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ADENOVIRUS AND OTHER DNA VIRUS VECTORS Adenovirus and Other DNA Virus Vectors 477 Molecular Modeling of Coagulation FVII Binding to Adenovirus Hexon Revealed a Binding Pocket for Coagulation Factor GLA Domain That Defines the Infectivity of Adenovirus-Coagulation Factor Complexes Eric E Irons,1 Justin W Flatt,2 Konstantin Doronin,3 Tara L Fox,2 Mauro Acchione,1 Phoebe L Stewart,2 Dmitry M Shayakhmetov.1 Department of Medicine, University of Washington, Seattle, WA; Department of Pharmacology, Case Western Reserve University, Cleveland, OH; 3Department of Molecular Microbiology and Immunology, St Louis University, St Louis, MO Adenoviruses (Ad) are promising vectors for therapeutic interventions in humans However, when injected into the bloodstream, Ad vectors can bind several vitamin K-dependent blood coagulation factors, which contribute to virus sequestration in the liver by facilitating transduction of hepatocytes Although both coagulation factors FVII and FX bind HAd5 hexon with very high affinity, only FX appears to play a role in mediating Ad hepatocyte transduction in vivo To understand the discrepancy between efficacy of FVII binding to the virus and its poor capacity at supporting Ad cell entry, we analyzed the HAdv5-FVII complex using high-resolution cryoelectron microscopy, followed by molecular dynamics flexible fitting (MDFF) simulations The results indicate that the FVII GLA-domain sits within a surface-exposed hexon trimer depression in a different orientation than found for FX, although several of the same hexon residues that are involved in mediating FX binding also mediate FVII binding to hexon (T423-E424-T425) Furthermore, we found that when bound to the WT hexon, two proximal FVII molecules interact via their serine protease (SP) domains and bury potential heparin sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface While FVII binds WT hexon with Kd=3nM, E424A or T423G-E424A amino acid substitutions reduced FVII binding 10-fold (Kd=30 nM) Surprisingly, despite the equal binding affinity of FVII for E424A and T423G-E424A hexon mutants, the vector possessing a two-amino acid substitution demonstrated superior cell transduction efficacy in the presence of FVII, when compared to either the WT or E424A mutant MDFF simulations of FVII binding to E424A and T423G-E424A hexon mutants revealed altered interfaces between hexon and FVII that could affect the orientation of FVII and, thus, the efficacy of SP domain dimer formation Because FX interaction with hexon does not lead to the formation of SP domain dimers that occlude HSPG cell receptor interacting sites, our data suggests that upon FVII binding to T423G-E424A, but not to the WT or E424A mutant, SP domain dimer formation is either ablated or inefficient, leading to highly effective virus-mediated gene transfer in the presence of FVII Thus, our structural, computational, and biochemical analyses revealed important roles for specific hexon amino acids that lead to different binding orientations for FX and FVII Gla domains, which lead to a drastic variation in cell-transduction efficacy of the resultant coagulation factor-Ad vector complexes Our study provides important information on the dynamic nature of the coagulation factor-Ad hexon interaction and may aid in the design of novel safe and effective Ad vectors, amenable to cell type-specific targeting after intravascular delivery 478 Identification of a Negative Regulator of TRPV1 Via an HSV Based cDNA Library Screen Bonnie L Reinhart,1 Asaff Harel,1 MingDi Zhang,1 James R Goss,1 William F Goins,1 Justus B Cohen,1 Joseph C Glorioso.1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA Chronic pain occurs when normal control of acute pain signaling is lost, resulting in reduced stimulus thresholds and hypersensitive sensory neurons The transient receptor potential vanilloid channel (TRPV1) is a pro-nociceptive cation channel that is activated in response to noxious stimuli such as capsaicin, heat, or low pH TRPV1 is functionally up-regulated in pathophysiological states such as painful diabetic neuropathy and chronic inflammation We have developed an HSV replication-based selection strategy to identify novel proteins that negatively regulate TRPV1 Over-expression of TRPV1 from an HSV vector causes calcium influx and cell death in the presence of capsaicin, thereby inhibiting virus growth Coexpression of a cDNA library and TRPV1 from the same backbone should allow replication and selection of only those vectors that express negative modulators of TRPV1’s response to capsaicin To create this library, we developed an HSV vector (2xTRPV1) that contained two essential modifications: 1) a Gateway expression cassette to facilitate insertion of a cDNA library under control of the CMV promoter, and 2) duplicate copies of the TRPV1 gene, under control of the viral TK promoter, to reduce the selection of TRPV1-defective mutants As anticipated, the 2xTRPV1 vector failed to replicate in the presence of capsaicin In contrast, a control vector that co-expressed the two TRPV1 genes and TRPV1-poreless, a dominant-negative mutant of TRPV1, was able to replicate in the presence of capsaicin A cDNA library derived from PC12 cells was introduced into the 2xTRPV1 vector backbone and screened for TRPV1 antagonists Viruses that escaped capsaicin-induced cell death were isolated and the corresponding cDNAs were sequenced Candidate cDNAs were introduced into a fresh selection vector and retested for TRPV1 inhibition One candidate gene, PP1, rescued virus growth in the presence of capsaicin to a level similar to that of TRPV1-poreless PP1 expressed from a replication-defective vector reduced capsaicin-induced calcium influx in rat primary dorsal root ganglion (DRG) neurons and, similar to a TRPV1-poreless vector, decreased sensitivity to thermal pain in Sprague-Dawley rats compared to a control vector expressing GFP These data suggest that our virus-based in vitro screen can be employed to identify negative in vivo modulators of TRPV1, providing insight into peripheral pain signaling and suggesting novel approaches for the control of primary hyperalgesia 479 Long Term Follow Up of Phase II Clinical Trial of a Granulocyte-Macrophage Colony-Stimulating Factor-Encoding, SecondGeneration Oncolytic Herpes Virus (Talimogene Laherparepvec, T-VEC) in Patients with Unresectable Metastatic Melanoma John Nemunaitis,1,2,3,4 Cynthia Bedell,1 Beena O Pappen,4 Staci Horvath,1 Neil Senzer.1,4 Mary Crowley Cancer Research Centers, Dallas; 2Medical City HCA, Dallas; 3Texas Oncology, P.A., Dallas; 4Gradalis, Inc, Dallas We previously conducted a multicenter phase II trial to assess the efficacy of T-VEC in advanced stage IIIc and IV melanoma patients (Senzer, Nemunaitis et al JCO 2009) Treatment involved intratumoral (IT) injection of up to mL of 106 pfu/mL of product followed weeks later by up to mL of 108 pfu/mL every weeks for up to 24 treatments Clinical activity assessed by RECIST tumor response, survival and safety were monitored Fifty patients (stages S184 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ADENOVIRUS AND OTHER DNA VIRUS VECTORS IIIc, n = 10; IVM1a, n = 16; IVM1b, n = 4; IVM1c, n = 20) received a median of six injection sets; 74% of patients had received one or more nonsurgical prior therapies for active disease, including dacarbazine/ temozolomide or interleukin-2 (IL-2) The overall initial response rate by RECIST was 26% (complete response [CR], n = 8; partial response [PR], n = 5) Remarkably, despite technology being limited to local-regional IT injection, both local and distal, non-injected, metastatic sites (including visceral) demonstrated response Overall survival was previously reported as 58% at year We now report ³ year follow up of a subset of all 15 patients managed at MCCRC Four of these patients were previously identified as having achieved complete response involving both local regional and metastatic disease sites including lung and liver Three of these patients remain alive 2528, 2031, and 1833 days, and one survived 868 days before disease related mortality No long term adverse events were identified in all 15 patients including the surviving ³ years Five year Kaplan Meier survival from time of first treatment of the 15 patients treated at MCCRC is shown below These results support long term durability of response without evidence of adverse toxicity detectable VRX-007 in their tumors, in contrast to modest levels of VRX-007 in tumors of animals treated with CP continuously These results imply that (1) long term immunosuppression by CP is not required for the CP-mediated enhancement of tumor growth control by VRX-007, and (2) that the CP may be acting as a chemotherapeutic agent in combination with VRX-007 but without increasing VRX-007 replication in tumors To further address these issues, we used a luciferase-expressing VRX-007 vector to view virus location and replication over time in HaK tumors via the IVIS in vivo imaging system We found that short-term CP treatment, dosed either one week before or one week after tumor infection, had an additive effect on VRX-007 tumor growth control even though virus replication in tumors was absent after one week Therefore, in this immunocompetent situation, the anti-tumor effects of VRX-007 and CP, alone or in combination, are exerted early after treatment and not involve long-term virus replication We have added radiation therapy as a third treatment modality to vector and CP therapies We found that tumor-specific irradiation has an additive effect with VRX007 therapy and further augments the inhibition of tumor growth, in both CP-treated and non-CP-treated animals, even though radiation does not lead to increased viral replication in tumors when compared to those treated with virus alone These results further suggest that long term viral replication is not the cause for tumor growth inhibition in our model This project aims to better understand the mechanism of action of VRX-007 and possibly enhancing its anti-tumor efficacy 481 Analysis of Host Cell and Viral Transcriptomes during Infection by Wild-Type Species C Ad6 and Species D Ad26 Mallory A Turner,1,2 Sean E Hofherr,3 Michael A Barry.2 Virology and Gene Therapy Program, Mayo Graduate School, Rochester, MN; 2Department of Medicine, Mayo Clinic, Rochester, MN; 3Laboratory Medicine, Children’s National Medical Center, Washington, DC 480 Combined Therapies with Oncolytic Adenovirus Suppress Tumor Growth in Hamsters Brittany A Young,1 Karoly Toth,1 Jacqueline F Spencer,1 William S M Wold.1 Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St Louis, MO The Syrian hamster model has been described and used by our laboratory and others for evaluating anti-tumor efficacy, toxicity and biodistribution of oncolytic adenovirus serotype (Ad5)-based vectors Hamster tumors and tissues are semi-permissive for Ad5 replication, which allows us to use permissive immunocompetent animals for our studies We have previously reported that the Ad5based vector (VRX-007) replicates and suppresses tumor growth when it is injected directly into subcutaneous tumors formed by injection of hamster HaK renal cancer cells We also reported that VRX-007 replication is increased and tumor growth is suppressed when hamsters are treated with using cyclophosphamide (CP) Effects that can be attributed to CP (an alkylating agent) include immunosuppression of the hamsters that results in increased VRX007 replication and oncolysis in tumors as well as a chemotherapeutic effect of CP on the tumor cells We now report that short-term CP treatment, given for only one week before VRX-007 injection into tumors, had a similar effect on enhancing the ability of VRX-007 to retard tumor growth as did long-term continuous CP treatment Further, hamsters treated with short-term CP therapy did not have Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy There are more than 57 human Ad serotypes, which fall into seven species with diverse cell and tissue tropisms: A, B, C, D, E, F, and G To date, species C Ad5 has dominated the field of Ad based therapeutics However, in recent years limitations to Ad5 have emerged, including pre-existing immunity, blood factor affinity, and off-target effects To combat these issues, many research groups are exploring the utility of vectors based on other Ad species and serotypes To better understand the differences in Ad serotypes, we compared the transcription and replication programs of of species D Ad26 and species C Ad6 in permissive cells qPCR for viral DNA showed that Ad6 and 26 have similar kinetics of viral DNA synthesis beginning between and 12 hours after cell binding TruSeq mRNA libraries were generated for samples at and 12 hours post infection and 101bp paired-end mRNA-seq was conducted 99.8% of 150 million reads were used to analyze both viral and host mRNAs Viral transcript sequences at hours showed induction of Ad ‘early’ gene transcripts (E1, E2, E3, E4) for both Ad6 and Ad26 However, viral transcriptional output varied between Ad6 and Ad26 at some sites Notably, Ad6 E1A transcripts at hours were about 4-fold higher than E1A transcripts of Ad26 Likewise, E3 total mRNAs varied between the two viruses in regions encoding homologous E3 proteins; fold changes varied between two and four E1B mRNAs were not significantly different between the two viruses Adenoviral transcripts at 12 hours post infection showed substantial increase in late gene transcripts (L1, L2, L3, L4, and L5) compared to the hour time point as expected In some cases Ad6 mRNAs exceeded those for Ad26 (L1, L3, L4) Interestingly, several presumptive early genes were not activated during the early phase, but were instead induced with the late genes mRNAs of the E2b region coding for DNA polymerase (pol) and the preterminal protein (pTP) were increased over 300fold at 12 hours compared to hours for both viruses The increase S185 ... mortality No long term adverse events were identified in all 15 patients including the surviving ³ years Five year Kaplan Meier survival from time of first treatment of the 15 patients treated at... the anti-tumor effects of VRX-007 and CP, alone or in combination, are exerted early after treatment and not involve long-term virus replication We have added radiation therapy as a third treatment... treatment modality to vector and CP therapies We found that tumor-specific irradiation has an additive effect with VRX007 therapy and further augments the inhibition of tumor growth, in both CP-treated
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