Epidermolysis Bullosa Simplex Patients Research Articles

Clinical and molecular features in a cohort of Middle Eastern patients with epidermolysis bullosa.

Epidermolysis bullosa (EB) features skin and mucosal fragility due to pathogenic variants in genes encoding components of the cutaneous basement membrane. Based on the level of separation within the dermal-epidermal junction, EB is sub-classified into four major types including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB) with 16 EB-associated genes reported to date. We ascertained a cohort of 151 EB patients of various Middle Eastern ethnic backgrounds. The cohort was comprised of EBS (64%, 97/151), DEB (21%, 31/151), JEB (12%, 18/151), and KEB (3%, 5/151). KRT14 and KRT5 variants were most common among EBS patients with 43% (42/97) and 46% (45/97) of EBS patients carrying mutations in either of these two genes, respectively. Truncal involvement was more common in KRT14-associated EBS as compared to EBS due to KRT5 mutations (p < .05). Mutations in COL17A1 and laminin 332-encoding genes were identified in 55% (10/18) and 45% (8/18) of JEB patients. Scarring alopecia, caries, and EB nevi were most common among JEB patients carrying COL17A1 mutations as compared to laminin 332-associated JEB (p < .05). Abnormal nails were evident in most DEB and JEB patients while poikiloderma was exclusively observed in KEB (p < .001). EB patients of Middle Eastern origin were found to feature specific phenotype-genotype correlations of relevance to the diagnosis and genetic counseling of patients in this region.

Allele-Specific Inactivation of an Autosomal Dominant Epidermolysis Bullosa Simplex Mutation Using CRISPR-Cas9.

Epidermolysis bullosa simplex (EBS) is a rare mechanobullous disease caused by dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) genes. Until now, there is no cure for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system raised hope for the treatment of EBS and many other autosomal dominant diseases by mutant allele-specific gene disruption. In this study, we aim to disrupt the mutant allele for the heterozygous EBS pathogenic variation c.449T>C (p.Leu150Pro) within KRT5. This mutation generates, naturally, a novel protospacer-adjacent motif for the endonuclease Streptococcus pyogenes Cas9. Thus, we designed a single-guide RNA that guides the Cas9 to introduce a DNA cleavage of the mutant allele in patient's keratinocytes. Then, transfected cells were single-cell cloned and analyzed by deep sequencing. The expression of KRT5 and KRT14 was quantified, and the keratin intermediate filament stability was assessed. Results showed successful stringent mutant allele-specific knockout. An absence of synthesis of mutant transcript was further confirmed indicating permanent mutant allele-specific inactivation. Edited EBS patient keratinocytes produced a lower amount of K5 and K14 proteins compared with nonedited EBS cells, and no disturbance of cellular properties was observed.

586 New class of mTOR inhibitor stabilizes intermediate filament networks in severe epidermolysis bullosa simplex keratinocytes

Epidermolysis Bullosa Simplex (EBS) is a rare genodermatosis caused by mutations in either KRT5 or KRT14. Severe EBS (i.e., EBS Dowling-Meara) is characterized by extremely fragile skin with frequent and severe herpetiform blistering. Genetic mutations cause poor keratin 5/14 dimerization resulting in mechanically weakened intermediate filament (IF) networks that collapse into intracellular aggregates upon stress (e.g., scratching, UV). Enhanced cellular removal and degradation of mutant keratins through stimulation of autophagy and the UPS system has the potential to strengthen the IF networks, reducing blistering and erosions. Previously, UPS cochaperones (e.g., 4-phenylbuterate) were shown to decreased keratin aggregation but also decreased keratinocyte adhesion and migration. The purpose of this research was to evaluate if BM-3103, (4-Hydroxy 4'-methyoxytolan), a known wound healing promoter, anti-inflammatory, and autophagy inducer, could remove mutant keratins/aggregates thereby strengthening the IF network without adversely affecting cell survival, adhesion, or migration. Assays tested on patient derived EBS keratinocytes included: (1) Assessment of cytotoxicity, adhesion, and migration effects of BM-3103 using MTT, LDH, and scratch assays. (2) Autophagy induction (LC3-II conversion) and keratin aggregation (heat stress assay). There were no signs of cytotoxicity, loss of cell adhesion or slowed migration. Cells were exposed to escalating concentrations of BM-3103 for 24 hrs then heat shocked (43°C for 30min) generating an abundance of aggregates visualized by immunofluorescent microscopy. Untreated keratinocytes displayed keratin aggregate formation in 72% of KRT5 and 87% of KRT14 mutant cells. Treatment with BM-3103 reduced the incidence of keratin aggregates to 16% (p<0.01) of KRT5 and 24% (p<0.01) KRT14 mutant cells. BM-3103 is the first compound identified that is capable of stabilizing IF networks in keratinocytes from severe EBS patients. A clinical trial is ongoing to assess if BM-3103, can heal blistering areas and strengthen EBS patient skin.

Generation of two induced pluripotent stem cell lines (UQACi002-A and UQACi005-A) from two patients with KRT14 epidermolysis bullosa simplex mutations

More than 107 pathogenic variations were identified in Keratin 14 gene (KRT14) in patients affected by epidermolysis bullosa simplex (EBS), a rare skin disease with still no curative treatment. Disease models as human induced pluripotent stem cells (hiPSCs) are promising tool for further advance the knowledge about this disorder and accelerate therapies development. Here, two hiPSC lines were reprogrammed from skin fibroblasts of two EBS patients carrying mutations within KRT14 by using CytoTune®Sendai virus. These iPSCs display pluripotent cell morphology, pluripotent markers expression, and the capability to differentiate into the three germ layers.

Generation of three induced pluripotent stem cell lines (UQACi003-A, UQACi004-A, and UQACi006-A) from three patients with KRT5 epidermolysis bullosa simplex mutations

Heterozygous mutations within Keratin 5 (KRT5) are common genetic causes of epidermolysis bullosa simplex (EBS), a skin fragility disorder characterized by blisters, which appear after minor trauma. Using CytoTune®Sendai virus, we generated three human induced pluripotent stem cell (iPSC) lines from three EBS patients carrying respectively the single heterozygous mutations in KRT5, c.449 T > C, c.980 T > C, and c.608 T > C. All lines display normal karyotype, expressed high levels of pluripotent markers, and can differentiate into derivatives of the three germ layers. These iPSCs are helpful for a better understanding of the EBS pathogenesis and developing novel therapeutic approaches.

A cell-based drug discovery assay identifies inhibition of cell stress responses as a new approach to treatment of epidermolysis bullosa simplex.

ABSTRACTIn the skin fragility disorder epidermolysis bullosa simplex (EBS), mutations in keratin 14 (K14, also known as KRT14) or keratin 5 (K5, also known as KRT5) lead to keratinocyte rupture and skin blistering. Severe forms of EBS are associated with cytoplasmic protein aggregates, with elevated kinase activation of ERK1 and ERK2 (ERK1/2; also known as MAPK3 and MAPK1, respectively), suggesting intrinsic stress caused by misfolded keratin protein. Human keratinocyte EBS reporter cells stably expressing GFP-tagged EBS-mimetic mutant K14 were used to optimize a semi-automated system to quantify the effects of test compounds on keratin aggregates. Screening of a protein kinase inhibitor library identified several candidates that reduced aggregates and impacted on epidermal growth factor receptor (EGFR) signalling. EGF ligand exposure induced keratin aggregates in EBS reporter keratinocytes, which was reversible by EGFR inhibition. EBS keratinocytes treated with a known EGFR inhibitor, afatinib, were driven out of activation and towards quiescence with minimal cell death. Aggregate reduction was accompanied by denser keratin filament networks with enhanced intercellular cohesion and resilience, which when extrapolated to a whole tissue context would predict reduced epidermal fragility in EBS patients. This assay system provides a powerful tool for discovery and development of new pathway intervention therapeutic avenues for EBS.

Identification of a de novo mutation in KRT5 gene underlying epidermolysis bullosa simplex by whole exome sequencing in a Vietnamese patient

Epidermolysis bullosa simplex (EBS) is a group of epidermolysis bullosa (EB) and accounts for 75-85% EB cases. Most EBS patients are caused by mutations in KRT5 or KRT14, encoding for keratin 5 and keratin 14, respectively, which impair the structural entirety of paired intermediate filaments expressed in the fracture of basal keratinocytes and subsequent blistering of the epithelium. This study aimed to identify the causative mutation in a Vietnamese EB case. Whole exome sequencing (WES) was performed in the affected individual and revealed a de novo heterozygous pathogenic mutation in exon 7 of KRT5 gene, resulting in an amino acid change at position 477, with glutamic acid to lysine substitution (p.E477K). The KRT5 p.E477K was strong associated with the very severe or lethal of generalized severe EBS (GS-EBS), characterized by the severe symptoms at birth, improving with age and evolution to palmoplantar keratoderma and nail dysplasia. Our finding will aid the molecular diagnosis, prognosis prediction of the patient with GS-EBS due to p.E477K and significant genetic counselling the family concerning the recurrence risk for future pregnancies.

Next-generation sequencing through multigene panel testing for the diagnosis of hereditary epidermolysis bullosa in Chinese population.

Epidermolysis bullosa (EB) is a heritable blistering disorder. We performed a next-generation sequencing-based multigene panel test and successfully predicted 100% of the EB types, including, 36 EB simplex (EBS), 13 junctional EB (JEB), 86 dystrophic EB (DEB), and 3 Kindler EB. Chinese JEB and recessive DEB (RDEB) patients have relatively mild phenotypes; for severe type separately accounts for 45.5% and 23.8%, respectively. We identified 96 novel and 49 recurrent pathogenic variants in 11 genes, although we failed to detect the second mutation in one JEB and five RDEB patients. We identified one novel p.E475K mosaic mutation in the clinically normal mother of one out of 13 EBS patients with KRT5 mutations, one recurrent p.G2034R mosaic mutation, and one novel p.G2043R mosaic mutation in the clinically normal relatives of two out of 19 dominant DEB patients. This study shows that next-generation technology could be an effective tool in diagnosing EB.

Generation of a human induced pluripotent stem cell line (UQACi001-A) from a severe epidermolysis bullosa simplex patient with the heterozygous mutation p.R125S in the KRT14 gene.

We have generated UQACi001-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with the generalized severe epidermolysis bullosa simplex phenotype (EBS-gen sev) and carrying the keratin 14 (K14) R125S mutation. Fibroblasts were reprogrammed using non-integrating Sendai virus vectors. The iPSC line displayed normal molecular karyotype, expressed pluripotency markers, is capable of differentiating into three embryonic germ layers and is genetically identical to the originating parental fibroblasts. The established iPSC model provides a valuable resource for studying the rare disease of epidermolysis bullosa simplex and developing new therapies as DNA editing by CRISPR/Cas9 technology.

An overview of the genetic basis of epidermolysis bullosa in Brazil: discovery of novel and recurrent disease-causing variants.

Epidermolysis bullosa (EB) is a genodermatosis that encompasses a group of clinically and genetically heterogeneous disorders classified in four major types: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and Kindler syndrome. Our aim was to characterize recurrent and novel mutations associated to EB in a sample of Brazilian patients. Eighty-seven patients (25 EBS, 4 JEB and 58 DEB) were studied. We performed a next-generation sequencing-based multigene panel through ion torrent technology including 11 genes: KRT5, KRT14, PLEC, TGM5, LAMA3, LAMB3, LAMC2, COL17A1, ITGB4, COL7A1, and FERMT1. A total of 72 different pathogenic or likely pathogenic variants were identified, 32 of them are novel. The causal variant was detected in 82 patients (efficiency of 94.3%). Pathogenic variants in the residue 125 of KRT14 were identified in 32% of all EBS patients. In DEB patients, four COL7A1 variants were quite frequent, some of them clustered in specific Brazilian regions. Our study extends the spectrum of known mutations in EB and describes, for the first time, the genetic profile of EB patients from Brazil.

Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

811 Cardiomyopathy in epidermolysis bullosa simplex patients with mutations in KLHL24 gene

811 Cardiomyopathy in epidermolysis bullosa simplex patients with mutations in KLHL24 gene

752 Simultaneous gene-editing and reprogramming of epidermolysis bullosa simplex fibroblasts

Epidermolysis bullosa simplex (EBS) is an autosomal dominant skin fragility disorder caused by mutations in either Keratin (K) 5 or K14. Although current therapy for EBS is limited to wound care, advances in reprogramming somatic cells into induced pluripotent stem cells (iPSC) offer the possibility of developing new approaches for EBS treatment. The iPSC-based therapeutic approach for EBS relies on the generation of patient-specific iPSCs, which then undergo genetic editing and differentiation into skin cells suitable for transplantation. One of the main hurdles in advancing the iPSC-based therapy into the clinic is the complexity of a multi-step manufacturing process to produce genetically corrected iPSC-derived epidermal sheets. To reduce the manufacturing complexity of an iPSC-based therapy for EBS, we have combined our previously presented high-efficiency RNA-based reprogramming approach with a specific CRISPR/Cas9-mediated knock-out of the mutant allele of the K14 gene into a one-step procedure. We established a fibroblast line from an EBS patient carrying a hot-spot heterozygous C373T mutation at codon 125 in K14 and successfully generated multiple isogenic EBS iPSCs lines with a specific knock out of the mutant K14 allele (KO EBS iPSC) within 4 weeks of initiating simultaneous gene editing and reprogramming. Simultaneous reprogramming and gene correction not only reduces the complexity of the iPSC-based therapy but also improves safety of the approach by avoiding lengthy cell culture periods, drug selection, and multiple sub-cloning steps. We are currently differentiating these KO EBS iPSCs into keratinocytes to determine if these gene edited iPSC-derived keratinocytes can be used for EBS therapy by employing a mouse xenograft model.

IL-6/IL-10 Ratio as A Prognostic and Predictive Marker of the Severity of Inherited Epidermolysis Bullosa.

Recent studies have shown that cytokines have an important role in the pathogenesis of inflammatory diseases and can be used as prognostic markers. To evaluate the IL-6/IL-10 ratio in patients with Inherited Epidermolysis Bullosa (EB) as a prognostic marker. Serum levels of IL-6 and IL-10 were measured in 13 patients with recessive dystrophic EB (RDEB) as well as 10 with EB Simplex (EBS), and in 18 healthy subjects. Receiver Operating Characteristics (ROC) analyses were used to assess the diagnostic accuracy of the IL-6/IL-10 ratio for detecting severe form of EB. The IL-6/IL-10 ratio was statistically higher in RDEB patients than in EBS patients and healthy subjects. The IL-6/IL-10 ratio significantly correlated with BEBS score. Our findings suggest that IL-6/IL-10 ratio >5.6 has a good diagnostic accuracy to identify patients with the highest severity of disease.

178 Cooperation of keratin mutations and posttranslational modifications

The skin fragility disorder Epidermolysis bullosa simplex (EBS) mostly results from KRT5 and KRT14 mutations which cause aggregation of the keratin cytoskeleton and render the skin sensitive to mild trauma, followed by recurrent blisters. The prevalence of more serious disease during warmer summer months in most EBS patients suggests that temperature-sensing mechanisms cooperating with keratin mutations contribute to this phenomenon. In order to dissect the underlying mechanism and to exploit this for a therapy approach, we analyzed several KRT5 and KRT14 mutations in a cell culture based heat stress assay. Hence, we identified a subset of KRT5 mutations forming extensive keratin networks with KRT14 at normal culture conditions but reversible aggregation of the keratin cytoskeleton upon short exposure to elevated temperature. This heat-induced aggregation of the keratin cytoskeleton subsequently led to impaired desmosomes and diminished intercellular adhesion. A phosphoproteomic approach revealed elevated Ser phosphorylation of mutant KRT5, but not KRT14, upon brief heat exposure, indicating the involvement of specific kinases in the heat-induced keratin aggregation. Currently, we are investigating kinase inhibitors to prevent the heat-induced filament collapse. Moreover, parthenolide, a plant sesquiterpene lactone that exerts anti-cancer and anti-inflammatory properties and modulates sirtuin function, was found to affect keratin organization, likely by reducing elevated Lys acetylation of mutant keratins. Most importantly, pre-treatment of a temperature-sensitive KRT5 mutant with parthenolide prevented heat-induced keratin aggregation and strengthened desmosome adhesion. Collectively, we show that keratin mutations promote select posttranslational modifications, partially in combination with elevated temperature. Our assay system and results enable the development of a rational therapy for the improvement of EBS.

Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

One amino acid deletion in collagen XVII-binding domain of plectin with a truncation mutation underlies epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a group of congenital skin fragility disorders characterized by intraepidermal skin detachment. PLEC, encoding plectin, is one of the causative genes responsible for EBS. Plectin deficiency is responsible for two autosomal recessive EBS subtypes: EBS with muscular dystrophy (EBS-MD) and EBS with pyloric atresia (EBS-PA). Plectin is a linker protein between hemidesmosomes and intermediate filaments. Plectin harbors a central rod domain which mediates homodimer formation. Plectin transcripts include both full-length and rodless forms. Previous studies indicate that loss of full-length plectin with the maintenance of rodless plectin leads to EBS-MD, whereas both full-length and rodless plectin isoforms are deficient in EBS-PA. However, a few EBS-MD cases have been reported to have in-frame deletions in the N-terminal globular domain of plectin. The Y domain of plectin (Arg618 to Val819) was implicated in binding to collagen XVII (COL17) in yeast two-hybrid assays. However the relevance of plectin-COL17 binding in EBS has not been uncovered. Here, we introduce an EBS patient harboring one amino acid deletion in the N-terminus globular domain (p.Phe755del) and a truncation mutation (p.Leu3065fs) in plectin. Plectin expression in skin specimens and cultured keratinocytes from the patient was diminished at the protein and the mRNA levels. COL17 expression was reduced at the protein level, whereas no pathogenic mutation was detected in the COL17A1 gene. Phe755, a deleted amino acid in one allele of the patient, is located in the Y domain of plectin (Arg618 to Val819). Our study is the first to demonstrate that defects of plectin-COL17 binding might contribute to the EBS phenotype.

167 Long-term in vivo correction of a recessive dystrophic epidermolysis bullosa phenotype using RNA trans-splicing repair

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.

169 Regeneration of a functional epidermis at a large, long-standing wound by gene-corrected autologous epidermal stem cells

164 CRISPR/Cas9-mediated gene repair in the COL7A1 gene S Hainzl, T Kocher, EM Murauer, F Larcher, M Steiner, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria, 2 Department of Dermatology, Paracelsus Medical University, Salzburg, Austria, 3 Epithelial Biomedicine Division, CIEMAT, Madrid, Spain and 4 3rd Medical Department, Paracelsus Medical University Salzburg, Austria, Laboratory for Immunological and Molecular Cancer Research, Salzburg, Austria The CRISPR/Cas9 system turned out to be a powerful tool for genome editing and is therefore a promising option for the specific repair of gene mutations causing the blistering skin disease epidermolysis bullosa (EB). We have exploited the CRISPR/Cas9-mediated homologydirected repair (HDR) approach for the correction of a homozygous mutation in COL7A1 exon 80, leading to a complete loss of type VII collagen within the basement membrane zone of the skin. We have predicted a guide RNA (gRNA) specific for intron 80 of COL7A1, which was then cloned either into a wild-type Cas9 dual vector system, inducing double strand breaks, or a D10A Cas9 dual vector system, causing single strand breaks within the target intron. Homology COL7A1 arms for HDR were cloned into a donor vector, including a selection cassette. Transfected patient keratinocytes were selected either via antibiotic selection or fluorescent-activated cell sorting (FACS). RT-PCR on genomic DNA of treated cells and subsequent restriction enzyme digest analysis of the resulting PCR products showed the genetic correction of the COL7A1 mutation. The mutation-specific enzymatic digest revealed the presence of 26% reverted alleles. Additionally, type VII collagen restoration was confirmed via Western blot analysis and immunofluorescence staining. Our data indicate that genome editing using the CRISPR/Cas9 system can be an elegant tool for the repair of genes involved in the severe skin disease epidermolysis bullosa. 165 Combining antisense molecules with splicing modulation for KRT14 repair in epidermolysis bullosa B Liemberger, C Arzt, S Hainzl, J Pinon Hofbauer, V Wally, EM Murauer, JW Bauer, J Reichelt and U Koller 1 EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria and 2 Department of Dermatology, Paracelsus Medical University Salzburg, Austria, Salzburg, Austria Epidermolysis bullosa (EB) is an inherited skin disease characterized by severe blistering of the skin and mucous membranes after minor mechanical trauma. In EB simplex (EBS) dominant mutations within KRT14 encoding keratin 14 lead to loss of integrity of the intermediate filament network within basal keratinocytes. In order to correct a dominant mutation in an EBS patient cell line, we exploited the RNA trans-splicing technology which utilizes the cell’s endogenous splicing machinery to facilitate a trans-splicing reaction between two RNAs, generating a new chimeric product at the pre-mRNA level. The designed RNA transsplicing molecule (RTM), containing a binding domain, splicing elements, and the wild-type KRT14 region to be introduced, has already been shown to be able to partially revert the EBS phenotype at the cellular level. In order to increase the repair efficiency mediated by RNA trans-splicing, antisense RNAs (asRNAs) specific for splice sites or splicing enhancer sequences within the KRT14 target pre-mRNA region were randomly generated with the aim of blocking these cis-splicing elements thereby increasing the trans-splicing rate. We analysed the functionality of 76 individual asRNAs by exploiting our fluorescence-based screening system, in which reconstitution of a GFP signal acts as a readout for accurate trans-splicing between a generated KRT14 minigene (KRT14-MG) and the RTM. Triple transfection experiments in HEK293 cells of the KRT14-MG, the RTM, and the most functional asRNA revealed an increase in trans-splicing efficiency of up to 7 fold as monitored by flow cytometry. Our data suggest that the inclusion of asRNAs can increase the trans-splicing efficiency to levels needed to overcome the EBS phenotype.