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

Abstract

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.

3D Macrocyclic Structure Boosted Gene Delivery: Multi-Cyclic Poly(β-Amino Ester)s from Step Growth Polymerization

“…Recessive dystrophic epidermolysis bullosa (RDEB) is one of the most severe subtypes of the rare debilitating skin disorder, which is caused by mutations in COL7A1 gene leading to absent, malformed or deficient type VII collagen (C7) and thus anchoring fibrils. , This results in the separation of the epidermis from the dermis following minimal trauma or friction. , Exon excision strategy has been developed as a promising approach to treat RDEB. For instance, exon 80 or exon 73, that are exons with high prevalence of mutations for the disease, have been successfully excised by the usage of CRISPR, achieving restoration of collagen VII levels .…”

Antibiotic-Free Gene Vectors: A 25-Year Journey to Clinical Trials

“…The transfection of embryonic stem-cell-derived neural stem cells (NSC) with a minicircle did not affect cell division kinetics, morphology or differentiation potential to astrocyte and neurons [ 50 ]. For a non-viral topical treatment of patients with Recessive Dystrophic Epidermolysis Bullosa (RDEB), a severe genetic skin disorder caused by mutations in the COL7A1 gene, RDEB skin cells were transfected with a minicircle expressing the large cDNA size of the COL7A1 gene (8.9-kb) to restore the expression and secretion of the structural protein-type-VII collagen (C7) at the dermal and epidermal junction [ 51 ]. The transfection of mesenchymal stem cells (MSCs) using a minicircle expressing the human angiopoietin 1 (ANGPT1) enhanced the MSC therapy by mediating a reduced pulmonary inflammation and lung permeability in lipopolysaccharide-induced acute lung injury (ALI) in mice [ 52 ].…”

Revertant Mosaicism in Genodermatoses: Natural Gene Therapy Right before Your Eyes

“…Most approaches under study for EB therefore focus on topical treatment [ 109 ], either by topical compound delivery (e.g., in vivo gene-addition by viral [ 110 ] or minicircle non-viral vectors [ 111 ], antisense oligonucleotides [ 112 ]), ex vivo gene-replacement or gene-correction followed by injection of corrected fibroblasts [ 113 ], or the transplantation of large epidermal sheets cultured from ex vivo -corrected keratinocytes [ 114 , 115 , 116 , 117 ], analogous to the treatment of full-thickness burn-wounds [ 118 ]. In 2017, Hirsch et al reported a 7-year-old boy with laminin 332-deficient JEB in whom almost the entire epidermis (~0.85 m 2 ) was replaced using exogenously corrected, autologous skin grafts [ 119 , 120 ].…”