Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

Practical considerations relevant to treatment with the gene therapy beremagene geperpavec-svdt for dystrophic epidermolysis bullosa.

BACKGROUND/PURPOSE: Dystrophic epidermolysis bullosa (DEB), a rare genetic skin disease caused by loss-of-function mutations in COL7A1, the gene encoding type VII collagen (COL7), is characterized by skin blistering, scarring, and extracutaneous manifestations that markedly reduce patient quality-of-life. Beremagene geperpavec-svdt ('B-VEC') is a gene therapy employing a non-integrating, replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding two copies of full-length human COL7A1 to restore COL7 protein after topical administration to DEB wounds. B-VEC was approved in the United States in 2023 as the first topical gene therapy and the first approved treatment for DEB. However, few providers have experience with use of this gene therapy. METHODS: Data was obtained through literature review and the experience of providers who participated in the B-VEC clinical study or initiated treatment after B-VEC approval. RESULTS: This review discusses the burden of disease, describes the clinical trial outcomes of B-VEC, and provides physician and patient/caregiver recommendations as a practical guide for the real-world use of B-VEC, which can be administered in-office or at the patient's home. CONCLUSIONS: By continuing to optimize the practical aspects of B-VEC administration, the focus will continue to shift to patient-centric considerations and improved patient outcomes.

Epidermolysis Bullosa: Two rare case reports of COL7A1 and EBS-GEN SEV KRT14 variants with review of literature.

EPIDERMOLYSIS: Bullosa is a rare hereditary skin condition that causes blisters. Genes encoding structural proteins at or near the dermal-epidermal junction are mutated recessively or dominantly, and this is the primary cause of EB. Herein, two Chinese boys were diagnosed with the condition, each with a different variant in a gene that serves as a reference for EB genetic counseling. Skincare significantly impacted their prognosis and quality of life. CASE PRESENTATION: Two Chinese boys, with phenotypically normal parents, have been diagnosed with distinct blister symptoms, one with Dominant Dystrophic Epidermolysis Bullosa and the other with a severe form of Epidermolysis Bullosa Simplex. The first patient had a G-to-A variant in the COL7A1 allele, at nucleotide position 6163 which was named "G2055A". The proband is heterozygous for Dystrophic Epidermolysis Bullosa due to a COL7A1 allele with a glycine substitution at the triple helix domain. A similar variant has been discovered in his mother, indicating its potential transmission to future generations. Another patient had severe Epidermolysis Bullosa Simplex with a rare c.377T > A  variant resulting in substitution of amino acid p.Leu126Arg (NM_000526.5 (c.377T > G, p.Leu126Arg) in the Keratin 14 gene. In prior literature, Keratin 14 has been associated with an excellent prognosis. However, our patient with this infrequent variant tragically died from sepsis at 21 days old. There has been a reported occurrence of the variant only once. CONCLUSION: Our study reveals that Epidermolysis Bullosa patients with COL7A1 c.6163G > A and KRT14 c.377T>A variants have different clinical presentations, with dominant forms of Dystrophic EB having milder phenotypes than recessive ones. Thus, the better prognosis in the c.6163G > A patient. Furthermore, c.377T>A patient was more prone to infection than the patient with c.6163G>A gene variant. Genetic testing is crucial for identifying the specific variant responsible and improving treatment options.

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites.

Methods for Decreasing Preweaning Mortality in a Fragile Mouse Model of Hypomorphic Collagen VII Deficiency.

Preweaning mortality is a widespread problem in laboratory mouse breeding, particularly in the case of fragile mouse models. While numerous studies explore alternative care methods to increase the survivability of common mouse strains, there remains a paucity of research into the care of mice with fragile health conditions that result from induced or natural genetic mutations. In this study, standard husbandry practices were enhanced by the addition of a softened diet, a nutritionally fortified dietary supplement, soft bedding, gentle handling techniques, decreased handling, lengthened weaning age, and dam productivity tracking. This alternative care plan was shown to increase the survival of a fragile recessive dystrophic epidermolysis bullosa mouse model, and some aspects could be used in developing a care plan for other fragile mouse strains.

Cyclization-enhanced poly(ß-amino ester)s vectors for efficient CRISPR gene editing therapy.

Among non-viral gene delivery vectors, poly(ß-amino ester)s (PAEs) are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. Over two decades, PAEs have evolved from linear to highly branched structures, significantly enhancing gene delivery efficacy. Building on the proven efficient sets of monomers in highly branched PAEs (HPAEs), this work introduced a new class of cyclic PAEs (CPAEs) constructed via an A2 + B4 + C2 cyclization synthesis strategy and identified their markedly improved gene transfection capabilities in gene delivery applications. Two sets of cyclic PAEs (CPAEs) with rings of different sizes and topologies were obtained. Their chemical structures were confirmed via two-dimensional nuclear magnetic resonance and the photoluminescence phenomena, and their DNA delivery behaviours were investigated and compared with the HPAE counterparts. In vitro assessments demonstrated that the CPAEs with a macrocyclic architecture (MCPAEs), significantly enhanced DNA intracellular uptake and facilitated efficient gene expression while maintaining perfect biocompatibility. The top-performance MCPAEs have been further employed to deliver a plasmid coding dual single guide RNA-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. In recessive dystrophic epidermolysis bullosa (RDEB) patient-derived epidermal keratinocytes, MCPAEs facilitated the CRISPR plasmid delivery and achieved efficient targeted gene editing in multiple colonies.

Advantages of whole-exome sequencing over immunomapping in 67 Brazilian patients with epidermolysis bullosa.

BACKGROUND: Epidermolysis bullosa (EB) is characterized by skin fragility and blistering. In Brazil, the diagnosis is usually obtained through immunomapping, which involves a skin biopsy. Most recently, whole exome sequencing (WES) has become an important tool for the diagnosis of the subtypes of EB, providing information on prognosis as well as allowing appropriate genetic counseling for the families. OBJECTIVE: To compare the results of immunomapping and molecular analysis and to describe the characteristics of a Brazilian cohort of patients with EB. METHODS: Patients were submitted to clinical evaluation and WES using peripheral blood samples. WES results were compared to those obtained from immunomapping testing from skin biopsies. RESULTS: 67 patients from 60 families were classified: 47 patients with recessive dystrophic EB (DEB), 4 with dominant DEB, 15 with EB simplex (EBS), and 1 with junctional EB (JEB). Novel causative variants were: 10/60 (16%) in COL7A1 associated with recessive DEB and 3 other variants in dominant DEB; one homozygous variant in KRT5 and another homozygous variant in PLEC, both associated with EBS. Immunomapping was available for 59 of the 67 patients and the results were concordant with exome results in 37 (62%), discordant in 13 (22%), and inconclusive in 9 patients (15%). STUDY LIMITATIONS: Even though EB is a rare disease, for statistical purposes, the number of patients evaluated by this cohort can still be considered limited; other than that, there was a significant difference between the proportion of types of EB (only one case with JEB, against more than 50 with DEB), which unfortunately represents a selection bias. Also, for a small subset of families, segregation (usually through Sanger sequencing) was not an option, usually due to deceased or unknown parent status (mostly the father). CONCLUSION: Although immunomapping has been useful in services where molecular studies are not available, this invasive method may provide a misdiagnosis or an inconclusive result in about 1/3 of the patients. This study shows that WES is an effective method for the diagnosis and genetic counseling of EB patients.

Ocular Gene Therapy in a Patient with Dystrophic Epidermolysis Bullosa.

Dystrophic epidermolysis bullosa is a rare genetic disease caused by damaging variants in COL7A1, which encodes type VII collagen. Blistering and scarring of the ocular surface develop, potentially leading to blindness. Beremagene geperpavec (B-VEC) is a replication-deficient herpes simplex virus type 1-based gene therapy engineered to deliver functional human type VII collagen. Here, we report the case of a patient with cicatrizing conjunctivitis in both eyes caused by dystrophic epidermolysis bullosa who received ophthalmic administration of B-VEC, which was associated with improved visual acuity after surgery.

Induced pluripotent stem cell (iPSC) line MLi005-A derived from a patient with dominant dystrophic epidermolysis bullosa (DDEB).

We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi005-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types.

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).

Exploring potential genes and mechanisms linking erectile dysfunction and depression.

Background: The clinical correlation between erectile dysfunction (ED) and depression has been revealed in cumulative studies. However, the evidence of shared mechanisms between them was insufficient. This study aimed to explore common transcriptomic alterations associated with ED and depression. Materials and methods: The gene sets associated with ED and depression were collected from the Gene Expression Omnibus (GEO) database. Comparative analysis was conducted to obtain common genes. Using R software and other appropriate tools, we conducted a range of analyses, including function enrichment, interactive network creation, gene cluster analysis, and transcriptional and post-transcriptional signature profiling. Candidate hub crosslinks between ED and depression were selected after external validation and molecular experiments. Furthermore, subpopulation location and disease association of hub genes were explored. Results: A total of 85 common genes were identified between ED and depression. These genes strongly correlate with cell adhesion, redox homeostasis, reactive oxygen species metabolic process, and neuronal cell body. An interactive network consisting of 80 proteins and 216 interactions was thereby developed. Analysis of the proteomic signature of common genes highlighted eight major shared genes: CLDN5, COL7A1, LDHA, MAP2K2, RETSAT, SEMA3A, TAGLN, and TBC1D1. These genes were involved in blood vessel morphogenesis and muscle cell activity. A subsequent transcription factor (TF)-miRNA network showed 47 TFs and 88 miRNAs relevant to shared genes. Finally, CLDN5 and TBC1D1 were well-validated and identified as the hub crosslinks between ED and depression. These genes had specific subpopulation locations in the corpus cavernosum and brain tissue, respectively. Conclusion: Our study is the first to investigate common transcriptomic alterations and the shared biological roles of ED and depression. The findings of this study provide insights into the referential molecular mechanisms underlying the co-existence between depression and ED.

Identification of a novel COL7A1 variant associated with dystrophic epidermolysis bullosa pruriginosa responding effectively to dupilumab.

BACKGROUND: Variants in COL7A1 cause an extremely rare and clinically heterogeneous syndrome known as dystrophic epidermolysis bullosa pruriginosa (DEB-Pr). Duplilumab, a fully humanized anti-IL-4Ra monoclonal antibody, can inhibit IL-4 and IL-13-driven signaling. METHODS: Ethical Compliance: Following our Institutional Review Board, genetic testing has been made available after completing a signed informed consent form. This article presents the case study of a DEB-Pr patient who received dupilumab therapy. Genomic DNA was extracted from the peripheral blood of the patient. RESULTS: The findings showed that a unique COL7A1 mutation was discovered in the patient who underwent genetic testing. As a result of the patient receiving dupilumab treatment, the individual reported experiencing significantly less itching and considerably improved erythema, less severe scales, crusts, and flattening of plaques. CONCLUSION: In conclusion, the current investigation showed that to the best of our knowledge, this is the first DEB-Pr patient with heterozygous COL7A1 (NM_000094.3:c.8110G>A [p. Gly2704Arg]) who responded positively to dupilumab treatment without experiencing any serious side effects.

Clinical and allelic heterogeneity in dystrophic epidermolysis bullosa- lessons from an Indian cohort.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is due to variation in the COL7A1 gene. The clinical phenotype and severity depends on the type of variation and domain of the affected protein. OBJECTIVES: To characterize the spectrum of COL7A1 variations in a cohort of DEB patients from India, to correlate these findings with clinical phenotypes and to establish a genotype-phenotype correlation. METHODS: This was a retrospective, observational study involving patients with DEB diagnosed on the basis of clinical manifestations, Immuno-fluorescence antigen mapping (IFM) and genetic analysis. A genotype-phenotype correlation was attempted and observations were further explained using IFM on skin biopsies and molecular dynamic simulations. Descriptive statistics were performed using SPSS version 20.0 with P values of <0.05 considered significant. RESULTS: We report 68 unrelated Indian DEB patients classified as RDEB-Intermediate (RDEB-I), RDEB-Severe (RDEB-S) or DDEB based on the EB diagnostic matrix, immunofluorescence antigen mapping and genetic data. Of 68 DEB patients, 59 (86.76%) were inherited in a recessive pattern (RDEB) and 9 (13.24%) in a dominant pattern (DDEB). Limbal stem cell deficiency was seen in four cases of RDEB-S very early in the course of the disease. A total of 88 variants were detected of which 66 were novel. There were no hotspots and recurrent variations were seen in a very small group of patients. We found a high frequency of compound heterozygotes (CH) in RDEB patients born out of non-consanguineous marriage. RDEB patients older than two years who had oral mucosal involvement, and/or deformities, were more likely to have esophageal involvement. Genotype phenotype correlation showed a higher frequency of extracutaneous manifestations and deformities in patients with Premature Termination Codons (PTCs) than in patients with other variations. Molecular simulation studies in patients with missense mutations showed severe phenotype when they were localized in interrupted regions of GLY-X-Y repeats. CONCLUSION: This large study of DEB patients in South Asia adds to the continually expanding genetic database of this condition. This study has direct implications on management as this group of patients can be screened early and managed appropriately.

Beremagene Geperpavec: First Approval.

Beremagene geperpavec-svdt (VYJUVEK™) is a topically applied, redosable, live, replication defective herpes simplex virus-1 (HSV-1) vector -based gene therapy that is being developed by Krystal Biotech to deliver functional human collagen type VII alpha 1 chain (COL7A1) genes in patients with both, dominant and recessive dystrophic epidermolysis bullosa. Beremagene geperpavec can transduce both keratinocytes and fibroblasts and restore functional COL7 protein. In May 2023, beremagene geperpavec received its first approval in the US for the treatment of wounds in patients ≥ 6 months of age with dystrophic epidermolysis bullosa with mutation(s) in the COL7A1 gene. A Marketing Authorization Application for beremagene geperpavec in Europe is planned for the second half of 2023. This article summarizes the milestones in the development of beremagene geperpavec leading to this first approval for dystrophic epidermolysis bullosa.

Management of acute sloughing of the esophageal lining in patients with dystrophic epidermolysis bullosa-A series of six pediatric patients.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a subtype of an inherited skin disorder characterized by skin and mucosal fragility due to collagen VII (COL7A1) gene mutations. Esophageal strictures leading to chronic dysphagia and acute episodes are well recognized complications within this subtype. Sloughing of esophageal mucosa and the treatment of this emergency have heretofore received limited attention in the EB literature. METHODS: We retrospectively reviewed the electronic medical records of the patients who had an acute episode of sloughing of the esophageal lining between 2008 and 2021 and extracted the information regarding their clinical presentation and management. RESULTS: Six patients out of 210 with recessive DEB severe (RDEB-S) (n = 4), RDEB intermediate (RDEB-I) (n = 1) and dominant DEB (DDEB) (n = 1) were identified. The mean age at the time of the episode was 2.7 years. All patients had early-onset severe gastroesophageal reflux. Clinically, they presented with a coughing episode (n = 6), hematemesis (n = 6), vomiting (n = 6), and choking (n = 3), followed by coughing up a string like tissue of variable length, part of the esophageal mucosal lining. Four patients recovered with medical management only, two patients required gastrostomy insertions for feeding due to severe persistent dysphagia and one also required a Nissen's fundoplication to manage severe reflux. One patient had aspiration pneumonia. CONCLUSIONS: Sloughing of varying lengths of segments of the esophagus is an emergency. The lining coughed up needs to be cut at the mouth not pulled and the emergency services called immediately for urgent assessment and management. Expert multidisciplinary care is needed to manage this rare but serious condition.

Phenotype and genotype correlation of inherited epidermolysis bullosa in Indonesia.

BACKGROUND: Inherited epidermolysis bullosa (EB) is a group of genodermatoses with considerable clinical and genetic heterogeneity. Clinical diagnosis of the EB subtypes is frequently imprecise and requires confirmation with genetic testing. There is still limited study using genetic testing to identify EB subtypes in Indonesia. This study aims to identify the pathogenic variants of inherited EB patients at the Department of Dermatology and Venereology, Universitas Padjadjaran-Dr Hasan Sadikin General Hospital in Bandung, West Java, Indonesia and to describe the correlation between the phenotype and genotype of our patients. METHODS: Twelve patients clinically diagnosed with EB were included in this study. Genetic testing was performed in collaboration with KK Women's and Children's Hospital, Singapore. RESULTS: Pathogenic variants were identified in the COL7A1 gene in seven patients, namely Dominant Dystrophic EB (DDEB) with mutation types c.5945G>T, c.6218G>A, Recessive Dystrophic EB (RDEB) c.2005C>T, c.6081dup, c.1268C>T, c.1784C>T which are all known mutations. Novel mutations were found in the COL7A1 gene in two patients namely DDEB c.6253G>T and RDEB c.6740C>T. Two EB Simplex (EBS) patients showed mutation KRT14 gene as c.356T>C, c.373C>T which are known mutation. In addition, a novel mutation in LAMA3 gene c.2649del was found in one Junctional EB (JEB) patient. CONCLUSION: The molecular diagnoses of 12 Indonesian EB patients were identified, of which three were novel pathogenic variants. Concordance between the initial clinical diagnosis and genetic testing was only 33%. This demonstrated the importance of early genetic testing for accurate diagnosis, prognostication, management and genetic counselling.