Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis Bullosa

Abstract

Functional defects in type VII collagen, caused by premature termination codons on both alleles of the COL7A1 gene, are responsible for the severe autosomal recessive types of the skin blistering disease, recessive dystrophic epidermolysis bullosa (RDEB). The full-length COL7A1 complementary DNA (cDNA) is about 9 kb, a size that is hardly accommodated by therapeutically used retroviral vectors. Although there have been successful attempts to produce functional type VII collagen protein in model systems of RDEB, the risk of genetic rearrangements of the large repetitive cDNA sequence may hamper the clinical application of full-length COL7A1 cDNA in the human system. Therefore, we used trans-splicing to reduce the size of the COL7A1 transcript. Retroviral transduction of RDEB keratinocytes with a 3' pre-trans-splicing molecule resulted in correction of full-length type VII collagen expression. Unlike parental RDEB keratinocytes, transduced cells displayed normal morphology and reduced invasive capacity. Moreover, transduced cells showed normal localization of type VII collagen at the basement membrane zone in skin equivalents, where it assembled into anchoring fibril-like structures. Thus, using trans-splicing we achieved correction of an RDEB phenotype in vitro, which marks an important step toward its application in gene therapy in vivo.

Concise Review: Transplantation of Human Hematopoietic Cells for Extracellular Matrix Protein Deficiency in Epidermolysis Bullosa

“…Therefore, expansion of such naturally corrected cells, for example, using induced pluripotency technology followed by parallel differentiation into both hematopoietic (for HCT) and skin cells (for local wound therapy) [46], may obviate the need for gene correction of the patient-specific cells altogether. For now, however, C7 gene augmentation, gene editing via homologous recombination, or C7 RNA transsplicing [27] using viral-mediated transgenesis of hematopoietic stem cells harvested from individuals with RDEB are more realistic means of providing autologous grafts and systemic treatment, whereby in principle the efficacy of HCT is preserved while the complications of allogeneic HCT are avoided.…”

“…This extracellular matrix protein produced by dermal fibroblasts and basal keratinocytes [20] is the major component of the anchoring fibrils (AFs) that extend from the lamina densa into the papillary dermis, thus providing the adhesion structures (''skin Velcro'') responsible for attachment of the epidermis to dermis [21]. [24][25][26][27][28][29][30]. As the lesions in RDEB are distributed over large areas of the body both externally (skin) and internally (the proximal and distal gastrointestinal sites), we reasoned that a systemic therapy providing durable correction of C7 deficiency would be preferred.…”

Gene Therapy for Skin Diseases

“…Similar to the approach used by Murauer et al (2011) for RDEB, Dallinger et al (2003) used a system to splice out specific mutant exons (spliceosome-mediated RNA trans-splicing, SMarT) to show correction of a particular collagen 17 mutation in nonlethal JEB keratinocytes in vitro.…”

“…However, this approach is mutation specific. Murauer et al (2011) performed retroviral transduction of RDEB keratinocytes with a 3 0 pre-trans-splicing molecule, which resulted in expression of C7 at the basement membrane as well as the formation of structures similar to AFs in vitro.…”

Spliceosome-Mediated RNA Trans-Splicing Facilitates Targeted Delivery of Suicide Genes to Cancer Cells

“…PTMs contain a binding domain (BD), which defines the target specificity, splicing elements for efficient trans-splicing, and a coding sequence that is desired to replace a part of the target pre-mRNA (3). In this manner, trans-splicing has already been successfully used to repair the mutated part of disease-causing genes, indicating its potential use in gene therapy for several genetic disorders in vitro and in vivo (4)(5)(6)(7).…”