Christina Gruber

A novel screening system improves genetic correction by internal exon replacement

Trans-splicing is a powerful approach to reprogram the genome. It can be used to replace 5′, 3′ or internal exons. The latter approach has been characterized by low efficiency, as the requirements to promote internal trans-splicing are largely uncharacterized. The trans-splicing process is induced by engineered ‘RNA trans-splicing molecules’ (RTMs), which target a selected pre-mRNA to be reprogrammed via two complementary binding domains. To facilitate the development of more efficient RTMs for therapeutic applications we constructed a novel fluorescence based screening system. We incorporated exon 52 of the COL17A1 gene into a GFP-based cassette system as the target exon. This exon is mutated in many patients with the devastating skin blistering disease epidermolysis bullosa. In a double transfection assay we were able to rapidly identify optimal binding domains targeted to sequences in the surrounding introns 51 and 52. The ability to replace exon 52 was then evaluated in a more endogenous context using a target containing COL17A1 exon 51–intron 51–exon 52–intron 52–exon 53. Two selected RTMs produced significantly higher levels of GFP expression in up to 61% assayed cells. This novel approach allows for rapid identification of efficient RTMs for internal exon replacement.

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

Patients suffering from recessive dystrophic epidermolysis bullosa (RDEB), a hereditary blistering disease of epithelia, show susceptibility to develop highly aggressive squamous cell carcinoma (SCC). Tumors metastasize early and are associated with mortality in the 30th–40th years of life in this patient group. So far, no adequate therapy is available for RDEB SCC. An approach is suicide gene therapy, in which a cell death-inducing agent is introduced to cancer cells. However, lack of specificity has constrained clinical application of this modality. Therefore, we used spliceosome-mediated RNA trans-splicing technology, capable of replacing a tumor-specific transcript with one encoding a cell death-inducing peptide/toxin, to provide tumor-restricted expression. We designed 3′ pre–trans-splicing molecules (PTM) and evaluated their efficiency to trans-splice an RDEB SCC-associated target gene, the matrix metalloproteinase-9 (MMP9), in a fluorescence-based test system. A highly efficient PTM was further adapted to insert the toxin streptolysin O (SLO) of Streptococcus pyogenes into the MMP9 gene. Transfection of RDEB SCC cells with the SLO-PTM resulted in cell death and induction of toxin function restricted to RDEB SCC cells. Thus, RNA trans-splicing is a suicide gene therapy approach with increased specificity to treat highly malignant SCC tumors. Mol Cancer Ther; 10(2); 233–41. ©2011 AACR.

The design and optimization of RNA trans‐splicing molecules for skin cancer therapy

Targeting tumor marker genes by RNA trans‐splicing is a promising means to induce tumor cell‐specific death. Using a screening system we designed RNA trans‐splicing molecules (RTM) specifically binding the pre‐mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB‐SCC). Specific trans‐splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3‐specific RTMs containing HSV‐tk were analyzed regarding their trans‐splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3‐MG). Expression of the chimeric SLCO1B3‐tk was detected by semi‐quantitative RT‐PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene‐mediated apoptosis in SLCO1B3‐MG expressing cells. The lead RTM also showed its potential to facilitate a trans‐splicing reaction into the endogenous SLCO1B3 pre‐mRNA in EB‐SCC cells resulting in tk‐mediated apoptosis. We assume that the pre‐selection of RTMs by our inducible cell‐death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.

Trans-splicing improvement by the combined application of antisense strategies.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

Epidermolysis bullosa - a group of skin diseases with different causes but commonalities in gene expression.

Epidermolysis bullosa (EB) is a group of hereditary skin disorders. Although each subtype is caused by mutations in genes encoding differentially located components of the skin, the resulting phenotype is similar. In this study, we investigated similarities in the gene expression profiles of each subtype on mRNA level. Type XVI collagen (COL16A1), G0/G1 switch 2 (G0S2), fibronectin (FN1), ribosomal protein S27A (RPS27A) and low density lipoprotein receptor (LDLR) were shown to exhibit corresponding changes in gene expression in all three EB subtypes. While COL16A1, G0S2 and FN1 are up‐regulated, LDLR and RPS27A mRNA levels are decreased. These data indicate that EB cells seem to take measures increasing their mechanical stability. Apoptosis is likely to be exacerbated, and migratory potential appears to be elevated. Protein degradation is hampered, and the release of fatty acids and glycerol is restricted, probably to save energy. These commonalities might benefit existing EB treatment strategies or could help to reveal new starting points for the treatment of EB in the future.

TGFβ-signaling in squamous cell carcinoma occurring in recessive dystrophic epidermolysis bullosa

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary skin disorder characterized by mechanical fragility of the skin, resulting in blistering and chronic wounds. The causative mutations lie in the COL7A1 gene. Patients suffering from RDEB have a high risk to develop aggressive, rapidly metastasizing squamous cell carcinomas (SCCs). Cutaneous RDEB SCCs develop preferentially in long-term skin wounds or cutaneous scars. Albeit being well differentiated, they show a more aggressive behavior than UV-induced SCCs. These findings suggest other contributing factors in SCC tumorigenesis in RDEB. Objective: To analyze factors contributing to RDEB tumorigenesis, we conducted a comprehensive gene expression study comparing a non-malignant RDEB (RDEB-CL) to a RDEB SCC cell line (SCCRDEB4) to achieve an overview on the changes of the gene expression levels in RDEB related skin cancer. Methods: We applied cDNA arrays comprising 9738 human expressed sequence tags (EST) with various functions. Selected results were verified by Real-time RT PCR. Results: Large-scale gene expression analysis revealed changes in the expression level of transforming growth factor β1 (TGFβ1) and several genes under the control of TGFβ for RDEB and SCCRDEB4 cell lines. Even untransformed RDEB keratinocytes show elevated levels of TGFβ1. Conclusion: Our findings demonstrate a prominent role of TGFβ-signaling in RDEB-related skin cancer. Once activated, TGFβ signaling either in response to wounding or in order to influence type VII collagen expression levels could facilitate cancer development and progression. Moreover, TGFβ signaling might also represent a potentially useful therapeutic target in this disease.

Transcutaneous Gene Gun Delivery of hNC16A Induces BPAG2-Specific Tolerance

Immune recognition and rejection of tissues expressing transfected genes is a major complication of gene replacement therapy for inherited genetic disorders. Owing to the high immunogenicity of human bullous pemphigoid antigen 2 (hBPAG2), the induction and maintenance of tolerance to this neo-antigen is essential to deliver the gene product to patients with epidermolysis bullosa junctionalis. In a skin grafting mouse model, we used gene gun transfection with a construct encoding hNC16A, the immunodominant domain of hBPAG2, to induce antigen-specific immune tolerance. Eighty percent of wild-type mice transfected with hNC16A showed long-term survival of skin grafts expressing hBPAG2. Tolerance was stable and transferable by T cells but not by B cells of tolerant mice to naive hosts. A dense Foxp3(+) regulatory T-cell (T(reg)) infiltrate was noticed in grafts of tolerant mice and depletion of these cells resulted in a loss of tolerance. Taken together, we show that long-lasting hBPAG2-specific tolerance was induced with gene gun delivery of hNC16A through a T(reg)-dependent mechanism. This is of relevance to patients undergoing gene therapy and has broader implications for the treatment of antigen-specific autoimmune diseases.

Pseudosyndactyly – an inflammatory and fibrotic wound healing disorder in recessive dystrophic epidermolysis bullosa

A genetic blistering skin disease, recessive dystrophic epidermolysis bullosa (RDEB), is marked by severe wound healing defects and finger contractures. The purpose of this investigation was to elucidate the mechanisms of impaired wound healing and pseudosyndactyly occurring in RDEB patients by studying the role of known inflammation and fibrosis markers in RDEB pseudosyndactyly tissue.

677 Low-dose calcipotriol as a therapeutic option to improve wound healing in epidermolysis bullosa

WOUND HEALING IN EPIDERMOLYSIS BULLOSA Christina Gruber,, Birgit Tockner,, Clemens Hüttner,, Dirk Strunk,, Cornelia Scharler,, Andrea Trost,, Martin Laimer,, Anja Diem,, Katharina Ude-Schoder,, John Common,, Angeline Tay,, Peter Hofbauer,, Martin Wolkersdorfer,, Johann Bauer,, Julia Reichelt,, Josefina Pinon Hofbauer,, Roland Lang, 1 Eb House Austria; Department of Dermatology, University Hospital of the Paracelsus Medical University, 2 Laboratory for Molecular Therapy, Eb House Austria; Department of Dermatology, University Hospital of the Paracelsus Medical University, 3 Spinal Cord Injury and Tissue Regeneration Center Salzburg (Sci-Trecs), Paracelsus Medical University (Pmu) Salzburg; Experimental & Clinical Cell Therapy Institute, 4 Research Program for Ophthalmology and Glaucoma Research; University Clinic of Ophthalmology and Optometry, Paracelsus Medical University (Pmu) Salzburg, 5 Landeskrankenhaus-Universitätsklinikum Salzburg; Department of Dermatology, 6 A*star; Institute of Medical Biology, A*star, 7 A*star; Institute of Medical Biology, 8 Landesapotheke Salzburg; Department of Production, Hospital Pharmacy, 9 Department of Dermatology ; University Hospital of the Paracelsus Medical University, 10 Salk; Eb-Haus, 11 University Hospital Salzburg, Paracelsus Medical University (Pmu) Salzburg; Department of Dermatology Introduction

Pseudosyndaktylie – eine entzündliche und fibrotische Wundheilungsstörung bei rezessiver Epidermolysis bullosa dystrophica

Die rezessive Epidermolysis bullosa dystrophica (engl.: dystrophic epidermolysis bullosa, RDEB) ist eine genetisch bedingte bullöse Dermatose und durch schwere Wundheilungsstörungen und Fingerkontrakturen gekennzeichnet. Der Zweck dieser Arbeit war es, die Rolle bekannter Entzündungs‐ und Fibrosemarker in Pseudosyndaktyliegewebe von RDEB‐Patienten zu untersuchen und so den Mechanismus der Wundheilungsstörung und der Pseudosyndaktylie bei RDEB zu beleuchten.