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115. Tumor Microenvironment-Targeting Hybrid Vector System Utilizing Oncolytic Adenovirus Complexed with pH-Sensitive and Bioreducible Polymer
Adenovirus Vectors and Other DNA Virus Vectors extracellular matrix (ECM) of pancreatic cancer and poor cancer specificity. The excess ECM prevents infiltration of drugs into the inner layer of the solid tumor. Therefore, novel treatment modalities that can specifically target the tumor and degrade the ECM are required for effective therapy. In the present study, we used ECMdegrading and Wnt signal-disrupting oncolytic adenovirus (oAd/ DCN/LRP) to achieve a desirable therapeutic outcome against pancreatic cancer. In addition, to overcome the limitations in systemic delivery of oncolytic Ad (oAd) and to specifically target pancreatic cancer, neurotensin peptide (NT)-conjugated polyethylene glycol (PEG) was chemically crosslinked to the surface of Ad, generating a systemically injectable hybrid system, oAd/DCN/LRP-PEG-NT. We tested the targeting and therapeutic efficacy of oAd/DCN/LRP-PEGNT toward neurotensin receptor 1 (NTR)-overexpressing pancreatic cancer cells, both in vitro and in vivo. The oAd/DCN/LRP-PEG-NT elicited increased NTR-selective cancer cell killing and increased transduction efficiency when compared with a cognate control lacking NT (oAd/DCN/LRP-PEG). Furthermore, systemic administration of oAd/DCN/LRP-PEG-NT significantly decreased induction of innate and adaptive immune responses against Ad, and blood retention time was markedly prolonged by PEGylation. Moreover, NTR-targeting oAd elicited greater in vivo tumor growth suppression when compared with naked oAd, and 9.5×106-fold increased tumor-to-liver ratio. This significantly enhanced antitumor effect of oAd/DCN/LRP-PEGNT was mediated by active viral replication and viral spreading, which was facilitated by ECM degradation and inhibition of Wnt signaling-related factors (Wnt, β-catenin, and/or vimentin) in tumor tissues. Taken together, these results demonstrate that oAd/DCN/ LRP-PEG-NT has strong therapeutic potential for systemic treatment of NTR-overexpressing pancreatic cancer, due to its NTR-targeting ability, enhanced therapeutic efficacy, and safety.
114. Replication-Defective HSV Vector Development for Targeted Gene Delivery
Bonnie Reinhart, Daniela Leronni, Justus B. Cohen, Joseph C. Glorioso Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA Gene therapy treatment strategies for Huntington’s disease (HD) depend on a vector that (i) allows targeted delivery of large therapeutic transgenes to specific cell populations and (ii) is replication-defective and non-cytotoxic. Thus, our goal is to develop a replication-defective HSV-based gene therapy vector to deliver therapeutic transgenes to the cells most affected by expression of the mutant Huntington gene (Htt). Strategies for full retargeting of HSV require virus detargeting from its cognate receptors (HVEM and nectin1), recognized by the virus attachment/entry component glycoprotein D (gD), and introduction of a new ligand into gD that allows entry through recognition of the corresponding cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptor GFRα1 we employed the ligand GDNF. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRα1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient J1.1-2 and B78H1 cells in a GFRα1-dependent manner. U2OS cells engineered to express GFRα1 demonstrated the most robust virus entry and spread, allowing us to create and propagate a fully retargeted virus that can no longer bind nectin1, gD(Δ38)_GDNF. Once the functionality of gD(Δ38)_GDNF was confirmed, we transferred this mutation to a replication-defective backbone lacking essential immediate early gene expression. Complementing cells were screened and those cells that best supported growth of the replication-defective backbone were selected. Virus stocks were propagated and tested for receptor specificity in vitro and experiments are currently underway to assess S48
GFRα1-specific entry in vivo. We anticipate that the use of neuronalspecific targeting ligands, such as GFRα1, in replication-defective backbones will promote homing of non-toxic HSV gene therapy vectors to cells affected in HD.
115. Tumor Microenvironment-Targeting Hybrid Vector System Utilizing Oncolytic Adenovirus Complexed with pH-Sensitive and Bioreducible Polymer Jung-Woo Choi1, Dayananda Kasala1, Chang Yoon Moon1, SooJung Jung1, Sung Wan Kim2, Chae-Ok Yun1 1 Department of Bioengineering College of Engineering Hanyang University, Seoul, Korea, Republic of, 2Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake, UT
Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)expressing cells, thereby demonstrating Ad-PPCBA’s ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA’s biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.
116. Overcoming CAR-Dependency of Oncolytic Adenovirus via Adenoviral Fiber Modification with Vesicular Stomatitis Virus Glycoprotein Epitope for Enhanced Antitumor Efficacy A-Rum Yoon, Jinwoo Hong, Chae-Ok Yun Department of Bioengineering College of Engineering Hanyang University, Seoul, Korea, Republic of
Utility of traditional oncolytic Ad has been limited due to low expression of coxackies and adenovirus receptor (CAR) in cancer cells which results in poor infectivity of Ads. Here with an aim of improving the efficiency of Ad’s entry to the cell, we generated a novel tropism-expanded oncolytic Ad which contains the epitope of vesicular stomatitis virus glycoprotein (VSVG) at the HI-loop of Ad fiber. We generated 9 variants of oncolytic Ads with varying linkers and partial deletion to the fiber. Only one VSVG epitope-incorporated variant, RdB-1L-VSVG, which contains 1 linker and no deletion to fiber, was produced efficiently. Production of 3-dimensionaly stable fiber in RdB-1L-VSVG was confirmed by immunoblot analysis. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
114. Replication-Defective HSV Vector Development for Targeted Gene Delivery
Bonnie Reinhart, Daniela Leronni, Justus B. Cohen, Joseph C. Glorioso Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA Gene therapy treatment strategies for Huntington’s disease (HD) depend on a vector that (i) allows targeted delivery of large therapeutic transgenes to specific cell populations and (ii) is replication-defective and non-cytotoxic. Thus, our goal is to develop a replication-defective HSV-based gene therapy vector to deliver therapeutic transgenes to the cells most affected by expression of the mutant Huntington gene (Htt). Strategies for full retargeting of HSV require virus detargeting from its cognate receptors (HVEM and nectin1), recognized by the virus attachment/entry component glycoprotein D (gD), and introduction of a new ligand into gD that allows entry through recognition of the corresponding cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptor GFRα1 we employed the ligand GDNF. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRα1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient J1.1-2 and B78H1 cells in a GFRα1-dependent manner. U2OS cells engineered to express GFRα1 demonstrated the most robust virus entry and spread, allowing us to create and propagate a fully retargeted virus that can no longer bind nectin1, gD(Δ38)_GDNF. Once the functionality of gD(Δ38)_GDNF was confirmed, we transferred this mutation to a replication-defective backbone lacking essential immediate early gene expression. Complementing cells were screened and those cells that best supported growth of the replication-defective backbone were selected. Virus stocks were propagated and tested for receptor specificity in vitro and experiments are currently underway to assess S48
GFRα1-specific entry in vivo. We anticipate that the use of neuronalspecific targeting ligands, such as GFRα1, in replication-defective backbones will promote homing of non-toxic HSV gene therapy vectors to cells affected in HD.
115. Tumor Microenvironment-Targeting Hybrid Vector System Utilizing Oncolytic Adenovirus Complexed with pH-Sensitive and Bioreducible Polymer Jung-Woo Choi1, Dayananda Kasala1, Chang Yoon Moon1, SooJung Jung1, Sung Wan Kim2, Chae-Ok Yun1 1 Department of Bioengineering College of Engineering Hanyang University, Seoul, Korea, Republic of, 2Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake, UT
Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)expressing cells, thereby demonstrating Ad-PPCBA’s ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA’s biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.
116. Overcoming CAR-Dependency of Oncolytic Adenovirus via Adenoviral Fiber Modification with Vesicular Stomatitis Virus Glycoprotein Epitope for Enhanced Antitumor Efficacy A-Rum Yoon, Jinwoo Hong, Chae-Ok Yun Department of Bioengineering College of Engineering Hanyang University, Seoul, Korea, Republic of
Utility of traditional oncolytic Ad has been limited due to low expression of coxackies and adenovirus receptor (CAR) in cancer cells which results in poor infectivity of Ads. Here with an aim of improving the efficiency of Ad’s entry to the cell, we generated a novel tropism-expanded oncolytic Ad which contains the epitope of vesicular stomatitis virus glycoprotein (VSVG) at the HI-loop of Ad fiber. We generated 9 variants of oncolytic Ads with varying linkers and partial deletion to the fiber. Only one VSVG epitope-incorporated variant, RdB-1L-VSVG, which contains 1 linker and no deletion to fiber, was produced efficiently. Production of 3-dimensionaly stable fiber in RdB-1L-VSVG was confirmed by immunoblot analysis. Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy