Development of Minicircle Vectors Encoding COL7A1 Gene with Human Promoters for Non-Viral Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions.

Identification of multi-exon deletion in the COL7A1 gene underlying dystrophic epidermolysis bullosa by whole-exome sequencing

Dystrophic epidermolysis bullosa (DEB) is a rare form of genodermatosis characterized by skin blisters, milia, scarring over the entire body, and nail dystrophy. In this study, a pedigree with one affected member with skin blisters, and a clinical diagnosis of epidermolysis bullosa who was a result of a non-consanguineous marriage, was investigated by whole-exome sequencing (WES). This survey revealed that the proband is a compound heterozygote for a previously reported heterozygous missense variant (c.6205C>T) and a heterozygous deletion of exons 13–24 in the COL7A1 gene. This study indicates that the use of WES along with copy number variation (CNV) analysis gives a higher diagnostic yield for such patients. Moreover, considering the autosomal recessive and dominant forms of the disease, both caused by variants in one gene, proper interpretation and classification of novel variants in heterozygous as well as homozygous states is always a major challenge.

A Novel Heterozygous Missense Mutation in COL7A1 Gene in Dystrophic Epidermolysis Bullosa

ABSTRACT Hereditary Epidermolysis Bullosa is categorized by blister formation on the skin which includes Dystrophic Epidermolysis Bullosa as one of its types. It is characterized by blister formation on hands, feet, knees, and elbows. The severe condition leads to vision loss and blemishes. The objectives included the identification of COL7A1 gene mutation for DEB susceptibility through sequence analysis of hot spot regions of 73, 74 and 75 exons. The experimental design included the enrolment of DEB-affected two families. The genetic analysis techniques included inorganic DNA extraction, Polymerase Chain Reaction and Sanger sequencing method. The study inferred the identification of novel missense mutation in exon 75 of COL7A1 gene at (c. 6223 G>T) where the aspartic acid is converted into tyrosine in the heterozygous condition in affected families. The recognized novel missense mutation is silent (heterozygous) which becomes severe when homozygous. However, whole-exome sequencing strategy may identify the causative mutations.

Signatures of Dermal Fibroblasts from RDEB Pediatric Patients

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a debilitating disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Mutations in the COL7A1 gene induce multiple abnormalities, including chronic inflammation and profibrotic changes in the skin. However, the correlations between the specific mutations in COL7A1 and their phenotypic output remain largely unexplored. The mutations in the COL7A1 gene, described here, were found in the DEB register. Among them, two homozygous mutations and two cases of compound heterozygous mutations were identified. We created the panel of primary patient-specific RDEB fibroblast lines (FEB) and compared it with control fibroblasts from healthy donors (FHC). The set of morphological features and the contraction capacity of the cells distinguished FEB from FHC. We also report the relationships between the mutations and several phenotypic traits of the FEB. Based on the analysis of the available RNA-seq data of RDEB fibroblasts, we performed an RT-qPCR gene expression analysis of our cell lines, confirming the differential status of multiple genes while uncovering the new ones. We anticipate that our panels of cell lines will be useful not only for studying RDEB signatures but also for investigating the overall mechanisms involved in disease progression.

Raloxifene and n-Acetylcysteine Ameliorate TGF-Signalling in Fibroblasts from Patients with Recessive Dominant Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disease caused by mutation of the COL7A1 gene. RDEB is associated with high levels of TGF-β1, which is likely to be involved in the fibrosis that develops in this disease. Endoglin (CD105) is a type III coreceptor for TGF-β1 and its overexpression in fibroblasts deregulates physiological Smad/Alk1/Alk5 signalling, repressing the synthesis of TGF-β1 and extracellular matrix (ECM) proteins. Raloxifene is a specific estrogen receptor modulator designated as an orphan drug for hereditary hemorrhagic telangiectasia, a rare vascular disease. Raloxifene stimulates endoglin synthesis, which could attenuate fibrosis. By contrast, the antioxidant N-acetylcysteine may have therapeutic value to rectify inflammation, fibrosis and endothelial dysfunction. Thus, we present here a repurposing strategy based on the molecular and functional screening of fibroblasts from RDEB patients with these drugs, leading us to propose the repositioning of these two well-known drugs currently in clinical use, raloxifene and N-acetylcysteine, to counteract fibrosis and inflammation in RDEB. Both compounds modulate the profibrotic events that may ultimately be responsible for the clinical manifestations in RDEB, suggesting that these findings may also be relevant for other diseases in which fibrosis is an important pathophysiological event.

A CASE REPORT OF AGGRESSIVE SQUAMOUS CELL CARCINOMA IN PATIENTS WITH RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA

Recessive dystrophic epidermolysis bullosa (RDEB) is an orphan genetic skin disease which is caused by mutations in COL7A1 gene. COL7A1 encodes collagen VII - a major component of anchoring fibrils which sustain the dermal-epidermal junction. Non-healing wounds typically are presented in patients with RDEB. They predispose cutaneous squamous cell carcinoma (SCC) development. RDEB-associated SCC is a rapidly growing tumor of aggressive nature which generally arises at young age and results in early mortality. This article reports 2 patients with RDEB-associated SCCs.