364. Cyclophosphamide Controls the Humoral Response to Oncolytic Viruses

STEM CELL THERAPIES II ml) (n=11) proteins were detected in supernatant from MV-Morateninfected neutrophils than the uninfected control or MV-WT (levels similar to the controls) (p<0.01). Only one of the molecules evaluated, TRAIL was secreted from MV-Moraten infected neutrophils without a concomitant up-regulation of TRAIL mRNA. Inhibiting protein synthesis with cycloheximide did not reduce MV-induced TRAIL secretion, suggesting that pre-formed TRAIL was secreted from granules. Hence, we evaluated neutrophil de-granulation by FACS in response to MV infection (n=7). A significant enhancement of CD66b and CD63 expression (specific granule and azurophil granule markers) on neutrophil cell surface confirmed that MV-Moraten induced neutrophil degranulation, whereas MV-WT did not. These data suggest that MV-Moraten can directly stimulate neutrophil immunoregulatory and anti-tumor functions causing both de-novo protein synthesis and degranulation. Our data provide the first documented example of a differential effect between an attenuated, oncolytic virus and its wild-type counterpart on the innate immune system. The contrast between the effects of MV-Moraten and MVWT in this regard are not simply due to the fact that MV-WT does not infect neutrophils since abundant MV-N RNA was detected in both cases. The distinct innate immune responses elicited by MVMoraten may play an essential role in oncolysis in-vivo and require merit further investigation during clinical studies.

364. Cyclophosphamide Controls the Humoral Response to Oncolytic Viruses

Kah-Whye Peng,1 Andrew Greenslade,1 Rae Myers,1 Emily K. Mader,1 Suzanne M. Greiner,1 Mark J. Federspiel,1 Stephen J. Russell.1 1 Molecular Medicine, Mayo Clinic, Rochester, MN.

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. Here we evaluated if cyclophosphamide, which kills proliferating lymphocytes, can be used to modulate these counterproductive antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to oncolytic measles virus or Vesicular Stomatitis Virus (VSV) were completely suppressed by a high dose cyclophosphamide regimen initiated one day before virus administration. Lower doses of cyclophosphamide slowed the kinetics of the primary antibody response and lowered maximum titers. When measles-immune or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with high dose cyclophosphamide, their anamnestic antibody responses were completely suppressed, and their antiviral antibody titers fell significantly below pre-booster levels. Antibody titers declined still further after additional exposures to the virus with concurrent high dose cyclophosphamide, at least as much as when cyclophosphamide was given without the virus booster. In immunocompetent tumor-bearing mice, VSV could be administered repeatedly by the intravenous route with cyclophosphamide without provoking an antibody response, and leading to more efficient virus delivery to sites of tumor growth. We conclude that cyclophosphamide should be used in combination with oncolytic virotherapy to control the antiviral antibody response.

365. Inhibition of Rho Associated Coiled-Coil Forming Kinase Increases Efficacy of Measles Virus Infection

Mateusz Opyrchal,1 Ianko Iankov,1 Cory Allen,1 Evanthia Galanis.1 Molecular Medicine, Mayo Clinic, Rochester, MN.

1

BACKGROUND: Attenuated measles virus (MV) is a promising oncolytic agent in cancer vibrotherapy and it has been shown to have robust antitumor effect against wide range of cancer cells both in vitro S142

and in vivo. MV strains exert their cytopathic effect by inducing cell fusion and syncytia formation. RhoA and its downstream effector, Rho associated coiled-coil forming kinase (ROCK) are known regulators of formation of actin cytoskeleton in cells. Regulation of RhoA and actin cytoskeleton is known to influence paramyxovirus associated cell fusion. The two isoforms of ROCK (ROCK1 and 2) are ubiquitously expressed in all human cells and the ROCK inhibitor, Y27632, has been shown to inhibit hepatic and prostate cancer metastases in mouse models. Y27632 and other specific ROCK inhibitors are being tested as anti-neoplastic agents. We hypothesized that the combination therapy of Y27632 and MV could improve MV mediated oncolysis through enhanced cell fusion and increase in syncytia size. RESULTS: The hypothesis was tested in vitro on three cancer cell lines. First we assessed the effect of Y27632 cell proliferation against prostate cancer (PC-3), glioblastoma (U251) and breast cancer (MDA-MB-231) cells. Concentrations with minimal effect on cell proliferation were employed in combination experiments (100 µM, 25 µM and 150 µM respectively). The effect of Y27632 on MV cytopathic ffect was explored next. Cells infected with MV while concurrently treated with Y27632 showed increased cytopathic effect of the virus as assessed by trypan blue exclusion assays. This effect was dependent on the concentration of the inhibitor in MDA-MB-231 cells. Next we tested the effect of the ROCK inhibitor on viral proliferation. In all three cell lines, there was a significant increase in viral proliferation after day two by at least one log or more as tested in one step viral growth curves. Viral titers correlated with increasing concentration of Y27632 up to 150 µM in MDA-MB-231 cells. Furthermore, treatment with Y27632 significantly increased syncytia size in MDA-MB-231 and PC-3 cells following MV infection, associated with cytoskeleton disruption as demonstrated by actin staining. Animal combination experiments are ongoing. CONCLUSIONS: Inhibition of ROCK by the specific inhibitor, Y27632, enhanced therapeutic efficacy of oncolytic effect of MV on prostate, breast and glioblastoma cancer cells. Increased killing effect correlated with higher viral titers and larger syncytia. Y27632 effect on cellular cytoskeleton likely accounts for the larger syncytia size. These results contribute to better understanding of the mechanism of MV infection and present new direction in improving efficacy of MV virotherapy.

Stem Cell Therapies II 366. Reprogramming Mouse iPS Cells as a New Approach To Correct the Stem Cell Defect in the Mpl-/- Mouse Model

Jackie Roy,1 Angela Epp,1 Junli Feng,1 Xiaoping Wu,1 Neil C. Josephson.1 1 Research Division, Puget Sound Blood Center, Seattle, WA.

Congenital Amegakaryocytic Thrombocytopenia (CAMT) is an inherited recessive stem cell disorder cause by loss of function mutations in Mpl, the receptor for Thrombopoietin. Infants with CAMT typically present with isolated thrombocytopenia in the perinatal period and over the course of months to years develop a lethal pancythopenia. The only effective treatment is allogeneic stem cell transplantation. Unfortunately, a significant number of patients do not have a suitable HLA matched donor and even patients with donors still face significant complications post-transplantation including graft rejection, graft versus host disease, and infectious risks from chronic immunosuppression. Restoration of Mpl expression by hematopoietic stem cell (HSC) gene therapy offers numerous advantages in the treatment of CAMT. However, the disease’s inherent HSC defect greatly limits the number and viability of marrow repopulating cells that could be isolated and corrected. An alternate strategy is to generate and correct patient-derived iPS cells which could then be differentiated into HSCs and used to rescue individuals with marrow Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy