title: Development of targeted therapy for bladder cancer mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences.
abstract: BACKGROUND
  The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers (including bladder cancer), while existing at a nearly undetectable level in the surrounding normal tissue.Single promoter vectors expressing diphtheria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superficial cell carcinoma of the bladder (treated with H19-DTA). However this targeted medicine approach could be limited, as not all cancer patients express high levels of H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4.
  
  
  METHODS
  H19 and IGF2-P4 gene expression was tested in samples of Transitional Cell Carcinoma (TCC) of the bladder by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR). The therapeutic potential of the double promoter toxin vector H19-DTA-IGF2-P4-DTA was tested in TCC cell lines and in heterotopic and orthotopic animal models of bladder cancer.
  
  
  RESULTS
  Nearly 100% of TCC patients highly expressed IGF2-P4 and H19, as determined by ISH and by qRT-PCR. The double promoter vector exhibited superior tumor growth inhibition activity compared to the single promoter expression vectors, in cell lines and in heterotopic and orthotopic bladder tumors.
  
  
  CONCLUSIONS
  Our findings show that bladder tumors may be successfully treated by intravesical instillation of the double promoter vector H19-DTA-P4-DTA.Overall, the double promoter vector exhibited enhanced anti-cancer activity relative to single promoter expression vectors carrying either gene alone.
authors.affiliation: The Hebrew University of Jerusalem, Biological Chemistry, Jerusalem 91904, Israel. dyamit@gmail.com
authors.forename: Doron
authors.initials: D
authors.lastname: Amit
authors.forename: Abraham
authors.initials: A
authors.lastname: Hochberg
mesh_terms: D000283:Administration, Intravesical; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004167:Diphtheria Toxin / Q000378:metabolism*; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D017403:In Situ Hybridization; D007335:Insulin-Like Growth Factor II / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D010446:Peptide Fragments / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D040521:Subcutaneous Tissue / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D023041:Xenograft Model Antitumor Assays
chemical_list: D004167:Diphtheria Toxin; C094335:H19 long non-coding RNA; D010446:Peptide Fragments; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; C029795:diphtheria toxin fragment A; D007335:Insulin-Like Growth Factor II; D008156:Luciferases