title: Transcriptional targeting of glioblastoma by diphtheria toxin-A driven by both H19 and IGF2-P4 promoters.
abstract: BACKGROUND
  The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as in vivo, in comparison to a single promoter construct in bladder carcinoma. Here we extended this approach to glioblastoma and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in samples of diffuse glioma.
  
  
  METHODS
  IGF2-P4 gene expression was tested by qRT-PCR as well.
  
  
  RESULTS
  Both H19 and IGF2-P4 transcripts were highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic glioblastoma animal model.
  
  
  CONCLUSIONS
  Our findings indicate antitumoral efficacy against glioblastoma of the targeted double promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in clinical trials is warranted.
authors.forename: Doron
authors.initials: D
authors.lastname: Amit
authors.forename: Imad J
authors.initials: IJ
authors.lastname: Matouk
authors.forename: Iris
authors.initials: I
authors.lastname: Lavon
authors.forename: Tatiana
authors.initials: T
authors.lastname: Birman
authors.forename: Jenifer
authors.initials: J
authors.lastname: Galula
authors.forename: Rasha
authors.initials: R
authors.lastname: Abu-Lail
authors.forename: Tamar
authors.initials: T
authors.lastname: Schneider
authors.forename: Tali
authors.initials: T
authors.lastname: Siegal
authors.forename: Abraham
authors.initials: A
authors.lastname: Hochberg
authors.forename: Yakov
authors.initials: Y
authors.lastname: Fellig