676 The dNTP Binding Efficiency of Adenovirus DNA Polymerase Contributes to Virus Replication Efficiency and Host Cell Tropism Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The Ameri[.]
ADENOVIRUS AND OTHER DNA VIRUS VECTORS III Conclusions: Systemic delivery of oncolytic viruses Ad.sTßRFc and mhTERTAd.sTßRFc induced significant inhibition of bone metastases and can be developed as a safe and effective approach for the treatment of established bone metastasis of breast cancer Significant reduction of tumor burden and osteoclast activation, and increased body weight gains were observed Oncolytic adenoviruses were safer than dl309, a wild type virus Slight elevation of liver enzyme activity was observed by Ad.sTßRFc that subsided with time Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 676 The dNTP Binding Efficiency of Adenovirus DNA Polymerase Contributes to Virus Replication Efficiency and Host Cell Tropism Cristina Capella-Gonzalez,1 Michael J Beltejar,1 Baek Kim,1 Stephen Dewhurst.1 Microbiology and Immunology, University of Rochester, Rochester, NY Mutations in the viral DNA polymerase of HIV-1 that reduce the efficiency of dNTP substrate utilization result in a selective loss of viral replicative activity in resting cells (which contain low dNTP concentrations), but not in rapidly dividing cells such as cancer cells (which contain high levels of dNTPs) This suggests a potential new approach to the development of oncolytic, conditionally replicating adenoviruses With this in mind, a series of site-directed mutations were introduced into the DNA polymerase of adenovirus type-5 (Ad5), in regions known to interact with the dNTP substrate or the template strand DNA – including highly conserved domains (motifs A and B and IxGG motif) and key consensus sequences within these domains (Kx3NSxYG within motif B) The introduction of even conservative mutations in these motifs, that are known to have only a modest (5-fold) effect on polymerase function in in vitro biochemical assays, had a dramatic effect on viral replicative capacity when studied in the context of an intact, full-length adenovirus genome Indeed, such mutations completely abrogated virus replication in 15 out of the 20 viral molecular clones constructed The five replication-competent molecular clones that were recovered were as follows: I664V, I664M, S259 ADENOVIRUS AND OTHER DNA VIRUS VECTORS III R665K (IxGG motif mutants) and C687S and M689V (motif A mutants) All replicated efficiently in HEK293 cells, but the I664M, R665K, and C687S mutants exhibited restricted replication in both A549 cells (a human lung carcinoma cell line) and Wi-38 cells (a primary human lung fibroblast cell line) The I664V mutant exhibited replication kinetics essentially identical to those of WT Ad5 in all of the cell lines tested, while the M689V mutant showed restricted replication in cancerous cells (A549), but efficient replication in primary cells (Wi-38) - even though dNTP concentrations in Wi-38 cells (0.1-0.2 µM) are substantially lower than in A549 cells (1-73.3 µM) or HEK293 cells (1.0-2.3 µM) Of note, the M689V mutant was also able to establish persistent, productive infection of Wi-38 cells, as reflected by the prolonged expression of an encoded GFP reporter gene – unlike the WT virus, which rapidly killed the Wi-38 cells We conclude that (i) even modest changes in the enzymatic activity of Ad5 DNA polymerase are sufficient to completely abrogate virus replication, and (ii) that a subset of polymerase mutations can alter virus host cell tropism, and virally-mediated cell killing These findings have implications for the future development of adenovirus vectors, for both gene transfer and oncolysis applications 677 In Vitro & Vivo Gene Targeting with Adenoviral Vector Conjugated Magnetic Nanobeads for Cardiac Regeneration in patients with TNBC The objective of this study was to determine the cytotoxic effects of an oncolytic herpes virus on TNBC and to discover the potential correlation of the MAPK pathway in sensitizing TNBC to oncolytic viral therapy METHODS: The cytotoxic effect of a replication-competent herpes virus (NV1066) containing a GFP protein expression cassette was tested on human TNBC cell lines MDA-MB-231, HCC1806, HCC38, HCC1937, and HCC1143 at various multiplicities of infections (MOIs) Proliferation assays were performed to assess cell doubling time Cytotoxicity was determined using a standard lactate dehydrogenase assay Viral replication was assessed via standard plaque assay Pre- and post-viral infection cell lysates were analyzed by Western blotting with antibodies against proteins of interest involved in significant oncogenic signaling pathways RESULTS: All cell lines exhibited similar proliferation rates with an average doubling time of 48 hours NV1066 infected, replicated in, and killed all TNBC cell lines GFP expression confirmed viral infection and replication by 24 hours and was proportionate to both viral dosages and cytotoxicity results At an MOI of 0.1, greater than 90% cell kill was achieved in cell lines MDA-MB-231, HCC1806, and HCC38 by day post-infection Even in less sensitive cells lines HCC1937 and HCC1143, NV1066 achieved greater than 70% cell kill by day post-infection at an MOI of 1.0 Yue Zhang,1 Wenzhong Li,1 Gustav Steinhoff,1 Nan Ma.1 Uni Rostock, Medicine Facult, Rostock, Germany Objectives: we try to test the concept whether delivery of magnetic nanoparticles (MNB) /Adenovirus-VEGF gene could regenerate infarcted hearts in the rat model under the control of external magnetic fields Methods: Adenovirus vector-gene was conjugated to MNBs with the Sulfo-NHS-LC-Biotin linker In vitro transduction efficacy of MNBS/Adenovirus was compared with Adenovirus alone in (MSCs) mesenchymal stem cells under magnetic field stimulation In vivo, in the rat myocardial infarction (MI) model, MNBS/AdenovirusVEGF complexes were injected intravenously and an epicardial magnet was employed to attract the circulating MNBS/Adenovirus complexes Results: In vitro, Compared with Adenovirus alone, MNBs/Adenovirus complexes had a 30-to 50-fold higher transduction efficiency under the magnetic field In vivo, the epicardial magnet effectively attracted MNBs/Adenovirus complexes and resulted in strong therapeutic gene expression in the ischemic zone of the heart Also compared to Adenovirus group, MNBs/Adenovirus group significantly improved left ventricular function (n=8-10, p