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213 Response of Drug Sensitive and Resistant Breast Cancer Cells to Combinatorial siRNA Therapy Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Ther[.]
CANCER-TARGETED GENE & CELL THERAPY: VIRAL VECTOR / SIRNA-MEDIATED THERAPIES the hepatocytes including mitochondrial injury, caspase activation, autophagy and apoptosis In certain cases the liver pathology progresses to hepatocellular carcinoma (HCC) We have previously developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant AAT-PiZ (Z-AAT) knockdown in the liver and increased expression of M-AAT While showing that artificial miRNAs have a minimal effect on the endogenous miRNA liver profile, the miRNA microarray analysis revealed that miR-1 expression was significantly altered in PiZ compared to age-matched non-treated BL/6 Importantly, miR1 is has been recently described to function as a tumor suppressor in various models, although thus far it has mainly been associated with lung cancer and prostate cancer However our preliminary data suggests that miR-1 is down-regulated in human HCC samples as well The human PiZ transgenic mouse model naturally develops liver tumors at months of age and by 16-19 months, 70% present tumors A longitudinal analysis of these mouse livers indicate that already in young PiZ mice (1-5 months), miR-1 expression is lower than in age-matched BL/6 Furthermore, analysis of older PiZ mice with HCC shows that miR-1 is significantly down-regulated in the liver tumors tissue (T) when compared to non-tumoral liver tissue (NTL) To determine if modulation of miR-1 had an effect in the progression of tumorogenesis in this model, we dosed the mice with a rAAV8 vector expressing a miR-1 antagonist In this model AAV-mediated miR-1 knockdown accelerated tumor progression, specifically we observed that 66% of PiZ mice injected at wk with AAV8-tuD-miR-1 developed tumors at months p.i., while PiZ normally start developing tumors by months of age only, and such tumor percentage is not reached before 16-19 months Finally, gene expression analysis revealed that validated miR-1 targets including Cyclin D1 are up-regulated in PiZ mice Ongoing miR-1 target predictions and validation will help define the precise role of miR-1 in the development of liver tumors in AAT deficiency background Dysregulation of miR-1 and its targets in AAT patient samples will ultimately be confirmed by miRNA and gene profiling 212 AAV Mediated IFNβ Gene Therapy of Orthotopic Xenograft Mouse Model of Invasive Glioblastoma multiforme Dwijit GuhaSarkar,1 James Neiswender,1 Miguel Sena-Esteves.1 UMASS Medical School, Worcester Introduction: Glioblastoma multiforme (GBM) is the most prevalent and aggressive form (WHO grade IV) of primary brain tumors Extensive angiogenesis, rapid and highly invasive growth, recurrence and high mortality are some of the characteristics of this disease IFN is a type I interferon that has been shown to have several anti-cancer properties through its pro-apoptotic, anti-angiogenic, cytostatic and immue-modulatory roles But, despite some success in interferon therapy in animal models, it failed to improve GBM patient survival in clinical trials The lack of efficacy in humans is thought to be due to its short half-life and considerable toxicity associated with high dose systemic infusion A low but continuous expression of interferon locally may be a solution to these limitations Hence, AAV mediated interferon gene therapy can be an effective approach Experiment: We have shown before that locally delivered AAVrh8/ human IFN gene therapy can regress an established orthotopic human GBM (U87-fluc-mCherry) in adult athymic nude mice Next, we were interested to see if we can replicate our success with an invasive and highly migratory GBM model Moreover, we wanted to know how late we can treat the tumor post-implantation and if the therapeutic effect of this treatment is stable or the tumors recur Human GBM8 glioblastoma cells are highly invasive in the mouse brain forming diffuse tumors.These cells are grown in culture as neurospheres in S82 defined serum-free medium and have a high percentage of CD133+ cells We modified GBM8 cells by lentiviral infection to create a line (GBM8-fluc-mCherry) that stably expresses firefly luciferase (fluc) and mCherry We tested the properties of the modified cells and confirmed that the modification does not affect its invasiveness or aggressiveness We then implanted these cells in the left striatum of 6-8 week-old athymic nude mice and treated them with AAVrh8/ CBA-IFN or empty vector control injecting locally at 2, and weeks post tumor implantation We assessed tumor growth in real time by live bioluminescence imaging of the GBM8-associated fluc expression weekly and euthanized the animals when they reached the humane endpoint We terminated the experiment after months We then performed post-mortem histological analysis of the brains from all the animals Result: We found that all the control group animals had to be euthanized before 50 days post tumor implantation with a median survival of 47days(empty vector) or 49days(untreated) In contrast, for the 2nd week treated group, we had to euthanize only out of a group of mice on 154th day and the rest of this group lived normally until the termination of experiment Importantly, we did not find any trace of tumor in the brains from any of these mice in post-mortem histological analysis The success of the therapy for the 3rd and 4th week treated groups were partial with median survivals of 66 and 110 days respectively, both significantly higher than controls but lower than 2nd wk group We conclude from this experiment, that AAV mediated IFN gene therapy with local injections can be effective with invasive GBM when treated early until it becomes too large or migrates beyond the physical reach of the local therapy 213 Response of Drug Sensitive and Resistant Breast Cancer Cells to Combinatorial siRNA Therapy Hamidreza Montazeri Aliabadi,1 Parvin Mahdipoor,1 Hasan Uludag.1 Chemical and Mterial Engineering, University of Alberta, Edmonton, AB, Canada Chemotherapy is an effective approach to curb uncontrolled proliferation of malignant cells; however, even the most recent molecularly-targeted drugs lose their effect as a result of resistance development in a relatively short period of time The inherent plasticity of transformed cells and diverse resistance mechanisms enable malignant cells to mount an effective resistance against the administered drugs Molecular changes in drug resistant cells are beginning to emerge, including adjustments in expression level of various proteins involved in cell survival and apoptosis We are exploring the potential of siRNA silencing as a new approach to induce apoptosis and/or overcome drug resistance in breast cancer cells This study was conducted to develop a global approach to identify crucial targets and deliver siRNAs to silence a combination of proteins in order to treat breast cancer by inducing apoptosis (independent of chemotherapy) and/or sensitize the resistant cells to chemotherapy Resistance was induced in two breast cancer lines by chronic exposure to gradually increasing doses of doxorubicin, and resistance induction was confirmed by a significant increase in IC50 of DOX An apoptosis microarray was used for analysis of mRNA levels of 84 apoptosis related proteins in wild type and resistant cells While different members of caspase and TNFR family were among down-regulated proteins, over-expressed proteins in both resistant cell lines included Bcl2, survivin, NFB, and Mcl1 Human Apoptosis siRNA library and kinase siRNA library were screened using PEILA2.1 as delivery system Those siRNAs were selected as hits which induced cell death in cancer cell lines without significant reduction in cell viability of skin fibroblast cells The “hits” in apoptosis library included BIRC 7, NFB, and Mcl1 Eight kinases were selected (based on the same criteria and potential synergistic effect with Mcl-1) In vitro evaluations revealed that among the Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy CANCER-TARGETED GENE & CELL THERAPY: VIRAL VECTOR / SIRNA-MEDIATED THERAPIES selected targets, MAP2K3 was the most potent in decreasing cell viability, while RPS6KA5 showed the most promising potential for simultaneous silencing with Mcl-1 The combination of Mcl-1 and RPS6KA5 was evaluated in vivo both as multiple intratumoral (1.5 g for each siRNA) and intraperitoneal (10 g each siRNA) treatments in a MDA-MB 435 WT xenograft model in NCR nu/nu nude mice In the intratumoral treatment, a significant inhibition of tumor growth was observed for Mcl-1 single silencing compared to scrambled siRNA and Mcl-1/RPS6KA5 double silencing compared to Mcl-1 silencing Intraperitoneal treatment showed a similar trend; however, only the combinational silencing of Mcl-1 and RPS6KA5 showed a significant inhibition of tumor growth This study demonstrated the potential for kinase silencing as a therapeutic strategy in breast cancer therapy, as well as the promising synergistic effect for Mcl-1 and kinase silencing 214 Combination Therapy with Novel Nanoparticle, SNS01-T, and Bortezomib Results in Synergistic Cytotoxicity In Vitro and In Vivo in Multiple Myeloma Catherine Taylor,1 Qifa Zheng,1 Zhongda Liu,1 Terence Tang,1 Sarah Francis,1 Richard Dondero,2 John Thompson.1,2 University of Waterloo, Waterloo, Canada; 2Senesco Technologies Inc., Bridgewater INTRODUCTION: SNS01-T is a novel nanoparticle that is designed to selectively initiate apoptosis in B-cell cancers such as multiple myeloma and B-cell lymphomas SNS01-T is comprised of a plasmid encoding a pro-apoptotic form of the eukaryotic translation initiation factor 5A (eIF5A) containing a single-point mutation that prevents hypusination, an siRNA that inhibits expression of eIF5A, including the pro-survival hypusine-eIF5A protein, and the polymer polyethylenimine that serves to assemble the nucleic acids into a nanoparticle SNS01-T is currently being investigated in a multi-site, open-label Phase1b/2a dose escalation study in patients with relapsed or refractory multiple myeloma (MM) or B cell Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy lymphoma Bortezomib is the first therapeutic proteasome inhibitor to be investigated in the clinic and has been approved in the U.S for the treatment of relapsed MM In this study we investigated the in vitro and in vivo anti-cancer activity of SNS01-T in combination with bortezomib METHODS: The cytotoxicity of the SNS01-T and bortezomib drug combination in MM cell lines was assessed using XTT assays Western blot analysis was used to determine if exposure to the drug combination resulted in changes in expression of apoptotic proteins To determine whether SNS01-T treatment increases the anti-myeloma activity of bortezomib in vivo, 0.375 mg/kg SNS01-T (intra-venous; 2x weekly) was combined with either 0.2 or 0.5 mg/kg bortezomib (intra-peritoneal; 2x weekly) in a RPMI 8226 xenograft model of multiple myeloma RESULTS: The combination of SNS01-T and bortezomib synergistically reduced viability of RPMI 8226 and KAS-6/1 MM cells in culture The reduction in cell viability in response to the drug combination was accompanied by increased cleavage of caspases and and reduced expression of myeloid leukemia cell differentiation protein (Mcl1), an anti-apoptotic Bcl-2 family protein In a xenograft model of MM, the drug combination was more effective at controlling tumor growth than either drug alone At the end of dosing, tumor growth was inhibited by 59 % (p = 0.002), 38 % (p = 0.003), and 89% (p