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CANCER TARGETED GENE THERAPY I tumors, compared to control Innate immune cells including macrophages and neutrophils were found to infiltrate at the regressing tumor site This study provides proof of principle that intratumoral IL-4Rα plasmid injection followed by systemic or intratumoral IL4 cytotoxin administration may be a useful strategy for the treatment of localized breast cancer 333 Enhancement of Myoblast Microencapsulation for Gene Therapy Anna Li,1 Feng Shen,1 Pasquale Cirone,2 Tao Zhang,1 Murray Potter,3 Patricia Chang.1 Pediatrics, McMaster University, Hamilton, ON, Canada; Biology, McMaster University, Hamilton, ON, Canada; Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada An alternative to viral-based gene therapy is to implant nonautologous cells genetically engineered to secrete the desired gene product These “universal” cells can be protected from the host’s immune response by microencapsulating them in biocompatible selectively permeable polymers Early studies used microencapsulated fibroblast cells to successfully deliver gene products However, proliferative cell lines such as fibroblasts are not ideal for long-term therapy because the limited space in microcapsules leads to overcrowding and cell death, and if cells escape from the microcapsules there is a risk of tumor formation Myoblasts have similar proliferative and stable long-term expression of target genes as fibroblasts, but they are also able to undergo terminal differentiation into myotubes, thus overcoming the problems of space limitation and tumorigenesis However, once encapsulated, myoblasts not proliferate and differentiate as well as when they are unencapsulated We have developed alginate-poly-L-lysinealginate (APA) microcapsules to deliver therapeutic gene products from genetically engineered myoblast cells Our previous work in vitro has proven that proliferation and differentiation of encapsulated myoblasts were significantly improved by inclusion of basic fibroblast growth factor (bFGF), insulin growth factor (IGF-II) and collagen within the APA-microcapsules (“enhanced” capsules) Here we report further characterization of the enhanced microcapsules in vitro as well as the growth, differentiation and clinical efficacy in vivo of C C 12 myoblasts encapsulated in enhanced APAmicrocapsules There was no difference in the rates of diffusion of human factor IX (65 KDa), murine IgG (150 KDa) and a lysosomal enzyme, b-glucuronidase (300 KDa), from engineered myoblasts in either classical or enhanced microcapsules For initial in vivo studies, the enhanced capsules were cultured in vitro for weeks before implantation into the peritoneal cavity of mice By day 14 postimplantation, most of the cells were found in clusters which reached 50μm in diameter The CPK activity and MHC staining (markers for differentiation) of the myoblasts and the cell number per capsule in the enhanced microcapsules indicated a higher degree of differentiation and proliferation when compared to the classic microcapsules Efficacy was tested in a breast cancer tumor model, using B16/neu tumor cell injection to induce solid tumors in mice C2C12 cells were engineered to secrete angiostatin, an inhibitor of endothelial cell proliferation that has been shown to inhibit growth of different types of tumors (including breast cancer) These cells were encapsulated in classical or enhanced microcapsules and then implanted into the peritoneum three days after tumor cells were injected Mice treated with enhanced APA-microcapsules had a 50% reduction in tumor volume at day 22 compared to mice treated with classical APA-microcapsules As well, myoblasts cell count per microcapsule recovered on day 22 was 3.4 times higher in enhanced versus classical microcapsules In conclusion, enhanced APA microcapsules improve the growth and differentiation properties of encapsulated myoblasts and increase clinical efficacy in vivo Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 334 AAV-Mediated Anti-Angiogenic Therapy of Pancreatic Cancer in an Orthotopic Metastatic Model in Golden Hamsters: Influence of Route of AAV Vector Administration Takuji Noro,1,3 Koichi Miyake,1 Noriko Suzuki,1 Eiji Uchida,2 Takeyuki Misawa,3 Yoji Yamazaki,3 Takashi Shimada.1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan; 2Department of Surgery, Nippon Medical School, Tokyo, Japan; 3Department of Surgery, Jikei University School of Medicine, Tokyo, Japan We examined the feasibility of AAV mediated systemic delivery of endostatin in gene therapy of metastases of pancreatic cancer Pancreatic cancer is highly invasive and often lethal No effective therapeutic strategies are available at this moment We have established an animal model of metastatic pancreatic cancer in which a pancreatic cancer cell line PGHAM-1 was inoculated into the pancreas of Syrian golden hamsters Transplanted cells grow rapidly and metastasize into liver An AAV vector expressing endostain (5 x 1010 particles) was injected intramuscularly into the left quadriceps (AAV-IM) or intravenously into the portal vein (AAV-IPV) These two routes of vector administration were evaluated by measuring various parameters of tumor development After intramuscular injection, the vector genome was localized only in the injected site and a modest increase in serum endostatin was detected The number of metastasis and the incidence of hemorrhagic ascites were decreased in the treated animals On the contrary, the serum concentration of endostatin was significantly increased after intraportal injection of the vector The significant anti-tumor effects were observed in all parameters including the size and microvessel density of the primary pancreas tumor, the size and number of liver metastasis, and incidence of hemorrhagic ascites One potential problem may be that the vector was distributed into systemic organs in addition to liver These results suggest that systemic delivery of endostatin represent a potentially effective treatment for pancreatic cancer and liver metastases The efficacy of AAV mediated expression of endostatin is influenced by route of vector administration Intraportal injection of AAV vector appears to be more effective for anti-angiogenic systemic gene therapy of cancer 335 An Anti-Vascular Approach to Gene Therapy: Exploiting Hypoxia Nicola Ingram,1 Colin D Porter.1 Gene Therapy Team, Cell and Molecular Biology, The Institute of Cancer Research, London, United Kingdom An anti-vascular approach to cancer gene therapy is an attractive way to eliminate solid tumours due to the dependence of a large number of cells on relatively few blood vessels The group is studying a number of ways to target a retroviral vector to the endothelial cells of tumour blood vessels and to limit extraneous expression.1, As part of this approach, the tumour-specific phenomenon of transient hypoxia in the microvasculature is being exploited to target transcription Previous studies have shown that a trimer of hypoxic response elements (HREs) from the phosphoglycerate kinase-I gene placed in a Moloney murine leukaemia viral LTR strongly induces expression in hypoxia compared to normoxia.³ We have similarly replaced the viral 3’ LTR enhancer with this HRE trimer and confirmed these results Histochemical titre was reduced 100-fold in normoxia, equivalent with enhancer deletion, but expression was strongly induced in 0.5% oxygen A 6-mer of HREs gave even higher levels of enzyme production and fully restored histochemical titre In addition, constructs with mutated HREs did not respond to hypoxia and behaved similarly to the vector with a deleted enhancer LTR hybrid vectors incorporating the 6-mer of HREs adjacent to endothelialS131 CANCER TARGETED GENE THERAPY I specific promoters were constructed to see if the HREs could provide a further level of specificity for hypoxic endothelial cells Constructs in which the endothelial specific promoter replaced the viral enhancer in the 3’ LTR showed greater histochemical titres than those that replaced both the viral enhancer and proximal promoter However, this was at the expense of a slight increase in basal expression The best levels of induction were obtained with the 6-mer of HREs adjacent to the prepro-endothelin-I and Flt-I promoters Current investigations are concentrating on evaluating the transcriptional specificity and therapeutic potential of HRE-containing constructs using a tumour xenograft model in vivo ¹ Mavria et al (2000) Gene Therapy 7: 368 ² Mavria et al (2001) Gene Therapy 8: 913 ³ Boast et al (1999) Human Gene Therapy 10: 2197 336 Telomerase-Directed Suicide Gene Therapy: An Analysis of hTR and hTERT Promoters Nicol Keith,1 Alan E Bilsland,1 Claire J Anderson,1 Aileen J Monaghan,1 Jane A Plumb Cancer Research UK Department of Medical Oncology, University of Glasgow, Cancer Research UK Beatson Labs, Glasgow, Scotland, United Kingdom A major challenge facing cancer therapy is to generate tumourspecific treatment strategies The level and frequency of telomerase activity in cancers reinforces the notion that telomerase has potential within anticancer strategies Numerous potential therapeutic strategies have been proposed with the telomere or telomerase enzyme as the molecular target One strategy is to exploit tumourspecific telomerase gene expression to target gene therapy vectors to cancer cells, thus causing rapid cell kill The hTR and hTERT promoters are interesting candidates for development of gene therapy systems since they should display activity over a range of malignancies Therefore, in order to model delivery of telomerase specific suicide gene therapy vectors, we have generated a family of adenovirus gene therapy vectors using the transcriptional regulatory regions from the telomerase hTERT and hTR genes to drive the bacterial nitroreductase gene, (NTR) The NTR gene bio-activates the prodrug CB1954 into an active cytotoxic alkylating agent We demonstrate by infection of human cancer and normal cells and Western blotting that NTR is expressed specifically in cancer cells and that transcription of NTR constructs was correctly initiated from both promoters Together, these data indicated that the cellspecific function of the telomerase promoters was retained in the adenovirus background Furthermore, a range of human cancer cells were specifically and efficiently sensitised to CB1954 in a promoterdependent and dose-dependent manner both in vitro and in vivo, showing specific hTR-NTR and hTERT-NTR mediated enhancement of CB1954 induced cytotoxicity in monolayers and in xenografts The cell lines efficiently targeted by this approach in monolayer experiments include drug resistant derivatives of the A2780 ovarian adenocarcinoma cell line that are known to tolerate the effects of a diverse range of DNA damaging drugs including the alkylating agent N-methyl-N-nitrosourea, the purine analogue 6thioguanine and the DNA crosslinker cisplatin Significantly, we include normal adult human epithelial cell strains and show that neither promoter construct resulted in enhanced toxicity to CB1954 in these cells or in normal foetal lung fibroblasts, despite high permissiveness for adenovirus infection Since telomerase targeted therapies should be defined by selectivity for cancer cells above normal cells, the insensitivity of normal adult cell strains to telomerase targeted therapeutics presents an interesting and encouraging finding Taken together, the present data show that the differential promoter activities of the hTR and hTERT promoters between normal and cancer cells are retained in viral models of gene S132 transfer and help to validate the general principle underlying a telomerase-directed approach in gene therapy In summary, it is clear that the addition of Ad-hTR-NTR and Ad-hTERT-NTR to a potential telomerase-specific anti-cancer armoury is an exciting prospect, although optimal systems for telomerase directed gene therapy will presumably require targeted delivery systems in addition to a clear understanding of the regulation of hTR and hTERT genes in target tumours 337 Antiangiogenic Gene Therapy Targeting the Endothelium-Specific Flk-1 and Tie-2: Inhibition of Tumor Angiogenesis and Growth in Prostate Cancer Sudhanshu P Raikwar,1,2,3 Thomas A Gardner,1,2,3 Chinghai H Kao.1,2,3 Urology; 2Microbiology & Immunology; 3Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, United States Prostate cancer is the most frequently diagnosed cancer and is increasingly recognized as a major health problem in the Western male population Lack of an effective treatment for patients with advanced, hormone-refractory prostate cancer, necessitates the development of novel gene therapy approaches Angiogenesis, the formation of new capillaries from the pre-existing vessels, is critical for tumor growth and metastasis Considering the importance of vascular growth in tumor progression, development of anti-angiogenic approaches targeting the tumor neo-vasculature may provide longterm, effective control of the disease Here we report on the development of replication-incompetent adenoviral vectors ADVsFlk-1 and ADV-sTie-2 expressing the soluble forms of dominant negative Flk-1 and Tie-2 endothelium-specific genes aimed at investigating the anti-angiogenic and anti-tumoral effect in vitro as well as in human prostate tumor xenografts in nude mice Replicationincompetent adenoviral vectors ADV-sFlk-1and ADV-sTie-2 were generated by conventional homologous recombination technique in human embryonic kidney 293 cells and amplified in PERC.6 cells The viruses were purified by two rounds of cesium chloride density gradient ultracentrifugation followed by extensive dialysis Human vascular endothelial cells (HUVECs) were transduced with either ADV-sFlk-1, ADV-sTie-2 or the control green fluorescent protein (GFP) expressing adenoviral vector ADV-GFP at a multiplicity of infection of 100 viral particles/cell The transduced HUVECs were subjected to an in vitro matrigel tube formation assay by plating on growth factor reduced matrigel in the presence of 100 ng/ml vascular endothelial growth factor and 10% bovine calf serum Tube formation was analyzed 48 hours post-transduction using normal and fluorescence microscopy The HUVECs transduced with ADV-sFlk1 vector revealed apoptosis induction and significant inhibition of endothelial cell differentiation and lack of any tubular network formation While the HUVECs transduced with ADV-sTie-2 revealed a lesser degree of apoptosis induction and consequently a greater degree of tubular network formation The HUVECs transduced with the control ADV-GFP displayed endothelial cell differentiation, GFP expression and extensive tubular network formation We next investigated the ability of ADV-sFLK1, ADV-sTie-2 and ADVGFP to inhibit angiogenic sprouting from microvascular endothelial cells in an in vitro 3D angiogenesis model using the mouse aortic ring culture Our results indicate significant reduction in angiogenic sprouting from the aortic rings transduced with ADV-sFLK-1 as compared with ADV-sTie2 while extensive sprouting and GFP expression was observed in the case of aortic rings transduced with ADV-GFP We are currently investigating the potential of ADVsFlk-1 and ADV-sTie-2 in mediating anti-angiogenic and growth inhibition responses in an androgen-independent human prostate tumor xenograft nude mouse model The outcome of these studies Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... CB1954 into an active cytotoxic alkylating agent We demonstrate by infection of human cancer and normal cells and Western blotting that NTR is expressed specifically in cancer cells and that transcription... adenoviral vectors ADVsFlk-1 and ADV-sTie-2 expressing the soluble forms of dominant negative Flk-1 and Tie-2 endothelium-specific genes aimed at investigating the anti- angiogenic and anti- tumoral... of adenovirus gene therapy vectors using the transcriptional regulatory regions from the telomerase hTERT and hTR genes to drive the bacterial nitroreductase gene, (NTR) The NTR gene bio-activates