Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

5'RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa.

Mutations within the COL7A1 gene underlie the inherited recessive subtype of the blistering skin disease dystrophic epidermolysis bullosa (RDEB). Although gene replacement approaches for genodermatoses are clinically advanced, their implementation for RDEB is challenging and requires endogenous regulation of transgene expression. Thus, we are using spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutations in COL7A1 at the mRNA level. Here, we demonstrate the capability of a COL7A1-specific RNA trans-splicing molecule (RTM), initially selected using a fluorescence-based screening procedure, to accurately replace COL7A1 exons 1 to 64 in an endogenous setting. Retroviral RTM transduction into patient-derived, immortalized keratinocytes resulted in an increase in wild-type transcript and protein levels, respectively. Furthermore, we revealed accurate deposition of recovered type VII collagen protein within the basement membrane zone of expanded skin equivalents using immunofluorescence staining. In summary, we showed for the first time the potential of endogenous 5' trans-splicing to correct pathogenic mutations within the COL7A1 gene. Therefore, we consider 5' RNA trans-splicing a suitable tool to beneficially modulate the RDEB-phenotype, thus targeting an urgent need of this patient population.

[Wound therapy with cold atmospheric plasma in severe recessive dystrophic epidermolysis bullosa : A pilot study]. / Wundtherapie mit kaltem Plasma bei Epidermolysis bullosa dystrophica : Eine Pilotuntersuchung.

BACKGROUND: Cold atmospheric pressure plasma (CAP) has antimicrobial and wound-healing properties. Patients affected by severe autosomal recessive dystrophic epidermolysis bullosa (RDEB) suffer from widespread, difficult-to-treat wounds, which require complex wound management. OBJECTIVE: In a pilot project, we investigated over a period of 5 months the response and tolerability of a CAP wound therapy in a 21-year-old and a 28-year-old female patient with severe generalized RDEB and following cutaneous squamous cell cancer (cSSC) in the older patient. MATERIALS AND METHODS: In both patients, diagnosis of RDEB was confirmed by molecular genetics. Individual- and patient-specific wound therapy was continued during the study period, and additionally CAP therapy with a dielectric barrier discharge (DBE) device was initiated. CAP treatment was performed for 90 s per wound and could be applied every day or every other day. Clinical evaluation included photographic documentation and regular interviews of patients and parents. RESULTS: CAP-treated wounds largely demonstrated improved wound healing and signs of a reduced bacterial contamination. Furthermore, CAP proved to prevent wound chronification. When applied on a polyester mesh, it was well-tolerated on most body sites. CONCLUSION: The introduction of CAP could improve the wound management of EB patients and should be evaluated in clinical studies. The effect of CAP on cSSC development should be particularly studied.

Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa.

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal-epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases.

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

Trans-splicing improvement by the combined application of antisense strategies.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

Pseudosyndactyly - an inflammatory and fibrotic wound healing disorder in recessive dystrophic epidermolysis bullosa.

BACKGROUND: A genetic blistering skin disease, recessive dystrophic epidermolysis bullosa (RDEB), is marked by severe wound healing defects and finger contractures. The purpose of this investigation was to elucidate the mechanisms of impaired wound healing and pseudosyndactyly occurring in RDEB patients by studying the role of known inflammation and fibrosis markers in RDEB pseudosyndactyly tissue. PATIENTS AND METHODS: We studied the expression of the fibrosis and/or inflammation markers tenascin-C, α-smooth muscle actin, transforming growth factor-ß1, interleukin-1ß, and interleukin-6 in scarring and nonscarring tissue from healthy donors and RDEB patients by semiquantitative real time-PCR and, where applicable, by immunoblots. Furthermore, the distribution pattern of α-smooth muscle actin and tenascin-C were assessed by immunofluorescence microscopy. RESULTS: Based on mRNA and protein analysis, we found upregulation of tenascin-C, interleukin-1ß, and interleukin-6 - but not of transforming growth factor-ß1 - in recessive dystrophic epidermolysis bullosa scar samples taken from pseudosyndactyly hands. Unexpectedly, α-smooth muscle actin was not upregulated. CONCLUSIONS: Our results confirm inflammation and fibrosis in recessive dystrophic epidermolysis bullosa, especially in scars, suggesting major roles for these processes in pseudosyndactyly. Our data therefore suggests the potential use of antiinflammatory and antifibrotic drugs in the prevention of pseudosyndactyly.

Orofacial Anomalies in Kindler Epidermolysis Bullosa.

Importance: Kindler epidermolysis bullosa is a genetic skin-blistering disease associated with recessive inherited pathogenic variants in FERMT1, which encodes kindlin-1. Severe orofacial manifestations of Kindler epidermolysis bullosa, including early oral squamous cell carcinoma, have been reported. Objective: To determine whether hypoplastic pitted amelogenesis imperfecta is a feature of Kindler epidermolysis bullosa. Design, Settings, and Participants: This longitudinal, 2-center cohort study was performed from 2003 to 2023 at the Epidermolysis Bullosa Centre, University of Freiburg, Germany, and the Special Care Dentistry Clinic, University of Chile in association with DEBRA Chile. Participants included a convenience sampling of all patients with a diagnosis of Kindler epidermolysis bullosa. Main Outcomes and Measures: The primary outcomes were the presence of hypoplastic pitted amelogenesis imperfecta, intraoral wounds, gingivitis and periodontal disease, gingival hyperplasia, vestibular obliteration, cheilitis, angular cheilitis, chronic lip wounds, microstomia, and oral squamous cell carcinoma. Results: The cohort consisted of 36 patients (15 female [42%] and 21 male [58%]; mean age at first examination, 23 years [range, 2 weeks to 70 years]) with Kindler epidermolysis bullosa. The follow-up ranged from 1 to 24 years. The enamel structure was assessed in 11 patients, all of whom presented with enamel structure abnormalities. The severity of hypoplastic pitted amelogenesis imperfecta varied from generalized to localized pitting. Additional orofacial features observed include gingivitis and periodontal disease, which was present in 90% (27 of 30 patients) of those assessed, followed by intraoral lesions (16 of 22 patients [73%]), angular cheilitis (24 of 33 patients [73%]), cheilitis (22 of 34 patients [65%]), gingival overgrowth (17 of 26 patients [65%]), microstomia (14 of 25 patients [56%]), and vestibular obliteration (8 of 16 patients [50%]). Other features included chronic lip ulcers (2 patients) and oral squamous cell carcinoma with lethal outcome (2 patients). Conclusions and Relevance: These findings suggest that hypoplastic pitted amelogenesis imperfecta is a feature of Kindler epidermolysis bullosa and underscore the extent and severity of oral manifestations in Kindler epidermolysis bullosa and the need for early and sustained dental care.

A novel screening system improves genetic correction by internal exon replacement.

Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5', 3' or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered 'RNA trans-splicing molecules' (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51-intron 51-exon 52-intron 52-exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement.

Novel and recurrent COL7A1 mutation in a Polish population.

Dystrophic Epidermolysis Bullosa (DEB) is a rare bullous genodermatosis caused by mutations in COL7A1, which encodes collagen type VII, the main component of anchoring fibrilis. DEB is inherited in an autosomal recessive and dominant manner, depending on the mutation type and localization. The aim of this study was to update the spectrum and frequency of COL7A1 mutations in a cohort of 42 Polish DEB patients. Using direct sequencing strategy we identified 25 different mutations, which gave us a detection rate of about 88%. In total, thirteen novel variants were identified, including three de novo mutations (p.G2680S, p.G2043R and p.Gly2064_Arg2069del). The panel of recessively inherited DEB causing recurrent mutations comprise of five variants: c.425A>G, c.682+1G>A, p.R2069C, p.W796X and, unreported before, c.7154delC, which accounts for about 59% of all mutated alleles in this group. In the dominant type of DEB, only p.G2043R was found to be recurrent and it was identified in 50% patients. Our results give further insight into the pathogenesis and epidemiology of DEB.

Recessive Dystrophic Epidermolysis bullosa due to Hemizygous 40 kb Deletion of COL7A1 and the Proximate PFKFB4 Gene Focusing on the Mutation c.425A>G Mimicking Homozygous Status

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

The international dystrophic epidermolysis bullosa patient registry: an online database of dystrophic epidermolysis bullosa patients and their COL7A1 mutations.

Dystrophic epidermolysis bullosa (DEB) is a heritable blistering disorder that can be inherited autosomal dominantly (DDEB) or recessively (RDEB) and covers a group of several distinctive phenotypes. A large number of unique COL7A1 mutations have been shown to underlie DEB. Although general genotype-phenotype correlation rules have emerged, many exceptions to these rules exist, compromising disease diagnosing and genetic counseling. We therefore constructed the International DEB Patient Registry (http://www.deb-central.org), aimed at worldwide collection and sharing of phenotypic and genotypic information on DEB. As of May 2011, this MOLGENIS-based registry contains detailed information on 508 published and 71 unpublished patients and their 388 unique COL7A1 mutations, and includes all combinations of mutations. The current registry RDEB versus DDEB ratio of 4:1, if compared to prevalence figures, suggests underreporting of DDEB in the literature. Thirty-eight percent of mutations stored introduce a premature termination codon (PTC) and 43% an amino acid change. Submission wizards allow users to quickly and easily share novel information. This registry will be of great help in disease diagnosing and genetic counseling and will lead to novel insights, especially in the rare phenotypes of which there is often lack of understanding. Altogether, this registry will greatly benefit the DEB patients.

Epidermolysis bullosa with late-onset muscular dystrophy and plectin deficiency.

Epidermolysis bullosa associated with muscular dystrophy is a rare, autosomal recessive form of epidermolysis bullosa simplex caused by mutations in the plectin gene, PLEC1. We describe a phenotypically mild case due to compound heterozygous mutations in PLEC1 (2677_2685del and the novel mutation Q1644X). Clinical features included mild skin blistering since birth, slowly progressive and late-onset upper limb-predominant weakness, facial weakness, ptosis, incomplete ophthalmoplegia, and paroxysmal atrial fibrillation.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa.

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Impact of low-dose calcipotriol ointment on wound healing, pruritus and pain in patients with dystrophic epidermolysis bullosa: A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Wound management is a critical factor when treating patients with the inherited skin fragility disease dystrophic epidermolysis bullosa (DEB). Due to genetic defects in structural proteins, skin and mucous epithelia are prone to blistering and chronic wounding upon minor trauma. Furthermore, these wounds are commonly associated with excessive pruritus and predispose to the development of life-threatening squamous cell carcinomas, underscoring the unmet need for new therapeutic options to improve wound healing in this patient cohort. Vitamin D3 is acknowledged to play an important role in wound healing by modulating different cellular processes that impact epidermal homeostasis and immune responses. In this study, we evaluate the safety and efficacy of low-dose calcipotriol, a vitamin D3 analogue, in promoting wound healing and reducing itch and pain in patients with DEB. METHODS: Eligible DEB patients, aged ≥ 6 years and with a known mutation in the COL7A1 gene, were recruited to a placebo-controlled, randomized, double blind, cross-over phase II monocentric clinical trial. Patients were required to have at least two wounds with a minimum size of 6 cm2 per wound. The primary objective was to evaluate efficacy of daily topical application of a 0.05 µg/g calcipotriol ointment in reducing wound size within a 4-week treatment regimen. Secondary objectives were to assess safety, as well as the impact of treatment on pruritus, pain, and bacterial wound colonization in these patients. RESULTS: Six patients completed the clinical trial and were included into the final analysis. Topical low-dose calcipotriol treatment led to a significant reduction in wound area at day 14 compared to placebo (88.4% vs. 65.5%, P < 0.05). Patients also reported a significant reduction of pruritus with calcipotriol ointment compared to placebo over the entire course of the treatment as shown by itch scores of 3.16 vs 4.83 (P < 0.05) and 1.83 vs 5.52 (P < 0.0001) at days 14 and 28, respectively. Treatment with low-dose calcipotriol did not affect serum calcium levels and improved the species richness of the wound microbiome, albeit with no statistical significance. CONCLUSIONS: Our results show that topical treatment with low-dose calcipotriol can accelerate wound closure and significantly reduces itch, and can be considered a safe and readily-available option to improve local wound care in DEB patients. Trial Registration EudraCT: 2016-001,967-35. Registered 28 June 2016, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001967-35/AT.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa.

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application.