title: 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.
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Peking
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koller
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hainzl
authors.affiliation: Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Sophie
authors.initials: S
authors.lastname: Kitzmueller
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kocher
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mayr
authors.affiliation: Department of Dermatology, University Medical Center, Freiburg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Nyström
authors.affiliation: Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS) Core Facility Microscopy, Paracelsus Medical University (PMU), Salzburg, Austria.|Department of Blood Group Serology and Transfusion Medicine, Paracelsus Medical University (PMU), Salzburg, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lener
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Julia
authors.initials: J
authors.lastname: Reichelt
authors.affiliation: University Hospital of Dermatology, Paracelsus Medical University Salzburg, Salzburg, Austria.
authors.forename: Johann W
authors.initials: JW
authors.lastname: Bauer
authors.affiliation: Research Program for Molecular Therapy of Genodermatoses, EB House Austria, Department of Dermatology, University Hospital of the Paracelsus Medical University, Salzburg, Austria.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Murauer