title: The design and optimization of RNA trans-splicing molecules for skin cancer therapy.
abstract: 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.
authors.affiliation: Division of Experimental Dermatology and EB House Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria. Electronic address: c.gruber@salk.at.
authors.forename: Christina
authors.initials: C
authors.lastname: Gruber
authors.forename: Ulrich
authors.initials: U
authors.lastname: Koller
authors.forename: Eva M
authors.initials: EM
authors.lastname: Murauer
authors.forename: Stefan
authors.initials: S
authors.lastname: Hainzl
authors.forename: Clemens
authors.initials: C
authors.lastname: Hüttner
authors.forename: Thomas
authors.initials: T
authors.lastname: Kocher
authors.forename: Andrew P
authors.initials: AP
authors.lastname: South
authors.forename: Helmut
authors.initials: H
authors.lastname: Hintner
authors.forename: Johann W
authors.initials: JW
authors.lastname: Bauer
mesh_terms: D017209:Apoptosis / Q000235:genetics; D014408:Biomarkers, Tumor / Q000096:biosynthesis / Q000235:genetics*; D004820:Epidermolysis Bullosa / Q000235:genetics / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D029362:Organic Anion Transporters, Sodium-Independent / Q000096:biosynthesis / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D012878:Skin Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000628:therapy*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D020040:Trans-Splicing / Q000235:genetics*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D029362:Organic Anion Transporters, Sodium-Independent; D012334:RNA, Neoplasm; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3
keywords: 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; BD; BP; Cancer gene therapy; DT-A; EB; EB-SCC; Epidermolysis bullosa; GAPDH; GCV; GCVTP; HSV; Herpes simplex virus – thymidine kinase (HSV-tk); IRES; MG; MMP9; MTT; PPT; RDEB; RNA trans-splicing; RNA trans-splicing molecule; RTM; RTM mutated 1, RTM mutated 2; RTM original; RTMm1, RTMm2; RTMmut; RTMorg; SCC; SLCO1B3; SMaRT; SS; STS; SqRT-PCR; Squamous cell carcinoma; TUNEL; binding domain; branch point; diphtheria toxin subunit A; epidermolysis bullosa; epidermolysis bullosa-associated squamous cell carcinoma; ganciclovir; ganciclovir triphosphate; glyceraldehyde 3-phosphate dehydrogenase; herpes simplex virus; internal ribosomal entry site; matrix metalloproteinase 9; minigene; poly pyrimidine tract; recessive dystrophic epidermolysis bullosa; segmental trans-splicing; semi-quantitative real time PCR; solute carrier organic anion transporter family member 1B3; splice site; spliceosome-mediated RNA trans-splicing; splicing deficient RTM; squamous cell carcinoma; terminal deoxynucleotidyl transferase dUTP nick end labeling; thymidine kinase; tk; β-subunit of human gonadotropin gene 6; βhCG6