A Gene Gun-mediated Nonviral RNA trans-splicing Strategy for Col7a1 Repair

Abstract

RNA trans-splicing represents an auspicious option for the correction of genetic mutations at RNA level. Mutations within COL7A1 causing strong reduction or absence of type VII collagen are associated with the severe skin blistering disease dystrophic epidermolysis bullosa. The human COL7A1 mRNA constitutes a suitable target for this RNA therapy approach, as only a portion of the almost 9 kb transcript has to be delivered into the target cells. Here, we have proven the feasibility of 5' trans-splicing into the Col7a1 mRNA in vitro and in vivo. We designed a 5' RNA trans-splicing molecule, capable of replacing Col7a1 exons 1-15 and verified it in a fluorescence-based trans-splicing model system. Specific and efficient Col7a1 trans-splicing was confirmed in murine keratinocytes. To analyze trans-splicing in vivo, we used gene gun delivery of a minicircle expressing a FLAG-tagged 5' RNA trans-splicing molecule into the skin of wild-type mice. Histological and immunofluorescence analysis of bombarded skin sections revealed vector delivery and expression within dermis and epidermis. Furthermore, we have detected trans-spliced type VII collagen protein using FLAG-tag antibodies. In conclusion, we describe a novel in vivo nonviral RNA therapy approach to restore type VII collagen expression for causative treatment of dystrophic epidermolysis bullosa.

Construction and validation of an RNA trans-splicing molecule suitable to repair a large number of COL7A1 mutations

“…RNA trans -splicing shows further advantages over gene replacement approaches such as increased efficiency of viral cell transductions due to reduced transgene size, the prevention of overexpression or ectopic expression of the transgene and the possibility to decrease dominant-negative gene products in dominantly inherited diseases. Furthermore, mRNA trans -splicing offers the opportunity for non-viral delivery into the skin using gene transfer methods such as the gene gun technology 23 or microneedles. 24 …”

“…Western blot analysis was performed as described previously. 23, 40 For detection of type VII collagen, the primary antibody rabbit anti-type VII collagen (kindly provided by Dr Alexander Nyström) was added to the blocking solution at a dilution of 1:3000 and incubated overnight at 4 °C, followed by incubation with the secondary antibody, anti-rabbit horse radish peroxidase (Dako, Glostrup, Denmark) 1:1000 in blocking buffer (Roche) for 1 h at room temperature.…”

“…12, 14, 15 (iv) The use of non-viral vectors such as minicircle DNA, or stabilized mRNA molecules for RTM delivery into the skin in vivo , circumvent the necessity of transplantations in ‘classical' ex vivo gene therapy. 16, 17, 18 …”

Novel and emerging therapies in the treatment of recessive dystrophic epidermolysis bullosa

“…demonstrating correction of an RDEB phenotype in vitro (126). In further work, a 5' RTM capable of replacing COL7A1 exons 1 to 15 in murine keratinocytes was injected into the skin of wild-type mice using a gene gun with vector delivery and expression in the skin (127).…”

RNA‐based therapies for genodermatoses

“…Depending on the disorder, either in situ administration or correction of epidermal stem cells and generation of skin grafts is conceivable. Most of the research on genodermatoses has so far focused on EB, for which RTMs have been successfully developed and optimized for preclinical studies on PLEC , KRT14 , COL17A1 and COL7A1 …”