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[.]
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ADENOVIRUS AND OTHER DNA VIRUS VECTORS III
Signifi cant 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
Conclusions: Systemic delivery of oncolytic viruses Ad.sTßRFc
and mhTERTAd.sTßRFc induced signifi cant inhibition of bone metastases and can be developed as a safe and effective approach for the treatment of established bone metastasis of breast cancer
DNA Polymerase Contributes to Virus Replication Effi ciency and Host Cell Tropism
Cristina Capella-Gonzalez,1 Michael J Beltejar,1 Baek Kim,1
Stephen Dewhurst.1
1 Microbiology and Immunology, University of Rochester, Rochester, NY.
Mutations in the viral DNA polymerase of HIV-1 that reduce the effi ciency 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 fi ve replication-competent molecular clones that were recovered were as follows: I664V, I664M,
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ADENOVIRUS AND OTHER DNA VIRUS VECTORS III
R665K (IxGG motif mutants) and C687S and M689V (motif A
mutants) All replicated effi ciently 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 fi broblast 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 effi cient 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
(1-7-3.3 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 refl ected 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 suffi cient 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
fi ndings 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
Yue Zhang,1 Wenzhong Li,1 Gustav Steinhoff,1 Nan Ma.1
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
fi elds Methods: Adenovirus vector-gene was conjugated to MNBs
with the Sulfo-NHS-LC-Biotin linker In vitro transduction effi cacy of
MNBS/Adenovirus was compared with Adenovirus alone in (MSCs)
mesenchymal stem cells under magnetic fi eld stimulation In vivo,
in the rat myocardial infarction (MI) model,
MNBS/Adenovirus-VEGF 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
effi ciency under the magnetic fi eld 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
signifi cantly improved left ventricular function (n=8-10, p<0.05)
assessed by pressure-volume loops after 4 weeks Although there was
no signifi cant difference in infarct size between two group (n=8-10,
p<0.05), wall thickness of remote area was signifi cantly reduced
in MNBs/Adenovirus (n= 6) compared with Adenovirus (n =8)
Meanwhile MNBs/Adenovirus group hearts exhibited higher capillary
density than Adenovirus group Conclusion: Magnetic targeting
enhances transduction effi ciency and improves heart function in vitro
and in vivo This novel method to improve gene therapy outcomes
offers the potential into clinical applications
Against Triple-Negative Breast Cancer
Sepideh Gholami,1 Chun-Hao Chen,1 Joshua S Carson,1 Dana
Haddad,1 Song Taejin,1 Joyce Au,1 Jun Kwong,1 Yuman Fong.1
1 Department of Surgery, Memorial Sloan-Kettering Cancer Center,
New York, NY.
BACKGROUND: Triple-negative breast cancer (TNBC) is one of
the most aggressive cancers, and so-called, as it lacks the expression
of common targets for breast cancer therapy: estrogen, progesterone,
and HER-2 receptor Moreover, overexpression of MAPK has been
correlated with increased resistance to chemotherapy and recurrence
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 signifi cant 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 confi rmed 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 6 post-infection Even in less sensitive cells lines HCC1937 and HCC1143, NV1066 achieved greater than 70% cell kill by day 7 post-infection at an MOI of 1.0
In the most sensitive cell line MDA-MB-231, p-MAPK signaling after 48 hours of infection with NV1066 was inversely correlated
to viral replication measured in plaque-forming units (R=-0.78, p<0.05)