749 transcriptional targeting of tumors with a novel tumor specific promoter the survivin gene promoter

749 Transcriptional Targeting of Tumors with a Novel Tumor Specific Promoter The Survivin Gene Promoter Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ����������� �!����� ��[.]

CANCER TARGETED GENE THERAPY II vivo, antisense (PS-ODN) and siRNAs were delivered by electroporation in pancreatic tumors xenografts into athymic mice The tumor growth was evaluated by tumor volume measurement during weeks Our results demonstrate that the antisense PSODN targeted specific k-ras mutation oncogene transcripts and reduced its expression The antitumor effect observed in vitro and in vivo was less than 50% The siRNAs appear to be more efficient with a long lasting effect in cell culture The best results were obtained with the recombinant vector expressing siRNA (k-Ras mut12) which induced an efficient and specific down-regulation and persistent oncogene expression The tumor growth of the pancreatic cancer cells (k-Ras mut12) targeted with specific siRNA was dramatically inhibited in vitro as well as in vivo In conclusion; these results indicate that the siRNA strategy is more efficient than antisense oligonucleotides and should be more investigated as a novel rationale for the targeting of pancreatic cancer therapy as effective as bcl-xl RNAi constructs Through BCL-XL downregulation via siRNA vectors, we have demonstrated a significantly improved response to CDDP in NSCLC in vitro These findings could have significant clinical applicability 748 siRNA Vectors Targeted to BCL-XL Significantly Reduce BCL-XL Expression and Enhance Response to Chemotherapy in NonSmall Cell Lung Adenocarcinoma Xiaobo Cao,1 Jonathan Daniel,1 Mustafa K Ozvaran,1 Steven Miller,1 Roy W Smythe.1 Thoracic and Cardiovescular Surgery, University of Texas M.D.Anderson Cancer Center, Houston, TX Introduction: An anti-apoptotic member of the bcl-2 gene family, bcl-xl, is strongly expressed in human non-small cell lung adenocarcinoma (NSCLC) specimens and cultured cell lines Previously, we have demonstrated that down-regulation of BCLXL protein expression can enhance chemosensitivity in NSCLC cell lines RNA interference (RNAi) has emerged as an effective tool for inhibiting gene expression in a specific fashion Effective inhibition of targeted RNA by siRNAs expressed from an RNA polymerase III vector based on an H1 or U6 promoter has been demonstrated The purpose of this study is to evaluate the effectiveness of hairpin siRNA directed against bcl-xl by using an H1 promoter based expression vector to downregulate BCL-XL expression and also to determine if this effect enhances response to conventional chemotherapy in vitro Methods: The human NSCL cell line H1299 was maintained in culture and utilized for these experiments Five expression vectors were generated by inserting five pairs of 64-nt oligonucleotides, each containing a 19-nt target which is included in both sense and antisense orientation, separated by a 9-nt spacer sequence H1299 was then transfected with these siRNA vectors using lipofectamine 2000 Seventy-two hours later, RNA and cell lysates were collected and BCL-XL expression was evaluated by quantitative RT-PCR and Western blot Additional members of the bcl-2 family, BAX, BAK and BCL-2, were also evaluated by Western blot and Super array A colorimetric assay (XTT) was used to assess cellular viability 72 hours following exposure of cells to siRNA and cisplatin (CDDP) Apoptosis was measured by Annexin V staining Results: Among the five siRNA vectors generated, two constructs were able to significantly decrease BCL-XL at the level of transcription (evaluated by RT-PCR) and protein expression (evaluated by Western blot) Expression of BAX, BAK, and BCL2 were unchanged Decreased BCL-XL expression led to apoptosis in H1299 cells (evaluated by Annexin V staining) The percentage of cells which stained positive were as follows: Control vector group – 12%, 30uM cisplatin group – 18%, BCL-XL siRNA vector group – 40%, and cisplatin plus BCL-XL siRNA vector group – 70% XTT assay demonstrated H1299 was sensitized to cisplatin after bcl-xl siRNA vectors were transfected Conclusion: siRNA vectors directed at bcl-xl demonstrate significant and specific reduction in BCL-XL protein levels in NSCLC Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 749 Transcriptional Targeting of Tumors with a Novel Tumor Specific Promoter-The Survivin Gene Promoter Zeng B Zhu,1 Bao G Lu,1 Ming H Wang,1 Lioudmila Kaliberova,1 Feng Z Li,3 Angel A Rivera,1 Parameshwar J Mahasreshti,1 Charles A Leath III,2 Sharmila K Makhija,2 David T Curiel.1 Division of Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL, United States; 2Department of Obatetrics & Gynecology, University of Alabama at Birmingham, Birmingham, AL, United States; 3Department of Pharmacology & Therapeutics, Roswell Park Institute, Buffalo, NY, United States Survivin, a novel member of the IAP (inhibitor of apoptosis) protein family, appears to be over-expressed in common cancers but not in corresponding normal adult tissues We hypothesized that the survivin promoter might be an excellent candidate to deliver transgenes to tumor cells Advantages of tumor-specific expression of transgenes include limitation of toxicity of therapeutic genes, such as herpes simplex virus thymidine kinase, and the potential for creation of a tumor-selective replicate-competent oncolytic virus To test this hypothesis, we measured the transcriptional activity of the survivin gene promoter in different tumor cell lines, including pancreatic cancer, breast cancer, ovarian cancer, and melanoma We also tested this promoter to see if it exhibited a “liver off” status in vivo using a mouse model A recombinant adenovirus (reAdGL3BSurvivin) was constructed by inserting a luciferase reporter gene and the survivin promoter, which drives the reporter expression, into the deleted E1 region of adenovirus type5 The tumor cell lines were infected with reAdGL3BSurvivin, and a human fibroblast and a mammary epithelial cell line were used as normal controls A reAdGL3BCMV, containing the CMV promoter, was used as a positive promoter control to normalize the luciferase levels To test the distribution of promoter activity, in vivo, a dose of 5x108 pfu of reAdGLB3Survivin or reAdGL3BCMV was injected intravenously into mice The major organs were harvested two days post-injection, and the luciferase activities were determined in major organs Luciferase assays revealed that the survivin promoter induced relatively high luciferase gene transcription in several cell lines including breast cancer, MDA-MB-361, ovarian cancer, OVCAR3, and melanoma, SK-Mel-28, 14.4%, 11%, and 16.8% of the activity of the CMV promoter, respectively The survivin promoter activities S289 CANCER TARGETED GENE THERAPY II in normal cell lines were less than 2% of CMV activity Luciferase activity in mouse liver induced by the survivin promoter was very low, only 0.07% (“liver off”) compared to that of CMV promoter The survivin promoter appears to be a tumor-specific promoter in some tumor cell lines with a very low expression in normal tissues based on the data of “tumor on” and “liver off” We conclude that the survivin promoter is a good candidate for transcriptional targeting in cancer gene therapy, especially, in breast, ovarian and melanoma cancers 750 Transferrin-Targeted, Cyclodextrin Polycation-Based Gene Vector for Systemic Delivery Nathalie C Bellocq,1 Mark E Davis,1 Heidrun Engler,2 Gregory S Jensen,1 Aijie Liu,1 Todd Machemer,2 Daniel C Maneval,2 Erlinda Quijano,2 Suzie Hwang Pun,1 Thomas Schluep,2 Shufen Wen.2 Insert Therapeutics, Inc., Pasadena, CA, United States; 2Canji, Inc., San Diego, CA, United States Successful delivery of nucleic acid therapeutics for the treatment of metastatic cancer requires a vehicle capable of localizing to numerous tumors areas from a systemic administration Non-viral vectors offer a viable alternative to viral vectors due to their low Tcell and humoral immunogenicity We have recently developed a new class of cyclodextrin-based polycations for use as systemic delivery vehicles The vector when assembled with nucleic acids forms stabilized, colloidal particles that can be modified with ligands for targeting to tumor cells Transferrin (TF) is a well-studied ligand for tumor targeting because the expression of the transferrin receptor is often elevated in cancer cells Here, transferrin-modified particles containing the p53 gene were formulated using our cyclodextrin polycation system and characterized The conjugated transferrin ligand retains high binding specificity to the transferrin receptor and in vitro delivery of the transferrin-modified particles to K562 cells demonstrated a transferrin-dependent increase in transfection efficiency over untargeted particles These particles were administered by low pressure tail vein injection to tumor-bearing (PC3 cells that are transferrin receptor positive and p53 null) mice The amount of p53 DNA delivered to the tumor was increased by more than two orders of magnitude by passive targeting due to PEGylation of the particles Incorporation of the transferrin targeting ligand did not significantly affect the p53 DNA levels in tumor over that of PEGylated particles (see Figure 1) However, p53 expression (as determined by RT-PCR analysis for p53 mRNA) in the tumor was achieved from systemic gene delivery and required the presence of the transferrin targeting ligand (see Figure 2) S290 751 Transcriptional Targeting of Gliomas Employing the Survivin Promoter Winan J Van Houdt,1,3 Yosef S Haviv,1,2 Xiaosheng Lei,1,2 Minghui Wang,1,2 Marijke Van Der Jagt,3 Daniel T Rein,1,2 Fengzi Li,5 Martine L M Lamfers,3,4 Clemens M F Dirven,4 David T Curiel,1,2 Zeng B Zhu.1,2 Division of Human Gene Therapy, Departments of Medicine, Pathology, and Surgery; 2Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL; 3Department of Medical Oncology, Division of Gene Therapy; 4Department of Neurosurgery, VUMC, Amsterdam, Netherlands; 5Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY Malignant gliomas are the most common neoplasm in the central nervous system (CNS) Despite some advances in treatment, the overall median survival is less than a year Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of malignant gliomas In this regard, gene therapy with adenoviral (Ad) vectors is a promising new modality for glioma A critical factor determining the utility of Ad vectors for cancer gene therapy is the selectivity of their transgene expression in cancer cells To this end, tumor- and tissue specific promoters (TSPs) are employed to achieve tumor-specific transgene expression However, currently available TSPs used in the context of Ad-based CNS targeting, are generally not tumor-specific but rather tissue specific In this context, survivin, a recently discovered member of the inhibitor of apoptosis protein (IAP) family, is highly expressed in human cancers including gliomas In contrast, survivin expression is low or undetectable in normal, differentiated human tissues Survivin expression in gliomas is associated with poor prognosis, shortened survival, resistance to chemo- and radiotherapy and accelerated rates of recurrence of the disease Therefore, survivin-based targeting of Ad vectors may be a meaningful strategy to address gliomas Thus, we hypothesized that the survivin TSP represents a novel and relevant transcriptional targeting strategy for glioma To test our hypothesis, we first tested survivin expression in gliomas Our initial findings indicated selective upregulation of survivin expression in glioma cell lines, both at the mRNA and protein levels, in contrast to its expression profile in non-malignant cell lines Next, we turned to evaluate the activity of the survivin promoter in a number of glioma cell lines To this end, we constructed an Ad vector, Ad.GL3B.survivin, encoding luciferase under the tight control of the survivin promoter When compared to several other TSPs, the survivin promoter showed a high level of activity in all the glioma cell lines, in the context of the Ad backbone We then tested the survivin TSP directly against the midkine and cox-2 promoters, previously shown to be of utility for transcriptional targeting of gliomas In these experiments, the survivin promoter showed dramatic Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... on” and “liver off” We conclude that the survivin promoter is a good candidate for transcriptional targeting in cancer gene therapy, especially, in breast, ovarian and melanoma cancers 750 Transferrin-Targeted,... acid therapeutics for the treatment of metastatic cancer requires a vehicle capable of localizing to numerous tumors areas from a systemic administration Non-viral vectors offer a viable alternative... resistance to chemo- and radiotherapy and accelerated rates of recurrence of the disease Therefore, survivin- based targeting of Ad vectors may be a meaningful strategy to address gliomas Thus,