48. MicroRNA-Regulated Transgene Expression System Reduces Unwanted Side Effects in Adenovirus Vector-Mediated Suicide Gene Therapy

Cancer - Apoptosis and Suicide: Viral Vectors 47. Antitumoral Efficacy and Neurotoxic Profile of Adenoviral Vectors Encoding Proapoptotic Genes To Be Used in Combination with Flt3L for Immunotherapy Against Glioblastoma

Marianela Candolfi,1 A. K. M. G. Muhammad,1 Mariana Puntel,1 Kurt M. Kroeger,1 Chunyan Liu,1 Gabrielle E. Ahlsadeh,1 Jonathan Lerner,1 Sharon Lee,1 David Foulad,1 Gwendalyn D. King,1 Katsuaki Sato,2 Pedro R. Lowenstein,1 Maria G. Castro.1 1 Gene Therapy Research Institute, Cedars Sinai Medical Center, Los Angeles, CA; 2Laboratory for Dendritic Cell Immunobiology, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Kanagawa, Japan.

Glioblastoma Multiforme (GBM) is the most common subtype of primary brain tumor in adults, and its prognosis is dismal. We assessed the efficacy and neurotoxicity of adenoviruses (Ads) expressing proapoptotic transgenes, i.e. HSV1-thymidine kinase (Ad-TK), TNF-α (Ad-TNF-α), FasL (Ad-FasL) or TRAIL (Ad-TRAIL) to be used for GBM immunotherapy in combination with the immunostimulant Flt3L (Ad-Flt3L). TK selectively kills rapidly dividing cells in combination with the prodrug ganciclovir (GCV), while TNF-α, FasL or TRAIL kill cells expressing the respective death receptor. Rats bearing intracranial CNS-1 tumors were injected intratumorally with the Ads expressing the proapoptotic transgenes 4 days after tumor implantation. We found that while rats bearing small tumors treated with saline, Ad-TNF-α and Ad-TRAIL succumbed due to tumor burden, Ad-TK and Ad-FasL inhibited tumor progression, and significantly improved the survival. However, when we used Ad-TK or Ad-FasL to treat larger tumors (day 9 after implantation), we found that alone; they are ineffective to improve survival. Less than 20% of rats treated with Ad-TK survived long term and none of the rats treated with Ad-FasL rats survived further than the saline group. Thus, we used the combination of Ad-TK or Ad-FasL with Ad-Flt3L which were injected intratumorally in rats bearing large tumors. Flt3L recruits and activates dendritic cells into the brain, eliciting antigen presentation. We found that although Ad-Flt3L poorly improved the survival of Ad-FasL-treated rats, it significantly increased survival when combined with Ad-TK; ~70% of long term survivors. The neuropathological analysis of naïve rat brains injected with these proapoptic viruses demonstrated that expression of FasL and TRAIL caused overt toxicity, leading to profuse infiltration of inflammatory cells, reduction in TH expression in the striatum, local hemorrhages and ventriculomegaly, while administration of Ad-TK did not significantly alter the structure of the normal brain and induced only a mild, transient local inflammation. Our results show that the combination therapy, Ad-Flt3L/Ad-TK plus GCV is the most efficient amongst the several proapoptotic approaches tested. Moreover, while intracranial expression of proapoptotic cytokines like TRAIL and FasL is very toxic to the normal brain, administration of Ad-TK does not induce overt neuropathological side effects. Our results warrant further development of this combination therapy for the future implementation of a clinical trial for GBM. Supported by NIH/NINDS R01 NS44556.01, R21-NSO54143.01; UO1 NS052465.01; RO3 TW006273-01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and R21 NS047298-01 to P.R.L; F32 NS058156.01 to M.C.

Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy

48. MicroRNA-Regulated Transgene Expression System Reduces Unwanted Side Effects in Adenovirus Vector-Mediated Suicide Gene Therapy

Fuminori Sakurai,1 Takayuki Suzuki,1,2 Masuo Kondoh,2 Kiyohito Yagi,2 Kenji Kawabata,1 Hiroyuki Mizuguchi.1,2 1 Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Ibaragi-City, Osaka, Japan; 2Graduate School of Pharmaceutical Sciences, Osaka University, Suita-City, Osaka, Japan. Adenovirus (Ad) vector-mediated suicide gene therapy using herpes simplex virus thymidine kinase (HSVtk) gene and ganciclovir (GCV) provides a promising therapeutic strategy for cancer; however, one of hurdles confronting Ad vector-mediated HSVtk/GCV system is severe hepatic damages due to unwanted HSVtk expression by Ad vectors in the liver. Ad vectors locally injected into tumors are drained into bloodstream and efficiently mediate hepatic transduction. In order to overcome this drawback, microRNA (miRNA)-regulated transgene expression system was inserted into Ad vectors. MicroRNAs are small 21-23-nucleotide noncoding RNAs expressed in a tissue-specific pattern. MicroRNAs form partial duplexes within the 3’ untranslated region (UTR) of targeted transcripts and repress the translation. We constructed a miRNA-regulated Ad vector by incorporating four tandem copies of sequence perfectly complementary to miR-122a into the 3’ UTR of firefly luciferase (Ad-L-mir122aT). miR-122a is abundantly expressed in hepatocytes. First, several types of cultured human cell lines were transduced with Ad-L-mir122aT, a conventional Ad vector expressing firefly luciferase (Ad-L), or a Ad vector containing the reverse sequence of miR-122a target sequence in the 3’ UTR (Ad-L-mirConT). Ad-L-mir122aT, Ad-L, and Ad-L-mirConT exhibited comparable transduction efficiencies in cells expressing negligible levels of miR-122a. On the other hand, transduction efficiency of Ad-L-mir122aT was significantly decreased, compared with Ad-L and Ad-L-mirConT in Huh-7 cells, which highly express miR-122a. Next, the Ad vectors were intratumorally injected into B16 tumor-bearing mice. Ad-L-mir122aT mediated transduction efficiencies comparable to Ad-L and Ad-L-mirConT in the tumors. In contrast, luciferase production by Ad-L-mir122aT in the liver was approximately 100-fold lower than those by Ad-L and Ad-LmirConT. These results indicate that incorporation of miR-122a target sequence into the 3’ UTR of transgene significantly repress the hepatic transduction following intratumoral administration, without disturbing transduction in tumors. We further constructed a miRNA-regulated Ad vector expressing HSVtk (Ad-tk-mir122aT) and intratumorally administered the Ad vectors into B16 tumor-bearing mice. Single injection of Ad-tk-mir122aT resulted in significant regression of the tumors. Antitumor effects of Ad-tk-mir122aT was almost comparable to those of Ad-tk-mirConT, which is a HSVtk-expressing Ad vector containing the reverse sequence of miR-122a target sequence. However, Ad-tk-mirConT exhibited apparent hepatotoxicity, assessed by histopathological analysis using liver sections. Elevation of glutamic-pyruvic transaminase (GPT) levels in serum and weight loss of mice were also found in Ad-tk-mirConT-injected mice. On the other hand, Ad-tk-mir122aT did not cause apparent hepatotoxicity. These results indicate that Ad vectors carrying miR-122a-regulated HSVtk expression system would be a powerful and safe vector for suicide gene therapy.

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Cancer - Targeted Gene Therapy: Clinical Trials and Large Animal Models Cancer – Targeted Gene Therapy: Clinical Trials and Large Animal Models 49. Pre-Clinical and Clinical Design of a Phase I-II of Gene Therapy for Pancreatic Cancer

Louis Buscail,1 Fabienne Vernejoul,2 Gilles Cambois,2 Fabian Gross,3 Barbara Bournet,1 Anny Souque,1 Hubert Lulka,1 Didier Drocourt,2 Stéphane Pyronnet,1 Gérard Tiraby,3 Pierre Cordelier.1 1 U858 I2MR, INSERM, Toulouse, France; 2Research and Development, Cayla/Invivogen, Toulouse, France; 3CIC Biothérapie, CHU Toulouse, Toulouse, France.

Background: Pancreatic cancer is one of the most aggressive and devastating human malignancies. Excepting surgical resection, there is no efficient treatment. The chemotherapeutic agent gemcitabine improves the patient’s clinical status but survival is not prolonged. Methods: DeoxyCytidine Kinase and Uridylate Monophosphate Kinase (DCK::UMK) fusion gene was designed to efficiently converts gemcitabine into its toxic phosphorylated metabolite. Somatostatin receptor subtype 2 gene (sst2) is a well characterized anticongene for pancreatic cancer, that targets multiple cancerous pathways such as altered cell proliferation, angiogenesis, invasion and apoptosis. Both antitumoral genes were driven by cellular promoters (GRP78 and GRP94) and associated in a bigenic expression vector pVivo (Cayla/ Invivogen). JetPEI polyethylenimine was used as synthetic carrier in these studies. Antitumor effect was tested in vitro in human (BxPc-3, MiaPaca2) pancreatic cancer cells and in vivo in an orthotopic transplantable model of pancreatic cancer established in hamsters. Results: gene transfer using DNA/Jet PEI complexes in pre-GMP conditions strongly sensitizes pancreatic cancer cells to gemcitabine toxic effect both in vitro and in vivo by dramatically reducing cell proliferation, pancreatic tumor growth (50% tumor regression) and metastatic progression. In addition, DNA/Jet PEI complexes toxicity and biodistribution were established in mice and hamsters. We are now setting up a phase I-II clinical trial to include advanced pancreatic cancer patients receiving intratumor injection of plasmid DNA-JetPEI complexes by means of endoscopic ultrasound (EUS) together with gemcitabine IV injection. Evaluation will include local and general tolerance, plasmid biodistribution, tumor volume (RECIST) and clinical benefit. Conclusions: The fusion gene DCK::UMK together with the sst2 gene and gemcitabine treatment strongly antagonize pancreatic cancer cells proliferation both in vitro and in vivo. A Phase I-II clinical trial is designed by sequential EUS-guided intra-tumor delivery of both genes in combination to gemcitabine.

50. Clinical Development of Novel “Bifunctional” shRNA Knockdown Therapeutics to Patient Specific Cancer Targets

John J. Nemunaitis,1,2,3,4 Phillip B. Maples,1,2 Donald D. Rao,2 Alex W. Tong,1,2 Padmasini Kumar,2 Joseph Kuhn,4 Neil Senzer.1,2,3,4 1 Mary Crowley Cancer Research Centers, Dallas, TX; 2Gradalis, Inc., Dallas, TX; 3Baylor Sammons Cancer Center, Dallas, TX; 4 Texas Oncology, P.A., Dallas, TX. It is our hypothesis that non targeted therapeutics shift the cancer fitness landscape only to enable cancer to re-evolve with the generation of a new resistance strategy and lower sensitivity to subsequent random target therapeutics (i.e. chemotherapy). However, in a robust system, e.g. cancer, oncogenic mutational events can also result in a limited number of dependent “high degree information transfer nodes” in the cancer signaling network thereby creating an “attack vulnerability”. We have created a novel process of identifying cancer relevant and potentially critical molecular targets in individual patients and have constructed corresponding RNA interference therapeutics, bifunctional shRNA, to test clinical impact. Malignant and non malignant tissue from 115 cancer patients were S20

harvested. Differentially overexpressed protein and correlated mRNA signals were determined in 34 patients. Prioritized proteins with cancer-relevant function and high connectivity (presumptive nodes) were then selected. Novel “bifunctional” shRNA constructs (n=3), comprised of both cleavage dependent and cleavage independent RISC (RNA induced silencing complex) mediated inhibition of the targeted homologous mRNA, were constructed and tested. The “bifunctional” shRNA produced >90% knockdown of the target protein at 48 hours after clonal cancer cell exposure. Correlative functional modulation (e.g., apoptosis) was observed. Duration of target knockdown, as measured by sequential Western blot and alkaline phosphatase reporter assay, demonstrated temporal advantage of bifunctional shRNA over siRNA (at optimal doses) beyond 48 hours and less off-target activity (5’ RACE assay). Subsequent treatment of malignant clonal cells with known high expression of the target protein confirmed enhanced cell death over time following exposure to the “bifunctional” shRNA. A clinical grade plasmid was engineered. Animal efficacy and toxicology testing are underway using the target vector packaged in a targeted liposomal-based nanoparticle for delivery. The clinical IND justifying patient treatment is in process. Updated results will be presented.

51. Long-Term Expression of Methotrexate Resistant Dihydrofolate Reductase after Lentiviral Stem Cell Transduction and Autologous Transplantation in Dogs

Jennifer L. Gori,1 Brian C. Beard,2 Christina M. Gooch,2 Debra Swanson,1 R. Scott McIvor,1 Hans-Peter Kiem.2,3 1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 3Medicine, University of Washington, Seattle, WA. We have previously demonstrated successful chemoprotection in mice from the antifolate agent methotrexate (MTX) that received congenic bone marrow lentivirally transduced to express methotrexate resistant dihydrofolate reductase (DHFR-L22Y) and green fluorescent protein (eGFP). We have now extended these studies to include autologous stem cell transplantation in dogs. We first generated lentivirus vectors that contain DHFR-L22Y and eGFP in three different configurations including: DHFR-ires-GFP and a DHFR-GFP fusion, each expressed from the human EF1-α promoter; and a two promoter construct (DHFR/GFP) in which DHFR-L22Y and eGFP are expressed by the human EF-1α and PGK promoters, respectively. CD34+ cells were obtained from dogs by apheresis following peripheral blood mobilization or by bone marrow aspiration. CD34+ cells were transduced overnight with or without a second 8 hour transduction at total multiplicities of infection ranging from 6 to 10. Dogs received a myeloablative dose of TBI (920 cGy) before the infusion of gene-modified cells. Gene transfer in peripheral blood has been evaluated at regular intervals by flow cytometry for the GFP marker and by real-time qPCR. The first dog was transplanted with DHFR-ires-GFP expressing cells and showed stable transgene expression >1% by flow cytometry and 2-8% marking by qPCR analysis. MTX with or without leucovorin rescue was administered in a single bolus dose at days 237, 299 and 390 days post transplantation. GFP expression increased in lymphocytes, granulocytes, platelets and red blood cells following two of the three MTX treatments (days 237 and 390). No pronounced cytopenia was observed after any of the MTX treatments. Initial integration analysis of total white blood cells (WBC) in dog #1 suggests that hematopoietic repopulation is polyclonal. In a second dog, cells transduced with either DHFR-GFP fusion or YFP alone were cotransplanted and both genes were detected early post-transplant (GFP 1% and YFP 3%, peaking at days 7 and 21, respectively). However, GFP expression fell below the limit of detection by day 40. This Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy