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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1 MEETING REPORT

8th International Conference on Oncolytic Virus Therapeutics

Barbara-ann Guinn1*, Lynne Braidwood2, Alan Parker3, Kah-Whye Peng4, Leonard Seymour5

1

Department of Life Sciences, University of Bedfordshire, Park Square, Luton, LU1 3JU, 2Virttu

Biologics, Glasgow, Scotland, 3Institute of Cancer & Genetics, University of Cardiff, Wales, U.K., 4

Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA, 5Department of

Oncology, University of Oxford, OX2 6HE, U.K.

*Corresponding Author:

Dr. Barbara Guinn, Department of Life Sciences, University of Bedfordshire, Park Square, Luton, LU1 3JU. Tel: +44 1582 743573 e.mail: [email protected]

Running title: OVC annual meeting, Oxford 2014 Keywords: oncolytic virotherapy, clinical trials, targeting delivery, imaging, virus genetic engineering Abbreviations used in this paper: aa: amino acid; Ad: adenovirus; APC: antigen presenting cell; BCG: Bacille de Calmette et Guérin; CAFs: cancer associated fibroblasts; CD: cytosine deaminase; CIS: Carcinoma in situ; CR: complete remission; CTC: circulating tumour cells; CTL: cytotoxic T lymphocyte; DC: dendritic cell; DNA-PK: DNA protein kinase; DR: durable response rate; FGF: fibroblast growth factor; FMT: Farmington; FS: frame shift; GBM: gliomablastoma multiforme; H-1PV: H-1 parvovirus; HCC: hepatocellular carcinoma; HDAC: histone deacetylase; HIF: hypoxia inducible factor; IC: intracranial; IFN: interferon; IHC: immunohistochemistry; IP: intraperitoneal; ISG15: IFNstimulated gene 15; IT: intratumoural; IV: intravenous; LPAIV: low pathogenicity avian influenza virus; MAVS: mitochondria antiviral signaling protein; MM: multiple myeloma; MRI: magnetic resonance imaging; MTS: Mitochondrial Targeting Sequence; MV: measles virus; NIS: sodium iodide symporter gene; NDV: Newcastle disease virus; NK: natural killer; NSCLC: non-small cell lung cancer; o-HSV: oncolytic-human simplex virus; OS: overall survival; OV: oncolytic virus; PD-1: Programmed death receptor-1; PDA: pancreatic ductal adenocarcinoma; PHD2: prolyl hydroxylase domain containing

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

2 protein 2; PI: propidium iodide; pfu: plaque forming units; RRV: retroviral replicating vectors; SC: subcutaneous; SD: stable disease; TAA: tumor associated antigen; TAP: Transporter associated with Antigen Processing; TMZ: temozolomide; UPR: unfolded protein response; vp: viral particles; VEGF: vascular endothelial growth factor; VSV: vesicular stomatitis virus; VV: vaccinia virus; VVPL: Viral Vector Production Laboratory; WT: wild type.

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

3 Abstract 8th International Conference on Oncolytic Virus Therapeutics meeting was held from 10 th – 13th April 2014 in Oxford, England. It brought together experts in the field of oncolytics from Europe, Asia, Australasia and the Americas and provided a unique opportunity to hear the latest research findings in oncolytic virotherapy. Presentations of recent work were delivered in an informal and intimate setting afforded by a small group of attendees and an exquisitely focussed conference topic. Here we describe the oral presentations and enable the reader to share in the benefits of bringing together experts to share their findings.

Introduction Many of the presentations showed a ground swell of excitement in the field of oncolytic viruses (OVs). Several clinical trials are now in progress and showing considerable promise. OVs have shown safety and efficacy in phase I/II clinical trials and in terms of responses there are a growing number of examples of patients achieving CR and extended survival. Now that oncolytic proof of principle has been established, we are entering a new era of OV development with greater focus on enhanced targeting to tumour tissue, improved replication in situ, spread of virus through tumour tissue and co-expression of transgenes which enhance anti-cancer activity. There has also been an acceptance that the immune system can often play an important role in effective OV killing of tumour cells, providing several new strategies for design of ‘oncolytic vaccines’. Finally, most of these advances are founded on basic science, and several presentations showed new insights into the mechanisms of selectivity and action of oncolytic viruses, and how tumour-associated changes often provide a molecular environment that encourages efficient virus replication and spread.

Immuno-virotherapy Kah Whye Peng, Mayo Clinic, USA described how during the first phase of OV infection the virus kills infected cells (the ‘oncolytic phase’) while often in a second phase the immune system comes to mop up dead and dying cells and presents antigens therein to stimulate the immune system (the ‘immune phase’) to cause further tumour cell death. Vesicular stomatitis viruses (VSV) has low virulence in the population but causes blistering disease in ungulates (cattles, horse, pigs), while human exposure most commonly causes flu-like symptoms. VSV has a broad tropism and so this virus has been modified to express interferon-beta (IFN and denoted VSV-IFN. A second virus was developed to also express the sodium iodide symporter gene, NIS (VSV-IFN-NIS). While both viruses express IFN at higher levels there remains a lack of VSV antibodies in the population. This low sero-prevalence makes VSV a suitable vector for systemic therapy. The Viral Vector Production Laboratory at the Mayo Clinic (VVPL; led by Mark Federspiel) makes VSV in a WAVE 3

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

4 bioreactor >99.5% purification with 30-50% yields and the Toxicology/Pharmacology Laboratory (Peng) is involved in the toxicology pharmacological assessment of VSV vectors prior to clinical trial. Mitesh Borad is the principal investigator for the clinical trials and is now overseeing a Phase I trial of intra-tumoural injections of patients with advanced hepatocellular carcinoma (HCC), who are refractory or intolerant to sorafenib and not eligible for transplant. Six patients have been treated to date, three at 105 TCID50 and three at 106 TCID50 dose. The clinical trial is also open for patients with liver metastases. VSV-hIFN has no NIS reporter so you cannot see extent of virus replication. Next generation VSV-IFN-NIS allows visualisation of the virus in multiple myeloma (MM) mouse models including human xenografts in SCID mice. The study has shown potency (VSV), specificity (IFN) and tracking (NIS imaging). They can see the virus is fast, following a single IV dose and then daily imaging within one day the virus has homed to tumour and you can see reduction in the NIS signal as the tumour is destroyed. Perivascular distribution of the virus at early time points, and every 6 hrs a rapid expansion is observed and by 48h, most of the infectious foci have coalesced. The speed of VSV spread in this model outpaces the immune system. 72hrs after administration the 5TGM1 tumour has been rapidly killed and the group showed that IFN is enhancing the anti-tumour immunity in mice. Neurotoxicity has not been seen in irradiated SCID mice given 108 TCID50 VSVmIFN-NIS, and when contrasted to immune competent mice, the effect is not as stark but still good. VSV-IFN-NIS translation in companion dogs, beagles, IV push 3-5mins VSV-hIFN-NIS diluted in saline, doses 108, 109, 1010, 1011 TCID50 per dose. Weight, pyruvate kinase (PK) levels, complete blood count, blood chemistry, coagulation, shedding of virus as detected by RT-PCR and inhibitor of virus replication (IVR) assays. Dose limiting toxicity was seen at 1011 – including serious vomiting and diarrhoea. A transient increase in alanine aminotransferase (ALT) liver enzyme levels were observed in both of the dogs which had T cell lymphoma and correlated with a more sustained shrinkage of tumour. Currently 2 x 1010 viral particles (vp) dose has been shown to be safe and minor increments in doses are now being tested. Richard Vile, Mayo Clinic, USA chose VSV as it is very sensitive to the type I IFN response, which is shut down in normal cells but not in tumour cells. This is only the case if tumour cells are truly and completely defective in all aspects of the IFN response. VSV will be an excellent oncolytic. In reality many tumour cells still have the ability to produce and/or respond to IFN. By encoding multiple TAA in VSV the Vile group found that they need to identify relevant tumour associated antigens (TAAs), release TAA for presentation to antigen presenting cells (APC), recruit and activate APC for presentation to TAA-specific T cells and then increase the frequency of fully activated T cells. Intravenous (IV) into B16 tumours has an oncolytic effect while depletion of CD4 cells led to removal of the effect, while the depletion of CD8 had a much smaller effect on tumour responses. VSV-TAA need all three self-antigens to generate a response. Systemic treatment of mice with IC tumours led to a significant improvement in survival, but the immunogenic targets differ between the 4

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

5 locations. IC studies demonstrated the important combinations of VSV were hypoxia inducible factor2 (HIF-2, SOX10, TYRP-1 and c-MYC – giving an IL-17 recall response. Subcutaneous (SC) then it was N-RAS, TYRP-1 and Cyt-c that generated the largest IL-17 response when compared to the IC combination. When the group looked at tumours from the different sites and examined their antigen expression they could see significant differences in the profiles of antigen expression even though B16 was used in the same strain of BL6 mice. The group demonstrated that it was the tumour microenvironment which imposed the phenotype on the tumour cells. Best survival was found with a combination of VSV-N-RAS+VSV-TYRP-1+VSV-CYT-C in IC tissues delivered as 12 intracranial (IC) injections. If they added in T cell costimulation the group showed you can move from extended survival to ≤80% cures. If the same tumour is then placed in different mice the group saw a difference in the combination of antigens which generated the best anti-tumour response. Avogadri and Wolchok (Avogadri and Wolchok 2012) showed that different antigens are needed to treat recurrent tumour compared to primary tumour. Recurrent B16 tumours can be treated with VSV-cDNA libraries which cross protect against a range of tumours. TYRP1 is an antigen from ASMEL which is expressed in B16 and is expressed in tumours in both locations. Akseli Hemminki, University of Helsinki, Finland showed data from the 290 patients who have been treated in an Advanced Therapy Access Program, which is not a trial, but through a process of individualized therapy, using ten different viruses. Patients showed mild side effects (grade 1-2) while some serious adverse effect were observed in about 10% of patients (such as emboli). Overall patients required much less chemotherapy and there were no treatment related deaths. There was also evidence of immune responses including vitiligo, a classic indicator of anti-tumour immunity. Lymphocyte numbers decreased in the blood 1d after treatment, when they increased in numbers in the tumour, but recovered within 1 week. There was an induction of anti-tumour T cells followed by trafficking to tumours and there was a high concordance between induction of anti-viral and antitumoural immunity.

In ovarian cancer patients the group could see benefits from oncolytic

virotherapy including major molecular responses, molecular complete remission (CR) and very long overall survival (OS). Cancer treatment with oncolytic Ad armed with different transgenes (GM-CSF, CD40L) with or without capsid modification were shown to be safe. Some patients benefitted from the therapy and Oncos Therapeutics Ltd has completed this Phase I and is now planning randomised Phase II. Adoptively transferred T-cells act as a catalyst for pre-existing T cells but fails as a therapy when there is lack of recruitment of transferred T cells to the tumour. Causes of this include anergy of transferred cells at the tumour, lack of human leucocyte antigen (HLA) in tumour cells and a lack of propogation of transferred cells within the tumour and lymph node. Solutions may include cytokines coded by virus to provide trafficking and activation signals. Jean Rommelaere, DKZ-Heidelberg, Germany described how H-1 parvovirus (H-1PV) is not associated with diseases in humans, it has been shown to be oncotropic and have oncolytic properties

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

6 in cell and animal models. In addition, it exerts immunomodulating effects which contribute to its capacity for tumor suppression. Cell transformation results in both quantitative and qualitative upmodulations of the cytotoxic activity of the parvoviral NS1 protein. This protein will kill tumour cells in a way which circumvents resistance unlike conventional anticancer treatments. The PDK1/PKC/PKB pathway represents a contributing factor for H-1PV oncotropism by enhancing the permissiveness of human cells for both NS1 production and functional activation through phosphorylation. The immunostimulating effects of H-1PV are mostly indirect and mediated by infected tumor cells. Compared to cells which are freeze-thawed or irradiated, H-1PV-infected tumor cell lysates are much more efficient at inducing dendritic cell (DC) maturation, tumor-associated antigen cross-presentation and tumor-specific cytotoxic T lymphocyte (CTL) activation, as measured by IFNγ production. Similarily, natural killer (NK) cell activation and anti-tumour responses are much enhanced when the target tumour cells are H-1PV-infected. The dual effect of H-1PV infection of tumor cells, namely direct oncolysis and immunostimulation, could also be demonstrated using patient pancreatic ductal adenocarcinoma cells (PDAC)-xenografted immunodeficient mice, which had been reconstituted with ex vivo PDAC-primed DC/T cells. Furthermore, immunodepletion experiments showed that in an immunocompetent rat model, H-1PV-mediated glioma suppression is in part CD8+ cell-mediated. Similarily in a mouse glioma model, MVMp-mediated cure leads to a tumour-specific T cell memory response. An ideal OV should be able to not only kill tumor cells but also to be produced in large amounts, resulting in virus spread and infection of tumour cells which were not hit by the primary infection. Forced passaging of H-1PV in human glioma cell cultures led to the isolation of virus mutants endowed with an enhanced capacity for production and propagation in these cultures. Molecular changes associated with H-1PV adaption to enable their efficient production in human glioma cells consisted of single amino acid (aa) substitution(s) displayed on the capsid surface which affected early interactions of the virus with putative intracellular receptor(s), and a deletion in the NS proteins affecting viral DNA replication and progeny virus production. H-1PV is the subject of an ongoing phase I/IIa clinical trial, ParvOryx01, in patients with recurrent gliomablastoma multiforme (GBM) progressing in spite of surgery and radio/chemotherapy. ParvOryx01 is the first oncolytic virotherapy clinical trial approved in Germany, and is composed of two arms differing in the first step of the treatment. In study group 1, the virus is applied intratumourally (IT)(single injection), while in study group 2, the virus is injected IV (5 daily infusions). After 9 days the tumour is resected and the same dose of virus is reapplied around the cavity. In the high dose group of IT treated patients, large necrotic areas were seen in two of the four resected

tumours.

Through

fluorescent

ins-situ

hybridisation

(FISH)

and

immunofluorescence/immunohistochemistry (IHC) analyses, there was dose-dependent detection of widespread IT virus replication and expression, and also glioma-infiltrating immune cells (in particular T-lymphocytes). Blood analyses gave evidence of transient viremia followed by virusneutralizing seroconversion and induction of T-cell responses mostly directed against viral NS 6

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

7 proteins. Remarkably, H-1PV proved able to cross the blood-brain barrier and efficiently penetrate gliomas after IV administration. While no toxicity was observed at the first three dose levels tested, more time is needed to draw a clinical lesson from the trial which is still in progress. Carolien Koks, KU Leuven, Belgium described her work on Newcastle disease virus (NDV) therapy for high grade glioma. In this work, she uses the murine GL261 glioma model, which is a fully immunocompetent, orthotopic tumour model. The group wanted to investigate the cell death pathways used by NDV in this model, as not many previous studies have investigated the pathways responsible for cancer cell demise following NDV treatment. They found an absence of Annexin V positivity in propidium iodide (PI) negative cells, but abundant Annexin V positivity in the PI positive fraction, both at early and at late time points after NDV infection, indicating that killing by NDV was of a necrotic and not an apoptotic nature. This was also clear from the morphology of infected cell cultures as viewed by microscopy. To further demonstrate this, they showed that indeed, inhibition of caspases did not rescue the cells from NDV-induced cell death. In fact, this action further diminished the viability of the cells, indicating the presence of necroptosis, a programmed form of necrosis. Accordingly, a specific inhibitor of necroptosis did restore cell viability. Together, this data demonstrated that NDV kills glioma cells in an apoptosis-independent fashion, with features of necroptosis. In their in vivo model, animals were challenged with tumour and treated IT with NDV or placebo after 7 days, when a tumour mass has been established in all mice. NDV therapy prolonged median OS of treated animals and cured 50% of established gliomas. There were no signs of virusinduced toxicity and no tumour recurrence for up to 100 days. Looking into the mechanism behind NDV therapy, they showed that NDV induces an immunogenic type of cell death in the GL261 cells, as seen by surface exposure of Calreticulin and release of the danger signal HMGB1. Simultaneously, infected cells upregulated their surface expression of tumour-associated antigens, thereby highlighting the immunogenicity of the cells. Secretion of ATP, another classical hallmark of an immunogenic type of cell death was absent in this model. The group suggests that this is due to the actively ongoing viral replication within the cells, which requires additional ATP to be used up. NDV therapy increases the brain infiltration of IFN+ secreting T cells and treated animals have less immunosuppressive myeloid derived suppresser cells (monocytic and granulocytic) present in their brains. This confirms the first hallmark of immunogenic cell death in vivo, namely a shift towards more immune activation. To investigate the importance of these activated T cells, RAG2-/- animals specifically lacking B and T cells were challenged with tumour cells and treated with NDV after 7 days. Though NDV could prolong median survival lightly but significantly, cure was only induced in fully immunocompetent animals. Untreated, immunocompetent animals also survived longer that their immunodeficient counterparts. These findings demonstrate that NDV therapy and the immune system seem to act together in a situation where either alone is not sufficient to induce a cure. Finally, long-term surviving immunocompetent animals rechallenged with GL261 cells resisted tumour outgrowth, showing the induction of a long-term anti-tumour memory response. In conclusion, though NDV was 7

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

8 shown to exert a (rather limited) cytotoxic effect on the murine glioma cells in vitro, the in vivo curative effect of NDV therapy depends mainly on the elicited cellular anti-tumour effects. The group are now looking at combination therapies. Martine Lamfers, Erasmus MC, Rotterdam, The Netherlands works with Delta24-RGD, which has a 24bp deletion in the Rb binding site of E1A, which makes it tumour selective, and an RGD modification leading to extended tropism to integrins. Delta24RGD is currently being tested in phase I/II clinical trials for glioblastoma. Oncolytic adenoviruses (Ad) lag behind other OVs with regards to their use as immunotherapeutic agents. To gain insight into this aspect, the group used an immune-competent orthotopic syngeneic mouse model C57BL/6 with the GL261 glioma cell line. Compared to human glioma cells, higher doses of virus are required to kill the murine GL261 cells. However, cytopathic effect-like morphology is observed, and up to 10-fold increase in E1A copy number is detected at 96 hours post-treatment. In vivo, staining for hexon was detected up to day 14 post IT virus injection, and neutralising antibodies were detected from day 3 post-injection. In this setting, Delta24-RGD cured 50% of mice using a dose of 108 vp. The group then used dexamethasone to knock out the immune system, resulting in complete loss of therapeutic efficacy of Delta24-RGD. Analysis of tumour lysates showed that the biggest player was IFN which was completely inhibited by dexamethasone. Delta24-RGD infection attracted NK cells, macrophages, CD4+ and CD8+ T cells to the tumour. Long term survivors were re-injected with tumour and were shown to reject it. Splenocytes from Delta24-RGD-treated mice recognise virus and tumour. In an OVA modeling system, the group investigated whether it was the IFN or the virus that caused the increased presentation of TAA to CD8+ T cells. It was found that the IFN was enhancing the response, which correlated with increased MHC class I expression on the tumour cells. Delta24-RGD has also been shown to interfere with DNA repair systems making cells more sensitive to standard of care agent temozolomide (TMZ). However, TMZ has pronounced effects on the immune system, which could hamper the viral-induced anti-tumour immune response. Therefore, the group tested different treatment regimens of combined Delta24-RGD+TMZ therapy. Administering TMZ prior to virus injection hampered the IT influx of DC, NK, CD4+ and CD8+cells, which was not the case when TMZ was administered post-virus injection. However, both combination treatment regimens significantly improved survival compared to TMZ alone. The TMZ post-virus regimen also prolonged survival significantly compared to virus only. In summary, Delta24-RGD injection into IC glioma triggers an innate Th1 immune response followed by an adaptive anti-tumour response, which is abrogated by high-dose dexamethasone treatment. Addition of TMZ to Delta24-RGD treatment increases the therapeutic efficacy of the virus, especially when administered post-virus injection.

Virus and the immune system Oncolytic cell killing strategies

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9 Paola Grandi, University of Pittsburgh, USA, described his work with GBM which has a median survival from diagnosis of 15 months. Overall strategies by the group include 1)

Tumour selective virus Human simplex virus-1 (HSV-1) infectivity based on tumour related plasma membrane targets (retargeted strategies: K-NT-scEGFR: Infection occurs only in cells expressing EGFR or EGFRvIII).

2)

Control of virus growth based on tumour-specific changes in metabolism particularly those essential to the tumour phenotype (e.g. miRNAs). ICP4-mir124: the virus replicate ONLY in the absence of mir-124 (cancer cells).

3)

Vector expression of genes that modify the extracellular matrix (vector armed with MMP9.

4)

Vector expression of genes that induce anti-tumour immunity.

Matteo Samuele Pizzuto, Imperial College London, England described recent work in which their group had demonstrated the ability of different avian influenza virus (AIV) to efficiently infect and induce high levels of apoptosis in pancreatic ductal adenocarcinoma (PDA) cells in vitro (Kasloff et al, 2014). These results had led to an investigation of the oncolytic potential of an engineered avian-origin influenza virus against PDA. They had focused on BxPC-3, a PDA cell line that was particularly sensitive to AIV infection, but which was previously shown resistant to other OVs (Murphy and others 2012). Like many other PDA cell lines, BxPC-3 are IFN-deficient due to the loss of genetic material within the chromosome arm 9P where the IFN genes are located. As such a conditionally replicating H7N3 low pathogeneticity AIV (LPAIV) was generated through the truncation of the viral NS1 protein, whose major role is to antagonize the host IFN-mediated antiviral response. Production of a protein of only 77 aa’s (NS1-77) resulted in an almost complete loss of the “effector domain” (ED) predominantly involved in IFN evasion and apoptosis modulation. The resulting H7N3 NS1-77 virus lost the ability to counteract the IFN-mediated antiviral response in IFN competent cells when compared to the wild type (WT) mainly because it was unable to limit posttranscriptional IFN-β induction. The effect of the NS1-77 truncation was shown to be strain specific as the difference in the IFN expression induced by the well characterized human H1N1 PR8 viruses with full length or truncated NS1 proteins was less pronounced. The H7N3 NS1-77 virus did not replicate efficiently in IFN competent embryonated chicken eggs (ECE), which represent a standard laboratory medium for propagation of influenza viruses, while it reached the same titre as the WT in 7 day old ECE, where the IFN system is not completely established. The truncated virus triggered higher levels of apoptosis compared to chemotherapeutic agents (Gemcitabine and Cisplatin) in BxPC-3 cells, but not in HPDE6 cells derived from normal human pancreatic ducts. Even more interesting was the fact that H7N3 NS1-77 virus induced significantly higher levels of cell death in BxPC-3 than the corresponding WT virus. Nevertheless, whilst the NS1-77 truncation conferred to the

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

10 H7N3 virus higher selectivity and further enhanced its apoptotic skills in BxPC-3 cells, it displayed the reduced replication observed in the parental virus. The idea then was that the virus was so efficient at promoting apoptosis during infection that it did not give enough time for its viral RNA to reach high copy number. Since the H7N3 virus triggered the intrinsic apoptotic pathway as shown by ICC targeting active caspases, to rebalance apoptosis induction in favour of virus replication their group investigated the viral PB1-F2 pro-apoptotic protein, which targets the mitochondrial membrane during infection causing depolarization of membrane potential (ΔΨm), release of cytochrome c (cyt c) and activation of caspase-9-mediated apoptotic pathway. A region near the C-terminus of PB1-F2 called Mitochondrial Targeting Sequence (MTS) is necessary and sufficient for its inner MM localization. Specific Leucine (Leu) residues within the MTS are considered to be responsible for mitochondrial targeting (Chen et al, 2010). Depending on the strength of the MTS region, PB1-F2 can also be found in the nucleus of infected cells, where it seems are important for virus replication as PB1-F2 has been previously suggested as an interaction partner of PB1, affecting viral polymerase activity. In addition to its role in inducing apoptosis, PB1-F2 has been shown to exert an IFN antagonist function by binding to the mitochondria antiviral signalling protein (MAVS), which, as a downstream adaptor for RIG-I, plays a central role in virus-triggered IFNβ induction. Therefore, any decrease of PB1-F2 mitochondrial targeting could reduce MAVS inhibition, increasing IFN expression by healthy cells during infection, hence contributing to selectivity and containment of the virus. Based on these considerations to rebalance apoptosis induction in favour of virus replication an H7N3 NS1-77 PB1F2 L75H virus with a decreased number of specific Leu within the PB1-F2 MTS was generated. PB1F2 L75H protein resulted in decreased mitochondrial targeting and corresponding increased virus replication and polymerase activity in permissive IFN deficient cells. H7N3 NS1-77 PB1-F2 L75H virus was still able to trigger higher levels of apoptosis in BxPC-3 cells compared to both chemotherapeutic agents and the corresponding full length NS1 virus. Moreover, the decrease in mitochondrial targeting reduced the viruses ability to antagonize IFN at the level of MAVS and further enhanced its preference to IFN deficient systems. In SCID mice bearing BxPC-3 derived solid tumours, H7N3 NS1-77 PB1-F2 L75H virus produced a significant reduction in tumour growth compared to the control within 14 days from the first administration. In conclusion, the group generated an engineered avian-origin H7N3 virus with enhanced selectivity, apoptotic potential and replication efficiency in IFN-deficient PDA cells which also displayed a beneficial effect in a xenograft model. The results obtained also underline the importance of strain specific characteristics like IFN-antagonism and/or apoptosis, emphasizing the possibility to modulate these features by manipulating virus genome to obtain optimal attenuation/replication and apoptosis/replication balance. Moreover, the data clearly show that PDA cells resistant to other OVs (Murphy et al, 2012) are effectively eliminated in vitro and in vivo using influenza viruses, stressing the idea that research in the area of oncolytic virology should be focused on a broad range of viruses characterized by

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Human Gene Therapy 8th International Conference on Oncolytic Virus Therapeutics (doi: 10.1089/hum.2014.118) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

11 specific cancer cell permissiveness, and that influenza virus could play a role in further studies on treatment of PDA. Hiroshi Nakashima, Brigham and Women’s Hospital, Boston, USA described the novel GADD34-armed oHSV1 vector NG34, which has been developed to improved its safety profile and efficacy. HSV therapy mostly uses oncolytic mutants which originally lacked ICP34.5, as its’ a specific determinant of neurovirulence by suppressing autophagic function in neurons. The protein GADD34 is a homologous protein to viral ICP34.5 to function PP1-binding for enhancing translation via dephosphorylation of eIF2 alpha. However it lacks the Beclin1 binding domain therefore it doesn’t restore the ICP34.5 associated neurovirulence. Hiroshi’s group had engineered 34.5 and UL39 double-null mutant HSV1 vector to express human GADD34 gene under the control of a gliomaactive nestin promoter, called NG34. The group showed that NG34 infection led to GADD34 expression in cells and reverses the phosphorylation of eIF2 in glioma cells. Thus GADD34 can functionally replace the viral ICP34.5 gene for enhanced oHSV1 replication. And, NG34 virus shows efficient cytotoxicity against primary GBM cells and didn’t show lethality at the dose wild-type HSV1 showed lethality after inoculating in mice brains. NG34 significantly prolongs the life of mice bearing an IC primary GBM. If TMZ is combined with NG34 then a synergy was shown using Chou Talaley plots at CI

8th international conference on oncolytic virus therapeutics.

The 8th International Conference on Oncolytic Virus Therapeutics meeting was held from April 10-13, 2014, in Oxford, United Kingdom. It brought togeth...
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