivery efficiency of KRT6A on of the callus on the siRNA-treated side, the ity ofthe transfected RNA, this is a safer option

, KRT6B , KRT5 KRT16 in vivo both IVT mRNAs andDNA , or KRT14 (72). Because siRNA may (72).siRNA Because KRT17 ) (77). A search on search A (77). ) ), epidermolytic This article is protected by copyright. All rights reserved. nucleotide polymorphisms that are in single totarget used are AONs disorder In this disease. Huntington’s disorder neurodegenerative genodermatoses, this approach isbeing evaluated for treatment ofother genetic conditions like the described yet for Although not knockdown mRNA to mutant utilized transcripts. be also can AONs COL7A1 siRNAs to knockdown endogenous mutation-independent strategy hasbeenproposed (79). strategyThe isbased on administration of These siRNA strategies have the disadvantage of being mutation-specific. To overcome this, a (80). collagen VII transcripts by depleting the mutant version. This would result in increased synthesis of wild-type type 79).rationale(78, The DEB there istochan improved topical delivery methods will be necessary. siRNAs have also been pursued for dominant intense pain experienced by thepatient due to other tissues complicate development of causal therapies. Here, the RNA-modulating therapies genodermatoses. The surface area oftheskin and the fact that manygenodermatoses also manifest in It isclearthat major advances are being made inthefield oftherapy development for Future perspectives andconcluding remarks on section in described the exon as same skipping by disadvantages AONs. the with comes but that manner, mutation-specific a in designed be also could AONs Alternatively, genodermatoses harboring single nucleotide polymorphisms withhigh minor allele frequencies. and patients, and could thusalso be relevant to several of the genes affected in dominant activating RNase H-mediated degradation (81). The approach is applicable to an array ofmutations Accepted than tools smaller use generally they because properties favorable have may review this in detailed Article cDNA that is not suppressed by the siRNA. siRNA. the by suppressed not is that cDNA cis COL7A1 with the mutation to knockdown the mutant mRNA by ge the ratio between mutant and wild-type mRNA in conjunction with a sequence-modified the injections was asignificant concern. Thus,

COL7A1 This article is protected by copyright. All rights reserved. Acceptedthese issues in the development ofthe exon skipping approach. Here, several stakeholder meetings challenges. In this regard, a lot can be learned from the way the DMD community is trying to solve product and must be evaluated separately. When patient numbers are very small, this poses obvious already small group ofpatients. Each therapeutic compound isconsidered a separate medicinal modulating therapies that are mutation-specific because they are only applicable to a subset of an which therapeutic approaches canbe tested is lim Another challenge is that genodermatoses are rare diseases, and therefore, the number ofpatients in anti-inflammatory oranti-fibrotic drugs tomaintain good quality oflife. also be necessary tocombine therapeutic approaches that restore protein function with, for instance, the therapeutic benefit is larger when patients are treated in the early stages of the disease (85). Itmay largely depend on the timing oftherapeutic interventions. In this regard, it is generally accepted that Consequently, itisforeseen that thepotential benefits even ofcausal RNA-based therapies will secondary self-perpetuatingmechanisms thatare not adirect cause ofthe genetic defect (84). For many disorders, the disease might evolve dueto inflammation andorgan damage, whichactivate Article determined for each disease, patient subset or even each individual patient independently. mutation, disease history anddisease modifiers. Ultimately, treatment cycles may have tobe the of nature the stability, protein as such variables many on depend regimens treatment However, DNA-based animal models areneeded. Animalmodels might alsoshed light on treatment cycles. knock-ins, which cannot be used to optimize RNAmodulating approaches (83). Thus, new genome can beused tostudy RNA-modulating therapies (82), andmany oftheexistingmodels are cDNA animal models arerequired. However, thereare relatively fewgenodermatoses animal models that made, thereis still much room forimprovement fo very small oligonucleotides topical delivery might beapossibility. Despite all theadvancements the traditional gene and cell therapy approaches. These properties facilitate systemic delivery. For ited. This is even more complicated for theRNA- r RNA-basedtherapies.r facilitate To good this,

This article is protected by copyright. All rights reserved. Accepted Acknowledgements on all varieties of RNA-based therapies. the other hand, yield greater clinical benefit. It istherefore imperative tocontinue pursuing research codon read-through therapies. Other approaches will likely take longer todevelop, butthey may on stop and AONs way, i.e. havepavedthe diseases inother studies where therapies are implementation then afew approaches will be undergoing clinical trials. The topcandidates for fast clinical where will RNA-based therapies for genodermatoses stand a decade fromIt now? islikely that by pharmaceutical companies The increased interest in rare diseases from newly formed disease-specific companies or history studies for genodermatoses,Article which potential therapies are entering the clinical trial stage, there is an increasing demand for natural therapies into the clinic because it makes it challenging todefine relevant trial endpoints. As more understanding of the natural progression of many genodermatoses will likely impede translation of as many patients as possible to participate in clinical trials (88). Additionally, the insufficient selecting to facilitate http://www.treat-nmd.eu/) disorders, neuromuscular for network infrastructure clinical trial registries for rare diseases like those coordinated by TREAT-NMD (aclinical trial it is critically important to be prepared for clinical trials. They further underscored the importance of been shown convincingly has for benefit clinical DMD once exon skipping AONs additional for trials have may smaller to it possible be imply that pharmaceutical companies todiscuss the challenges andopportunities (86, 87). These discussions havebeen organized with academicresearchers, regulators,patient representatives,and is making the future brighter for affected individuals and their families. their and So individuals affected for brighter future the making is are nowbeing planned and pursued (89). multiple AONs. The discussions also made clear that that made clear also discussions AONs. The multiple

large This article is protected by copyright. All rights reserved. Accepted statement that she will receive noshare or royalties from thispatent. inventors, AMGP,andJB, PvdA areeligible to receive ashare and/or royalties. AMGP hassigned a AMGP, andPvdA areemployed by UMCG,whichhol SAB ofProQR andPhilae Pharmaceuticals. Remuneration for these activities goes to the LUMC. JB, Global Guidepoint, GLCConsulting, Deerefield Cons a share of any royalties. AAR also acts as an ad hoc consultant for PTC Therapeutics, BioMarin, the patents have been licensed toBioMarin. Asco-inventor onsome of the patents, AARisentitled to AAR isemployed by LUMC, which holds patents on exon skipping technology forDMD. Some of Disclosures Action BM1207 ofCOST (www.exonskipping.eu). part PCvdA are (ZonMW, grant 90715614) toPCvdA, respectively, also sponsored this research. AMGP, AAR,JB, AMGP, EMM, and AN, the Netherlands Organisation for Health Research andDevelopment Articleand EMM. Grants for the Dutch Butterfly Child Foundation (Vlinderkind), DEBRAInternational to AAR AN,AMGP, to (SpliceEB) Diseases onRare Research for ERA-Net the ofErare-2, frame the Research andDevelopment (ZonMW), andAustrian Science Fund(FWF; ProjectI1175-B13) under Federal Ministry for Education and Research (BMBF), the Netherlands Organisation for Health stimulating discussions on IVTmRNA therapy. The work wassupported by agrant from theGerman Center, the Netherlands, for editing themanuscript and Dr Markus Mezger University of Tübingen for We thank Jackie Senior, UMC Groningen, and Dr. Willeke van Roon, Leiden University Medical project. the approved the submitted and final versions; OB, ANwr PCvdA, EMM, AAR,AMGP, UK, OB, PP,JB,

ANdesigned and prepared figures; AN coordinated ulting, Grunenthal andBioClinica and ison the ds patents on exonskipping forDEB.Asco-

ote andcritically revised the review, and This article is protected by copyright. All rights reserved. Accepted Chamcheu C,Adhami J M, V Siddiqui I Mukhtar V, H.Cutaneous cell- andgene-based 8. Limberis MP.Phoenix rising: gene therapy 7. Iriyama S,Tsunenaga M,Amano S,AdachiE. Key role ofheparan sulfate chains in assembly 6. Sriram G, Bigliardi P L, Bigliardi-Qi M.Fibroblast heterogeneity and its implications for 5. genotypic and Phenotypic H. Tsao NV, Grishin ML, Murray I, R Sadreyev D, J Feramisco 4. Article Has C, Nystrom A. Epidermal Basement Memb 3. Castori M, Morrone A, Kanitakis GrammaticoJ, P.Genetic skin diseases predisposing to 2. Ishida-YamamotoIgawa A, S.Genetic skin diseases related todesmosomes and 1. References Therapy Therapeutic Mechanisms andStrategies. therapies for inherited and acquired skin disorders. In: Templeton N S, (ed.) Gene and Cell Sinica 2012: 44: 632-640. 1091–1122. of anchoring complex at the dermal-epidermal junction. ExpDermatol 2011: 20: 953-955. engineering organotypic skin models invitro. Eur Cell J Biol 2015: 94: 483-512. database. Invest J Dermatol 2009: 129: 2628-2636. analyses of genetic skin disease through the Online Mendelian Inheritance in Man (OMIM) 2015: 76: 117-170. basal cell carcinoma. Eur DermatolJ 2012: 22: 299-309. corneodesmosomes. Dermatol J Sci2014: 74: 99-105.

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USA: CRC Press, 2015: This article is protected by copyright. All rights reserved. 1 DeLuca M, Pellegrini G, Mavilio F.Gene therapy ofinherited skin adhesion disorders: a 11. Droz-Georget Lathion S,Rochat A,Knott G, 10. 8 HouY,Guey L T, Wu T, et al. Intravenously administered recombinant human type VII 18. Chamorro C,Mencia A,Almarza D,etal.Geneediting forthe efficient correction of a 17. Izmiryan A,Danos O,Hovnanian A.Meganuclease-mediated COL7A1gene correction for 16. Maeder M L,Gersbach C A. Genome-editing technologies for gene and cell therapy. Mol 15. Chamcheu C, WoodJ GS, Siddiqui I A,et al. Progress towards genetic and pharmacological 14. Cavazza A, Mavilio F.Gene therapy ofskin adhesion disorders (mini review). Current 13. Mavilio F,Pellegrini G,Ferrari S, et al. Correction of junctional epidermolysis bullosa by 12. Accepted Article Sallach DiPasqualeJ, G,Larcher F,et al. Tropism-modified vectors AAV overcome barriers to 9. successful cutaneous therapy. Mol Ther 2014: 22: 929-939. recessive dystrophic epidermolysis bullosa mice. Invest J Dermatol 2015. collagen derived from chinese hamster ovary Cells reverses the disease phenotype in Molecular therapy Nucleic acids 2016: 5: e307. recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes. recessive dystrophic epidermolysis bullo 2016:Ther 24: 430-446. 2012: 21: 481-489. therapies for keratin genodermatoses: current perspective andfuture promise. Exp Dermatol pharmaceutical biotechnology 2012:13: 1868-1876. transplantation of geneticallymodified epidermal stem cells. Nat Med 2006: 12: 1397-1402. critical overview. Dermatol Br J 2009: 161: 19-24. safe ex vivo gene therapy. EMBOmolecular medicine 2015: 7: 380-393. sa. J Invest Dermatol 2016: 136: 872-875. et al. A single epidermal stem cell strategy for

7 Wenzel D,Bayerl J, Nystrom A,Bruckner 27. This article is protected by copyright. All rights reserved. Accepted Kuhl T, Mezger M,Hausser I, Handgretinge 26. Pasmooij A M, Pas HH, Deviaene F C,Nijenhuis M, Jonkman MF. Multiple correcting 25. Kiritsi D,Garcia M,Brander R,etal. Mechanismsof natural gene therapy indystrophic 24. Watt H,Wright SA,Dayal J S,etal.Lysy 23. Article Tolar BlazarJ, B R, Wagner J E. Concise review: Transplantation of human hematopoietic 22. Worth A BoothC, J, Veys P.Stem celltransplantation for primary immune deficiency. 21. McCrudden M T, McAlister E,Courtenay Gonzalez-Vazquez A J, P, Singh R, T Donnelly R 20. Wang Ghasri X, P,Amir M, etal. Topical application ofrecombinant type VII collagen 19. Science translational medicine 2014: 6: 264ra165. Genetically corrected iPSCs as cell therapy for recessive dystrophic epidermolysis bullosa. in dystrophic epidermolysis bullosa. Mol Ther 2015: 23: 1368-1379. local concentrations of intradermal MSCsrest Hum Genet2005:77: 727-740. COL17A1mutations inpatients with revertant mosaicism ofepidermolysis bullosa. Am J epidermolysis bullosa. J Invest Dermatol 2014. One 2015: 10: e0137639. membrane andis reduced in patients with recessive dystrophic epidermolysis bullosa. PLoS 29: 900-906. cells for extracellular matrix protein deficiency inepidermolysis bullosa. Stem Cells 2011: Current opinion in hematology 2013:20: 501-508. F. Microneedle applications in improving skin appearance. ExpDermatol 2015: 24: 561-566. 21: 1335-1344. incorporates into the dermal-epidermal junc tion and promotes woundclosure. Mol Ther 2013: l hydroxylase 3 localizes to epidermal basement r R,Bruckner-Tuderman L,Nystrom A.High ore skin integrity andfacilitate wound healing -Tuderman L,MeixnerA,Penninger M.J

6 Lehmann J,Schubert S, Emmert S. Xer 36. van denAkker PC,van Essen A KraakJ, M M, et al. Long-term follow-up of patients with 35. Schwieger-Briel A, Weibel L, Chmel N, et al. A COL7A1variant leading to in-frame 34. This article is protected by copyright. All rights reserved. Accepted LuQL,Cirak S,Partridge T. What can we 33. R,Krieg Kole AM.Exonskipping therapy 32. Article Turczynski M, S,Titeux Tonasso L,Hovnanian A.Exon skipping restores functional type VII 31. Turczynski M, S,Titeux Pironon N,Hovnanian A. Antisense-mediated exon skipping to 30. Sawamura GotoM, NishieD, al. W,et Targeted skipping of asingle exon harboring a 29. Umegaki-Arao N,Pasmooij AM,Itoh M,et al. Induced pluripotent stem cells from human 28. 12: 867-872. interdisciplinary patient care, andnovel therapeutic approaches. J Dtsch Dermatol Ges 2014: database andunusual phenotype-genotype correlations. Dermatol J Sci 2009: 56: 9-18. recessive dystrophic epidermolysis bullosa in the Netherlands: expansion of the mutation DermatolBr J 2015. skipping ofexon15attenuates disease severity DMD? Molecular therapy Nucleic acids 2014: 3: e152. drug delivery reviews 2015: 87: 104-107. collagenin dystrophic epidermolysisbul reframe transcripts. Methods inmolecular biology 2012:867: 221-238. 2620. for selective dystrophic epidermolysis bull premature termination codon mutation: implications and potential for gene correction therapy medicine 2014: 6: 264ra164. revertant keratinocytes for thetreatment of losa. Invest J Dermatol 2013: S144-S144. osa patients. Invest J Dermatol 2006: 126:2614- oderma pigmentosum: diagnostic procedures, learn from clinical trials of exon skipping for for Duchenne muscular dystrophy. Advanced in recessive dystrophic epidermolysis bullosa. epidermolysis bullosa. Science translational

This article is protected by copyright. All rights reserved. Accepted Mearini G,Stimpel D, Kramer E,et al. Repair of Mybpc3 mRNA by 5'-trans-splicing in a 44. Liu LuoX, M, Zhang LN, et al. Spliceosome- 43. ChaoH,Mansfield S G, Bartel RC, et al 42. Monjaret F,Bourg N,SuelL,etal. Cis-sp 41. Article Philippi S,Lorain S,Beley C,etal.Dysferlin rescue by spliceosome-mediated pre-mRNA 40. Avale ME, Rodriguez-Martin GalloT, J M. Trans-splicing correction of tau isoform 39. Koo WoodM ClinicalT, J. trials using antisense oligonucleotides induchenne muscular 38. van PuttenM, Young C,van denBerg S,etal. Preclinical studies on intestinal administration 37. e102. mousemodel ofhypertrophic cardiomyopathy. Molecular therapy Nucleic acids 2013: 2: 1116-1123. function to polarized human cystic fibrosis airway epithelial cells. Hum Gene Ther 2005: 16: adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator spliceosome-mediated RNA trans-splicing. NatMed 2003: 9: 1015-1019. 2014: 22: 1176-1187. molecule combine with efficiency insplice 2015: 24: 4049-4060. trans-splicing targeting introns harbouring weakly defined 3' splice sites. Hum MolGenet imbalance in a mouse model of tau mis-splicing. HumMol Genet 2013: 22: 2603-2611. dystrophy. Hum GeneTher 2013: 24: 479-488. dystrophy. Molecular therapy Nu of antisense oligonucleotides asamodel for oral delivery fortreatment ofduchenne muscular cleic acids 2014:cleic acids 3:e211. . Phenotype correction of hemophilia Amice by osome-mediated RNA trans-splicing. Mol Ther licing andtranslation of thepre-trans-splicing mediated RNAtrans-splicing with recombinant

This article is protected by copyright. All rights reserved. Accepted Peking U,Hainzl P,Koller S,etal. A 54. Bauer J W, Murauer E M, Wally V, Koller U.RNA trans-splicing forgenodermatoses. 53. Koller U,Hainzl S, Kocher T, et al. Trans-splicing improvement by thecombined application 52. Wally High-throughput U,Bauer K V JW screening forhighly functional RNA-trans-splicing 51. Murauer EM, Koller U,Hainzl S, Wally Bauer V, W. Areporter-basedJ screen to identify 50. Wally V, Klausegger A, Koller U,et al. 5' trans-splicing repair of the PLEC1 gene. J Invest Article49. Murauer E M, Gache Y, Gratz I et K, al. Functional correction of type VII collagen 48. Wally Brunner V, M, Lettner etT, al. K14 mRNA reprogramming fordominant 47. Koller U,Wally V, Mitchell LG,et al. Anovel screening system improves genetic correction 46. Berger A,Lorain S, Josephine C,et al. Repair of rhodopsin mRNA by spliceosome-mediated 45. strategy forCol7a1 repair. Molecular therapy Nucleic acids 2016: 5: e287. Methods in molecular biology 2013:961: 441-455. of antisense strategies. International journal of molecular sciences 2015: 16: 1179-1191. Genetic Diseases: Rijeka, 223-240, 2011:223-240. molecules: correction ofplectin inepidermolysis bullosa simplex. .In: D P-K,ed. Human 2013: 24: 19-27. potent 3' trans-splicing molecules for endogenous RNA repair. Human gene therapy methods Dermatol 2008: 128: 568-574. expression in dystrophic epidermolysis bullosa. Invest J Dermatol 2011: 131: 74-83. epidermolysis bullosa simplex. Hum MolGenet 2010: 19: 4715-4725. by internal exon replacement. Nucleic acids research 2011: 39: e108. 2015: 23: 918-930. RNA trans-splicing: a new approach for autoso Gene gun-mediated nonviral RNAtrans-splicing mal dominant retinitis pigmentosa. Mol Ther

3 Landre J BP,Hewett PW,Olivot M, etJ 63. This article is protected by copyright. All rights reserved. Accepted Jirikowski GF,Sanna PP, Maciejewski-Lenoir 62. PardiN, Muramatsu H,Weissman D,Kariko K. 61. Thess A, Grund S,Mui B L, et al. Sequen 60. SahinU,Kariko K, mRNA-basedTureci O. 59. Article Yamamoto A,Kormann M, Rosenecker J, Rudolph C.Current prospects for mRNAgene 58. Tahara M, Pergolizzi RG,Kobayashi H, et al. Trans-splicing repair of CD40 ligand 57. Mansfield SG, Clark RH, Puttaraju M, et al. 5' exon replacement and repair by spliceosome- 56. Koller U, Wally V, Bauer W, J Murauer E M. Considerations for a successful RNA trans- 55. 2002: 86: 540-552. large amounts of pancreatic-type ribonuclease (RNase 1). Journal of Cellular Biochemistry 996-998. in Brattleboro rats: intrahypothalamic injection of vasopressin mRNA. Science 1992: 255: modified nucleosides. Methods in molecular biology 969: 2013: 29-42. 1464. modifications enables an effective protein therapy in large animals. Mol Ther 2015: 23: 1456- Nature reviews Drug discovery 2014:13: 759-780. Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV 2009: 71: 484-489. delivery. European journal ofpharmaceutics andbiopharmaceutics :official journal of immunodeficiency. NatMed 2004: 10: 835-841. deficiency results in naturally regulated correction of a mouse model of hyper-IgM X-linked mediated RNAtrans-splicing. Rna2003:9: 1290-1297. splicing repair of genetic disorders. Molecular therapy Nucleic acids 2014: 3: e157. ce-engineered mRNA without chemical nucleoside al. Human endothelial cells selectively express therapeutics--developing anewclass ofdrugs. Invitro transcription of long RNAcontaining D, Bloom FE.Reversal of diabetes insipidus

This article is protected by copyright. All rights reserved. Mahiny DewerthA, Mays AJ, LE,etal. 71. Osborn M J, Starker C G,McElroy AN,et al. TALEN-based gene correction for 70. lung nterstitial disease. Pediatric research 2016: 79: 34-41. Griese M,Lorenz E,Hengst M, et al. Surfactant proteins in pediatric i 69. Kormann M S, Hasenpusch G,Aneja M K, et al. Expression of therapeutic proteins after 68. Probst WeideJ, B,Scheel B, etal. Spontane 67. Kariko Buckstein K, M, NiH,Weissman 66. YounH,Chung K. Modified mRNAJ for to plasmid asanalternative DNA(pDNA) 65. Harder SchroderJ, J M. RNase 7, a novel i 64. 3 Knobel M, O'Toole EA, Smith Keratins FJ. andskin disease. Cell Tissue Res 2015:360: 73. GuoS,Kemphues par-1,K J. agene required for establishing polarity inC.elegans embryos, 72. 4 Leachman SA, Hickerson R P,Schwartz ME, Accepted74. Article mRNA corrects SP-B deficiency. Natu epidermolysis bullosa. Mol Ther 2013: 21: 1151-1159. delivery ofchemically modified mRNA inmice. Nature biotechnology 2011:29: 154-157. in vivo isnucleic acid-specific, saturable and ion dependent. Gene Ther 2007: 14: 1175-1180. Immunity 2005:23: 165-175. receptors: the impact of nucleoside modification and the evolutionary origin ofRNA. 15: 1337-1348. transcript replacement and vaccination therapy. Expert opinion on biological therapy 2015: healthy human skin. Journal ofBiol phase Ib trial of an inherited skin disorder. Mol Ther 2010: 18: 442-446. 583-589. encodes aputative Ser/Thr kinase that is asymmetrically distributed. Cell 1995: 81: 611-620. ogical Chemistry 2002: 277: 46779-46784. re biotechnology 2015:33: 584-586. D. Suppression of RNArecognition by Toll-like ous cellular uptake ofexogenous messenger RNA Invivo genome editing using nuclease-encoding nnate immune defense antimicrobial protein of et al. First-in-humanet al. mutation-targeted siRNA

This article is protected by copyright. All rights reserved. Accepted Bruckner-Tuderman L, McGrath J 83. Nystrom Buttgereit A, BaderM,et al.RatJ, model fordominant dystrophic epidermolysis 82. Kay C,Skotte NH,Southwell AL,Hayden MR.Personalized gene silencing therapeutics 81. Fritsch A,Spassov S, Elfert S, et al. Dominant-negative effects of COL7A1mutations can be 80. Morgan CP,Allen DS,Millington-Ward S,O'Dwyer GE,PalfiA,Farrarmutation- G A J. 79. Article Pendaries V, Gasc G, Titeux M, Tonasso L, Mejia J E, Hovnanian A. siRNA-mediated allele- 78. Atkinson SD,McGilligan E,LiaoH,et V 77. Thomson FuDJ, C,Lunny DP,etal.Keratin 9is required for the structural integrity and 76. Leslie Pedrioli DM, FuD J,Gonzalez-Gonzal 75. PLoS One2013: 8: e64243. bullosa: glycine substitution reduces collagen VII stability andshows gene-dosage effect. for Huntington disease. Clinical genetics 2014: 86: 29-36. 30256. rescued by controlled overexpression of norm Dermatol 2013: 133: 2793-2796. independent therapeutic strategy fordominant dystrophic epidermolysis bullosa. J Invest InvestJ Dermatol 2012: 132: 1741-1743. specific inhibition of mutant type VII collagen in dominant dystrophic epidermolysis bullosa. 131: 2079-2086. siRNA forkeratin 5mutations in epidermolysis bullosa simplex. Invest J Dermatol 2011: terminaldifferentiation palmoplantar ofthe 2012: 132: 1627-1635. based therapeutics for a dominant-negative epidermal fragility disorder. InvestJ Dermatol bullosa: update 2010. J Invest Dermatol2010: 130:1485-1488. A, Robinson EC, Uitto AnimalJ. models of epidermolysis epidermis. Invest J Dermatol 2014: 134: 754-763. al collagen VII. Biol Chem J 2009: 284: 30248- al. Development ofallele-specific therapeutic ez E, et al.ez Generic E, and personalized RNAi-

This article is protected by copyright. All rights reserved. Accepted 1. Table and figure legends Table https://clinicaltrials.gov/. 2016. Accessed April 28, 2016. 89. Rodger S,Lochmuller H, Tassoni A,et al. The TREAT-NMD careand trial site registry: an 88. Article Aartsma-Rus A,Ferlini A, Goemans N,et al. Translational and regulatory challenges for 87. Muntoni F,Guicheney P,Voit 158thT. ENMC international workshop on congenital 86. PeesC,Laccone F,Hagl M, Debrauwer V,Mo 85. Nystrom A,Thriene K, Mittapalli V, et al. Losartan ameliorates dystrophic epidermolysis 84. 2013: 8: 171. online registry to facilitate clinical research for neuromuscular diseases. Orphanet J Rare Dis exon skipping therapies. Hum GeneTher 2014: 25: 885-892. The Netherlands. Neuromuscular disorders : NMD2009: 19: 229-234. muscular dystrophy(Xth international CM adults with Marfan syndrome. American The journal of cardiology 2013:112: 1477-1483. on the size of the ascending aorta in an unselected cohort of children, adolescents, andyoung 1228. bullosa and uncovers new disease mechanisms Summary ofthepresent status of RNA-based therapies for treatment ofgenodermatoses. D workshop) 8th-10th February 2008 Naarden, ser E, Michel-BehnkeI. Usefulness of losartan . EMBO molecular. EMBO medicine 2015: 7:1211-

Stop codon Stop read-through information Supplemental specific approaches can be found in their specific sections in the text and in Figures S1-S5. major mechanisms of the RNA-based therapy strategies presented in this review. More detail on the Investigation of significantly less toxic non-aminoglycosides with stop codon read-through capacity is ototoxicity. irreversible and renal involve that side-effects known have Aminoglycosides Thus, the potential of read-through therapies for genodermatoses has already been recognized. collagen anddeposition of theprotein at the dermal cancer (7). Furthermore, for RDEB, with xeroderma pigmentosum- agenodermatosis acco synthesis of xeroderma pigmentosum complementation group Cprotein in fibroblasts from patients for genodermatoses, aminoglycoside and non-aminoglyco studies on read-through-based therapies have been optimism (4-6). Aminoglycosides are the most prominent inducers ofPTC read-throughmost and Clinical trials performed in other genetic diseases, e.g. cystic fibrosis and DMD, have led to cautious read-through occurs, and translation of mRNA cont recognition. Asa result, the PTC is ignored, error-prone translation induced, i.e. translational PTC molecule drugs can create structural alterations in the ribosomal RNAthat disrupt normal PTC symptoms, chemically enhanced translational read-through of PTC is an attractive option. Small RDEB (2,3),inwhicharelativelyminor increase of all inherited genetic disorders (1). For ge Mutations converting anamino acid into apremature termination codon (PTC) are found in one-third This article is protected by copyright. All rights reserved. Accepted Article Figure 1. RNA-based therapy approaches for genodermat

aminoglycoside treatment led to nodermatoses, such as xeroderma pigmentosum and in protein synthesis significantly improves disease performed withthese compounds (1). Specifically -epidermal junction zone in skin equivalents (8). inues beyond the PTC (Fig.1 andFig. S5) (1). mpanied by a10,000-fold increased risk ofskin

side treatments have been shown to increase oses. Graphic summary ofprinciples and de novo synthesis of type VII This article is protected by copyright. All rights reserved. Acceptedeffect; it rather aims tocorrect mutant RNAs. RNA editing is aphysiological post-transcriptional genodermatoses. Therapeutic RNAediting doesnot employmolecules RNA to exert a therapeutic introduce it, although it is currently invery early stages andnot yet, to our knowledge, studied for RNA editing is an approach that could have future therapeutic value. Therefore we will briefly RNA editing mutation. through as therapy must bedetermined separately for each compound for each disease and each through compounds (16). Therefore, thefeasibility andefficacy ofinduced-translational read- PTC after the stop codon –iscrucial in determining the read-through potential of stop codonsand read- only thenature of thestop-codon itself, butalso the flanking sequence –especially the+1position functionality ofproteins, especially proteins with translation (i.e. insertion of another amino acid than the wild-type one) may significantly impact the leading to either loss- orgain-of-function of the protein. Additionally, an increased error rate during forced read-through of the mutated codon may evoke identity change ofthe original amino acid only avery limited number oftarget transcripts for inducing thePTC compounds. Furthermore, Articleuse of read-through therapies. When nonsense-mediated mRNA isvery efficient, there will be no or There are intrinsic factors related to the nature of the mutation and mutated proteins that may limit the through potential in agenodermatosis setting (15). studywith this compound withseveral (14) and further studies are needed to determine theusefulness of ataluren for genodermatoses. One a recent phase III trial in DMD, the primary endpoint was not met, except in aprespecified subgroup from theEuropean Medicine Agency for the use in ambulant DMD patients 5 years and older (13). In muscular dystrophy (DMD) (6, 12), for which it has received conditional marketing authorization been tested in clinical trials for multiple diseases. Development is most advanced for Duchenne being pursued (1,9).One example isthe compound COL7A1 and complex folding andorganization. Furthermore, not PTC124 (Ataluren/Translarna)PTC124 (10,11) that has KRT6A

stop codons could not confirm its read- This article is protected by copyright. All rights reserved. Accepted 59-69. 38: 2011: journal respiratory European The fibrosis. cystic mutation nonsense Wilschanski M, Miller L L, Shoseyov D, et 4. 2015. attenuates disease 15 exon severity inrecessive bullosa. Br dystrophic epidermolysis J Dermatol Schwieger-Briel A, Weibel L, Chmel N, et al. 3. 867-872. J Ges care, patient 12: therapeutic andnovel Dtsch Dermatol approaches. 2014: interdisciplinary diagnostic procedures, S, J,Schubert S. Emmert Lehmann pigmentosum: Xeroderma 2. diseases. Pharmacologytherapeutics& 2012: 136:227-266. in recode to inherited mutations nonsense therapies JP. Pharmaceutical H L, Dougherty Lee 1. Supplementary references whether it could have therapeutic potentialArticle for genodermatoses. questions regarding the true clinical potential of this approach has been performed domain toguide RNAs,i.e.complementary seque RNA catalytic the out by conjugating carried have been bases adenosine specific to ADAR enzymes different effects ontheresulting protein. Successful attempts todirect thecatalytic activity ofthe transitions and, consequently, tocreate different RNA versions of single RNA transcripts with subsequently regarded as a guanosine (G), RNAediting has the potential to induce many A>G (adenosine ADAReditases the called enzymes of I-editing, the deamination of adenosine (A)-bases into inosine (I)-bases, which is executed by agroup thousands of genes (reviewed in (17)). The best-known example of endogenous RNA editing is A-to- process that increases transcript diversity by enzymatically modifying bases of RNA-molecules in in vitro to correct a stop codon in the RNA editing exist and the future studies will tell A COL7A1 leadingvariant to in-frame skipping of deAminase acting on RNA). As the inosine is the oninosine As RNA). acting deAminase al.Chronic ataluren (PTC124) treatment of nces to the target sequence (18).For instance,

CFTR gene (19). Many This article is protected by copyright. All rights reserved. Acceptede81302. inpatients with nonsense production mutation Finkel R S, Flanigan K M, Wong B, et al. Phase 2a study of ataluren-mediated dystrophin 12. 47: 430-444. adult andfemale to healthy male administration andmultiple-dose single- following nonsense suppressor, mutation a nonaminoglycoside HirawatS, Welch E M, Elfring G L, et al.Safety, tolerability, and pharmacokinetics of PTC124, 11. 407-425. 64: 2013: ofmedicine Annualreview suppression. Peltz SW, Morsy M, Welch E M, Jacobson A. 10. 1653-1660. 21: intheTher ATM gene. 2013: Mol nonsense types all mutations of through of three read induce L, JungME, R, al. Du seriesDamoiseaux et Anew small weight compounds of molecular 9. Articlebullosa. Mol Ther 2014: 22: 1741-1752. epidermolysis dystrophic for therapeutic implications codons: termination premature overcoming by VII collagen type full-length restore al.Aminoglycosides et X, J,Wang J,Weinstein Cogan 8. termination codons. ProcNatl AAcadSciS 2013: U 110: 19483-19488. pigmentosumxeroderma group C cells induced premature bytranslational readthrough of C, Kuschal J DiGiovanna J, SG,Khan Gatti R 7. dystrophinopathy. Musclenerve & 2014: 50: 477-487. Finkel R, K, Bushby Wong B, et al. Ataluren 6. 67: 771-780. 2010: Annals neurology dystrophy. of muscular in Duchenne V, al. of L, Malik et Rodino-Klapac codons R,Gentamicin-inducedL Viollet readthrough stop 5. Duchenne muscular dystrophy. PLoS One 2013: 8: 2013: One PLoS dystrophy. muscular Duchenne volunteers. Journal of clinical 2007: volunteers. pharmacology treatment ofpatients with nonsense mutation Ataluren asan agent fo A, Kraemer K H. Repair of UV photolesions in

r therapeutic nonsense This article is protected by copyright. All rights reserved. Acceptedthe interacting exon to be skipped during splici performance. (2) After intracellular uptake undergo in-frame exonthat contains amutation, in casethis a pre-terminal stop codon (PTC). AONsThe Figure S1. Principle AON-based of therapy. Supplemental figure legends. 2013: 110:18285–18290. by site-directe regulator conductance transmembrane cystic within fibrosisthe al.Correction MF, of et mutations Montiel-Gonzalez 19. in Nuclei trans. hADAR2 SchneiderMF, etal.Optimal guideRNAsfor 18. 21:549-559. 2015: Medicine in Molecular Trends disease. andrewiring inhuman recoding, CH.RNArewriting, E,Jamieson JiangQ,Melese MA, Zipeto 17. Article 164-169. 48: 2000: neurology Annals of dystrophy. muscular Duchenne of aminoglycoside-inducedspecificity of stop potential condon implications for readthrough: treatment ShirtsM BH, Howard T, L M, FlaniganK Petros RF,M, Gesteland JF. Atkins Sequence 16. efficacy of PTC124 (Ataluren) inadiverse array of S T, al. S, codon Torrie P, McElroy Nomura L read-through et termination premature Alack of 15. https://ptcbio.com. Accessed April 21, 2016. 14. 25: NMD 5-13. disorders : gene. Neuromuscular 2015: dystrophin mutation anonsense in the from aged and patients 5years ofambulant with resulting older Duchenne treatment muscular dystrophy Haas M, Vlcek V, Balabanov P, et al. Europe 13. in silico in and in vitro c Acids Res 2014:42:e87. 2014:42:e87. Res c Acids selection to find one AON, or a combination of AONs, with the best the of with AONs, combination a one AON,or to find selection in vitro (1) Small AONs (20-30 bp) are designed to target an target to Small (20-30designed are (1) AONs bp) ng. A mature mRNA lacking the targeted exon is reporter assays. PLoS biology 2013: 11: e1001593. e1001593. 11: 2013: biology PLoS assays. reporter or an Medicines Agency review of ataluren for the re-directing deaminase activity of hADAR1and in vivo d RNA editing. Proc. Natl. Acad.Sci.U.S.A. Natl. Proc. d RNAediting.

AONs bind pre-mRNA the bind causing AONs to with the mutation.with After uptakecellular specifically the siRNA/AON binds to the mutation-containing dominanta nucleotide mutation or polymorphism to target single AONis designed or siRNA gene knockdown therapy. andAON-based mutant of siRNA S4.Figure Principle genomic of the DNA.. repair a sustained DNA) enables minicircle or vector (e.g. from viral DNAtemplate a together with cells uptake in after CRISPR-Cas9,or which II: Gene-editing usingIVT-mRNA encoding tailored translated into wild-type protein after therapy arerelevant for genodermatoses. I: Transcript replacement, where the IVT mRNA is of mRNA-based IVT approaches mRNATwo the (4) and digested purified. DNAtemplate the used for template or codon-optimized for improved translation. (2) RNApolymerases, usually SP6or T7, are product or aplasmid can be used for mRNA transcription. The template can either be awild-type therapy. of IVTmRNA-based S3. Figure Principle type protein. endogenous mRNA andthewild-type portion provided by theRTM, results in translation of wild- mediated formationa corrected of hybrid transcript.fusion The transcript,comprising partsof the This article is protected by copyright. All rights reserved. Accepted Articlebinding domain (BD)ofthe RTMbinds to a pre-mRNA, of assembly During cDNA. the of part internal an or 5’, 3’ the contain can RTM mRNA Figure S2. Principle of removed exonwhile retaining itsfunctionality. assembled, which allows translation of ashorter protein missing theamino acids encoded by the trans in vitro -splicingmolecule (RTM) encoding parts ofthe transcript that need to becorrected. The transcription. (3) After completion of transcription a 5’ cap is added to the mRNA, trans -splicing-based therapy. in vivo uptake into cells to transiently restore protein synthesis. the target intron/exon region, inducing nucleases such as zinc finger nucleases, TALEN (1) A DNA template, a sequence-verified PCR sequence-verified a ADNAtemplate, (1)

Trans

-splicing isbased on delivery ofapre- trans A specific A specific -splicing- in cis

This article is protected by copyright. All rights reserved. the mutation and sequence. is induced and translation of full-length proteins increased. The efficacy ofthe treatment depends on premature stop of protein translation. After treatment with a small molecule, readthrough of the PTC wild-type transcript results inafull-length protein. Translation of the mutated transcript leads to specificity of the codon-anticodon pairing and thus drive error-prone translation. Translation of the treatment with small molecule drugs. moleculesThe bind tothe ribosomal RNA andreduce Figure S5. Principle stop of codonreadthrough. effect. to relatively higher levels of wild-type protein being translated, thus diluting thedominant negative cleavage of the targeted mRNA. As aconsequence the wild-type mRNA transcript is enriched leading promoting cleavage the of siRNA-boundmRNA by AONs,it activates RNaseH RNases.For mRNA. ForsiRNA, this binding induces the forma therapy RNA-based Table 1 Table mediated gene gene mediated Trans readthrough replacement IVT mRNA-IVT IVT mRNA-IVT knockdown transcript- AcceptedStop codon Article gene/allele mediated mediated therapy Mutant editing AON -splicing ehns fato Dlvr Avnae Lmttos ies Key references Disease Limitations Advantages Delivery Mechanismaction of mRNA transcripts drive to transcripts mRNA part, carrying a mutation, carryingpart, a nucleases together with a a with together nucleases Introduction of corrected corrected of Introduction Targeted-degradation of Targeted-degradation In situ expression of wild-type wild-type of expression mRNAExchange an of nue edhog of Induced readthrough IVT mRNA coding for coding mRNAIVT transcription activator- like, or CRISPR-Cas9like, or optimizedfinger, zinc using the endogenous using the by its wild-type copy nue kpigof skipping Induced splicing machinery splicing mutant transcripts proteins in vivo in proteins DNA template uae exon mutated genewith repair PTCs intraepidermal skin cells and skin cells and (correction of (correction of / intradermal Intradermal Intradermal Intradermal Intradermal re-grafting) re-grafting) (Topical) Systemic Systemic Systemic Topical Ex vivo Ex vivo Oral 3. Advanced clinical development for for development clinical 3. Advanced 3. Advanced clinical development for for development clinical 3. Advanced 1. Deliveredtransgene shorteris than 1. Systemicadministration multiple/ 2. Multiple routesadministrationof 4.toxicity Lowand limited adverse safer than vector-based approaches vector-based than safer 2. Natural regulation transgene of full-length(facilitates transcript 2. Transient activitymaking it 2. Natural regulation gene-of 2. Natural regulation gene-of 1. Natural regulation gene-of 3. Natural regulation gene-of 3. Applicabledominantlyfor 1. 1. Can be used for dominant 1. Correctposttranslational 1. Systemic administration Systemic 1. routes of administration of routes events in clinical trials other geneticdiseases other geneticdiseases cloning and delivery)cloning and inherited disorders 3. Non-integrative 2. Non-integrative expression keptexpression expression keptexpression expression keptexpression expression kept modification expression expression disorders nuclease activity and viral 1. Safetyissuesfromviral 1.Multiple administration dependentdifficult to and dependentdifficult to and tissue specific translation translation specific tissue high achieve to is needed (unspecific splice events) splice (unspecific 3. Increased translational translational Increased 3. vector (stable integration trans-splicing efficiency) trans-splicing trans-splicing molecule and protein expression expression protein and 2. Delivery to the skin the2. Delivery to 2. Deliveryskinthe to 3. Inabilityregulateto 1. Not suitable for all 2. Safety issues from from issues Safety 2. 1. Safety issues from from issues Safety 1. vector based DNA vector based 2. Effectmutation 2. Effectmutation template delivery 2. Only for PTCs for Only 2. exons and genes and exons 1. Painful drug administration inaccuracy Toxicity 1. predict predict levels tion of a RNA-induced silencing complex (RISC) The readthrough of aPTC can beenhanced by the

keratoderma, EB pigmentosum, Pachyonychia epidermolytic palmoplantar Xeroderma congenita, congenita, EB EB EB EB - Kormann et al., et al., 2011 (51) Kormann - Murauer Murauer et al., 2013 (34) (38) al., 2014 Koller et (20) 2009 et al., van Akker den 2012(15) al., et Turczynski Gotoet al., 2006 (14) (9) Aartsma-Rus, 2010 Kuschal et al., et al., 2013 (69) Kuschal et al., 2014 (70) Cogan et al., 2012 (62) Pendaries (59) et al., 2012 Leslie Pedrioli et al., 2010Leachman (58) Atkinson et al., 2011 (61) Mahiny et al., et al., 2015 (55) Mahiny (54) 2013 al., Osborn et

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