FAST TRACK USE OF H19 REGULATORY SEQUENCES FOR TARGETED GENE THERAPY IN CANCER Patricia OHANA 1 , Osaat BIBI 1 , Imad MATOUK 1 , Carol LEVY 2 , Tatiana BIRMAN 3 , Ilana ARIEL 3 , Tamar SCHNEIDER 1 , Suhail AYESH 1 , Hilla GILADI 2 , Morris LASTER 1 , Nathan DE GROOT 1 and Abraham HOCHBERG 1 1 Department of Biological Chemistry, Institute of Life Sciences, Hebrew University, Jerusalem, Israel 2 Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel 3 Department of Pathology, Hadassah University Hospital, Jerusalem, Israel We present a tumor gene therapy approach based on the use of regulatory sequences of the H19 gene that are differ- entially expressed between normal and cancer cells. We constructed expression vectors carrying the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5-flanking region of the H19 gene. The cell killing activity of these constructs was in accordance with the relative activity of the H19 regulatory sequences in the transfected cells. We evaluated the therapeutic potential of the gene expression constructs driven by H19 regulatory sequences in an animal model of bladder cancer induced by subcutaneous injection of syngeneic bladder tumor cell lines. Intratumoral injection of these constructs caused a significant suppression of subcu- taneous tumor growth, with no obvious toxicity toward the host. © 2002 Wiley-Liss, Inc. Key words: H19 gene; regulatory sequences; gene therapy; bladder cancer; DT-A gene; HVS-tk gene One of the most serious problems in cancer therapy is the obvious necessity to limit the action of the drug(s) used in the therapy to tumor cells only, in order to prevent damage to healthy tissue. The approach used in our work on cancer therapy is based on the use of regulatory sequences of genes that are differentially expressed between normal and cancer cells, to drive the expression of a toxin gene in tumor cells only. The H19 gene is highly expressed in embryonic tissues, ex- pressed at low levels (or not at all) in normal adult tissues and expressed in tumors derived from tissues (such as bladder) that exhibited the gene during embryonic development. 1–3 H19 RNA is not present in normal adult bladder tissue but can be detected in bladder cancer cells in over 75% of bladder tumors; however, the relative number of H19-expressing cells is reduced during tumor differentiation. 4 The change in H19 RNA level may be caused by a change either in the level of H19 transcription or in H19 RNA stability or both. 5 We have previously reported that transcription of the human H19 gene is under the control of regulatory sequences downstream to the coding sequence. 6 We demonstrated that luciferase activity could be selectively expressed in cells transfected with a plasmid carrying the luc gene linked to the 814 bp 5'-flanking region of the H19 gene. 6 Our results showed that elements responsible for basal promoter activity are contained within the 85 bp upstream region and that cell specificity is mainly conferred by the 814 bp upstream sequences. 7 . We established a murine in vivo model for bladder carcinoma in which the dynamics of H19 expression and tumor progression could be correlated. 8 . Mice develop solid invasive bladder cancer after 20 –30 weeks of N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) treatment after hyperplasia and dysplasia of the urothe- lium. 8 We showed that H19 is expressed in the preneoplastic phase during BBN-induced tumor evolution. 8 Moreover, when tumor formation is inhibited by the anticancer drug halofuginone, H19 expression is extinguished. 9 Halofuginone also exerts anticancer effects on tumors formed by subcutaneous transplantation of MBT2-t50 murine bladder carcinoma cells (H19-positive cells) into syngeneic mice. 9 Tumor cell-specific gene therapy might be accomplished with a construct carrying H19 regulatory sequences that determine the tissue-specific expression of a toxin or suicide enzyme gene. We therefore constructed expression vectors containing “therapeutic genes” such as the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5'-flanking region of the H19 gene. Here we present evidence that the constructs expressing either the toxin or the suicide gene driven by the H19 regulatory se- quence could selectively exert their cytotoxic effects in H19- positive cells. The cell killing activity of these constructs was in accordance with the relative activity of the H19 regulatory se- quences in the transfected cells. Intratumoral injection of these constructs expressing the therapeutic genes caused significant sup- pression of subcutaneous tumor growth with no obvious toxicity toward the host. MATERIAL AND METHODS Cell culture, transfection and luciferase assay The human bladder carcinoma cell lines T24P and RT112, the hepatocellular carcinoma cell lines HepG2 and Hep3B, the pri- mary fibroblast cell line IMR-90 and the embryonic kidney cell line 293 were obtained from the American Type Culture Collec- tion (ATCC; Rockville, MD). A highly metastatic variant (MBT2- t50) of the MBT2 murine bladder carcinoma cell line was kindly provided by Dr. O. Medalia (Sackler Medical School, Tel-Aviv University, Tel-Aviv, Israel). The cells were grown as previously described. 10 A total of 0.4  10 6 cells were plated in a 6-well Nunc multidish (30 mm). Transient transfections were carried out using the cal- cium phosphate precipitation method as previously described. 10 Cells were harvested after 48 hr, and luciferase activity was measured using the Promega (Madison, WI) kit Luciferase Assay System (E-1500). Light output was detected using a Lumac Bio- counter apparatus (The Netherlands). Protein content was mea- Grant sponsor: German-Israeli-Palestinian Trilateral Authority, spon- sored by the Deutsche Forschungs Gemeinschaft (DFG); Grant sponsor: KERYX Biopharmaceuticals; Grant sponsor: The Public Committee for the Designation of Public Funds; Grant number: 90/2000. *Correspondence to: Department of Biological Chemistry Institute of Life Sciences, Jerusalem 91904, Israel. Fax: +972-2-5610250. E-mail: pohana@mail.ls.huji.ac.il Received 29 June 2001; Revised 7 September, 12 November 2001; Accepted 16 November 2001 Published online 7 February 2002 Int. J. Cancer: 98, 645– 650 (2002) © 2002 Wiley-Liss, Inc. DOI 10.1002/ijc.10243 Publication of the International Union Against Cancer