Amphotropic Murine Leukemia Virus Research Articles

Targeted Suicide Gene Therapy with Retroviral Replicating Vectors for Experimental Canine Cancers.

Cancer in dogs has increased in recent years and is a leading cause of death. We have developed a retroviral replicating vector (RRV) that specifically targets cancer cells for infection and replication. RRV carrying a suicide gene induced synchronized killing of cancer cells when administered with a prodrug after infection. In this study, we evaluated two distinct RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) in canine tumor models both in vitro and in vivo. Despite low infection rates in normal canine cells, both RRVs efficiently infected and replicated within all the canine tumor cells tested. The efficient intratumoral spread of the RRVs after their intratumoral injection was also demonstrated in nude mouse models of subcutaneous canine tumor xenografts. When both RRVs encoded a yeast cytosine deaminase suicide gene, which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil, they caused tumor-cell-specific 5-FC-induced killing of the canine tumor cells in vitro. Furthermore, in the AZACF- and AZACH-cell subcutaneous tumor xenograft models, both RRVs exerted significant antitumor effects. These results suggest that RRV-mediated suicide gene therapy is a novel therapeutic approach to canine cancers.

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.

EXTH-65. INSERTION OF MICRORNA TARGET SEQUENCES INTO RETROVIRAL REPLICATING VECTORS EFFECTIVELY RESTRICTS TRANSGENE EXPRESSION AND VIRAL REPLICATION IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Abstract Prodrug activator gene therapy with a retroviral replicating vector (RRV) has shown a highly favorable safety profile and long-term survival in early-phase trials for recurrent high-grade glioma. Overall endpoints were not met in a recent Phase 3 trial, but highly statistically significant survival was observed in prespecified patient subgroups as compared to randomized matched control patients receiving standard-of-care treatments, and further clinical evaluation is being focused on these subgroups. Additional strategies to enhance therapeutic potency may require two or more RRVs to deliver multiple transgenes simultaneously, but RRVs encoated with the same envelope protein will compete for the same cellular receptors, interfering with efficient co-infection. The current clinical vector (formerly Toca511, now DB107) is encoated by amphotropic murine leukemia virus (MLV) envelope, which binds to inorganic phosphate transporter PiT-2/SLC20A2. To switch RRV tropism, we developed RRVs pseudotyped with a heterologous envelope from Gibbon ape leukemia virus (GALV), which utilizes an alternative phosphate transporter, PiT-1/SLC20A1, for cell entry. Efficient co-infection of established and primary human glioblastoma cells with MLV- and GALV-pseudotyped RRV was achieved, without receptor competition. However, human hematopoietic stem/progenitor cells (HSPC) also express high levels of PiT-1, which may increase potential genotoxicity of GALV-pseudotyped RRV. Accordingly, as a novel strategy to restrict gene expression and replication in HSPC, we have developed and tested new RRV designs incorporating microRNA target sequences (miRT). Insertion of miRT recognized by hematopoietic lineage-specific microRNA-142-3p resulted in complete suppression of RRV replication in primary human CD34+ HSPC, with the percentage of infected cells below 1% after vector inoculation at multiplicities of infection (MOI) 0.05 and 0.1, and remaining below 3% even after inoculation at MOI 0.5 and 1.0. In vivo models have been developed for on-going studies to evaluate miRT-mediated restriction strategies for avoidance of potential genotoxicity by RRV in normal hematopoietic cells.

Antivirus activity, but not thiolreductase activity, is conserved in interferon-gamma-inducible GILT protein in arthropod

We have previously reported that gamma-interferon inducible lysosomal thiolreductase (GILT) functions as a host defense factor against retroviruses by digesting disulfide bonds on viral envelope proteins. GILT is widely conserved even in plants and fungi as well as animals. The thiolreductase active site of mammalian GILT is composed of a CXXC amino acid motif, whereas the C-terminal cysteine residue is changed to serine in arthropods including shrimps, crabs, and flies. GILT from Penaeus monodon (PmGILT) also has the CXXS motif instead of the CXXC active site. We demonstrate here that a human GILT mutant (GILT C75S) with the CXXS motif and PmGILT significantly inhibit amphotropic murine leukemia virus vector infection in human cells without alterning its expression level and lysosomal localization, showing that the C-terminal cysteine residue of the active site is not required for the antiviral activity. We have reported that human GILT suppresses HIV-1 particle production by digestion of disulfide bonds on CD63. However, GILT C75S mutant and PmGILT did not digest CD63 disulfide bonds, and had no effect on HIV-1 virion production, suggesting that they do not have thiolreductase activity. Taken together, this study found that antiviral activity, but not thiolreductase activity, is conserved in arthropod GILT proteins. This finding provides a new insight that the common function of GILT is antiviral activity in many animals.

Efficient Gene Delivery to Quiescent Interleukin-2 (IL-2)–Dependent Cells by Murine Leukemia Virus-Derived Vectors Harboring IL-2 Chimeric Envelopes Glycoproteins

AbstractInterleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2–displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2–displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2–displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2–dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Reactive-oxygen-species-mediated mechanism for photoinduced antibacterial and antiviral activities of Ag3PO4

Cubic-shaped Ag3PO4 crystals with a mean size of 1 μm were synthesized by a precipitation method from a mixed solution of AgNO3, Na2HPO4, and triethanolamine. The antibacterial activities against Escherichia coli, Listeria innocua, and Pseudomonas syringae DC3000 in both the absence and presence of Ag3PO4 under dark conditions and in the presence of Ag3PO4 under red-light (625 nm) and blue-light (460 nm) irradiation were examined. The concentrations of reactive oxygen species (ROS) were also measured in the antibacterial action of the Ag3PO4 against Escherichia coli. The photoinduced enhancement of the Ag3PO4 antibacterial activity under blue-light irradiation is explained by the formation of ROS during the antibacterial action of the Ag3PO4. Moreover, the antiviral activity of Ag3PO4 against amphotropic 10A1 murine leukemia virus enhanced under blue-light irradiation via ROS production. These results provide an insight into extended bio-applications of Ag3PO4.

Astrocyte Infection Is Required for Retrovirus-Induced Spongiform Neurodegeneration Despite Suppressed Viral Protein Expression.

The ability of retroviruses (RVs) to cause neurodegeneration is critically dependent upon two activities of the envelope protein (Env). First, Env facilitates viral genome delivery to CNS target cells through receptor binding and membrane fusion. Second, Env expression within one or more targets indirectly alters the physiology of certain neurons. Although the major Env expressing CNS cell types have been identified for many neurovirulent RVs, it remains unresolved, which targets play a causal role in neuropathogenesis. Moreover, this issue is complicated by the potential for post-infection virus suppression. To address these questions we explored herein, whether and how cryptic neurotropism differences between ecotropic and amphotropic murine leukemia viruses (MLVs) impacted neurovirulence. Neurotropism was first explored ex vivo using (1) acute primary glial cell cultures and (2) neural progenitor cell (NPC)- neural stem cell (NSC) neural sphere (NPH) chimeras. These experiments indicated that primary astrocytes and NPCs acutely restrict amphotropic but not ecotropic virus entry. CNS tropism was investigated using NSC transplant-based Cre-vector pseudotyping wherein mTmG transgenic fluorescent protein reporter mice revealed both productive and suppressed infection. Cre-pseudotyping with FrCasE, a prototypic neurovirulent ecotropic virus, identified glia and endothelia, but not neurons, as targets. Almost two-thirds (62%) of mGFP+ cells failed to show Env expression, suggesting widespread virus suppression. To circumvent RV superinfection interference confounds, targets were also identified using ecotropic packaging NSCs. These experiments identified known ecotropic targets: microglia, oligodendrocyte progenitor cells (OPCs) and endothelia. Additionally, one third of mGFP+ cells were identified as protoplasmic astrocytes, cells that rarely express virus in vivo. A CNS targeting comparison between isogenic ecotropic (FrCasE) and amphotropic (FrAmE) viruses showed a fourfold higher astrocyte targeting by FrCasE. Since ecotropic Env pseudotyping of amphotropic virus in the CNS dramatically exacerbates neurodegeneration, these results strongly suggest that astrocyte infection is a major disease requirement. Moreover, since viral Env protein expression is largely subdetectable in astrocytes, minimal viral protein expression appears sufficient for affecting neuronal physiology. More broadly, these findings raise the specter that subdetectable astrocyte expression of exogenous or endogenous RVs could play a major role in human and animal neurodegenerative diseases.

Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein

Replication-deficient retroviral (RDR) vectors have been generally used for gene therapy, but clinically beneficial transduction efficiency is difficult to achieve with these vectors. In recent times, attention has been focused on the use of murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vectors. RCR vectors have been shown to achieve efficient tumor reduction in a wide variety of cancer models. Most RCR vectors have been developed from amphotropic 4070 A MLV env, which is broadly applied in basic research. In this study, we generated RCR vectors based on Moloney MLV by replacing the native env gene in a full-length viral genome with the 10A1 env gene. 10A1 MLV can infect a wide variety of cells. Unlike amphotropic MLV, the 10A1 MLV can use amphotropic MLV receptor Pit2 or gibbon ape leukemia virus (GaLV) receptor Pit1. The resulting construct MoMLV-10A1-EGFP was able to replicate in 293 T, NIH3T3, and Mus dunni cells. To evaluate the potential of MoMLV-10A1 vector as a therapeutic agent, we incorporated the yeast cytosine deaminase (CD) suicide gene into vectors. The resulting vector MoMLV-10A1-CD could inhibit the growth of human 293T cells upon 5-fluorocytosine (5-FC) administration. In addition, to lyse tumor cells by syncytium, MoMLV-10A1-R(-)-EGFP was generated by replacing wild-type 10A1 env with the 16-amino acid R peptide-truncated 10A1 env gene. Syncytium formation was observed in the TE671 human tumor cells, 293 T and PG13 cells upon transfection of the MoMLV-10A1-R(-)-EGFP vector. This result suggests that replication of this vector could be oncolytic in itself. We also found that syncytium could contribute to enhance cell-to-cell transmission of the retroviral vectors. Our results thus show that the MoMLV-10A1 vectors can be potentially useful for cancer gene therapy.

Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications.

AlphoidtetO-type human artificial chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally artificial vector, alphoidtetO-HAC, can be transferred and maintained in human iPSCs as an independent autonomous chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications.

Dual-vector prodrug activator gene therapy using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have achieved efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here, we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.

Abstract 5927: Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy

Abstract Introduction/Aim: Retroviral replicating vectors (RRVs) afford a unique strategy for gene therapy targeting cancer cells. By “activating” RRVs with prodrug activator suicide genes, these RRVs become strong gene delivery vehicles that replicate selectively in cancer cells, and stably persist through proviral genomic integration, leading to inducible cell death upon prodrug administration. This strategy has shown highly promising clinical results in early phase trials (published), and is currently being evaluated in an international Phase III clinical trial for patients with recurrent high-grade glioma. Recently, we have also applied RRV-mediated gene therapy to preclinical models of ovarian cancer. The aim of this current study was to evaluate feasibility and efficacy of combination suicide gene therapy using two RRVs encoated (‘pseudotyped') with different envelope proteins and delivering different prodrug activators for the treatment of ovarian cancer. Methods: Viral spread was monitored in established and primary patient-derived ovarian cancer cell lines (SKOV3, OCI-P5X, OCI-C5X) over the course of ≥27 days using amphotropic murine leukemia virus (AMLV) envelope- and gibbon ape leukemia virus (GALV) envelope-pseudotyped RRVs delivering either fluorescent protein reporter genes (GFP, mStrawberry) or prodrug activator genes (yeast cytosine deaminase, E. coli nitroreductase), either individually or in combination, at various multiplicities of infection (MOI). Flow cytometry for GFP, mStrawberry, and Gag viral protein was used to determine transduction levels and transgene expression levels of RRVs at serial time points. Each therapeutic RRV was tested by MTS assay for its ability to induce cytotoxicity upon exposure to different concentrations prodrug. To determine viral integration and vector stability, PCR of genomic DNA extracted from infected cells was performed using RRV specific primers, and qRT-PCR was performed to determine vector copy number per cell. Results: All ovarian cancer cell lines tested could be >80% transduced by AMLV- and GALV-pseudotyped RRVs, both individually and in combination, within 6-24 days even after initial inoculation at MOI 0.001, with replication kinetics varying between different cell lines. Both RRVs could stably integrate into the target cell genome without evidence of transgene deletion. MTS assays demonstrated that prodrug activated cytotoxicity led to efficient killing of RRV-transduced ovarian cancer cells in vitro, with synergistic cytotoxicity observed at lower prodrug concentrations after combined treatment. Conclusion: RRV-mediated prodrug activator gene therapy may be an effective approach for efficiently and selectively inducing cytotoxicity in ovarian cancer cells. These data support further evaluation of this strategy through translational studies in vivo. Citation Format: James Grosso, Sara Collins, Aki Inagaki, Brian Slomovitz, Noriyuki Kasahara. Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5927.

Abstract A42: Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors

Abstract Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. An amphotropic murine leukemia virus (MLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is now being investigated in combination with Toca FC (extended-release 5-FC) in multi-center Phase IIB / III clinical trials for patients with recurrent high-grade glioma (clinicaltrials.gov: NCT02414165). RRV-mediated prodrug activator gene therapy represents the ultimate form of “intracellular” chemotherapy, generated selectively and directly from within the infected cancer cells themselves, without incurring systemic toxicity. Moreover, preclinical data support subsequent activation of the immune system selectively against the cancer. We further propose that combination with additional therapies may be desirable to optimize treatment outcomes. Here we first evaluated two different RRVs derived from MLV and gibbon ape leukemia virus (GALV), and expressing either the same fluorescent reporter gene (MLV-GFP and GALV-GFP, respectively), or different reporter genes (MLV-GFP vs. GALV-RFP; MLV-RFP vs. GALV-GFP), in different types of human cancer cells. Individually, RRVs expressing the same reporter gene efficiently infected human glioma, prostate cancer, and ovarian cancer cells, and showed efficient replication and spread in culture. When marked with different fluorescent reporter genes, it was found that MLV-GFP can spread in cells that had been pre-transduced with GALV-RFP, but not in MLV-RFP pre-transduced cells. Similarly, GALV-GFP can spread in MLV-RFP pre-transduced cells, but not in GALV-RFP pre-transduced cells. This mutually exclusive infection pattern is likely due to the phenomenon of receptor interference, which occurs when vectors derived from the same viral strain compete for binding to cell surface receptors, resulting in superinfection resistance. Notably, however, replication and spread of either RRV in culture was not affected by pre-transduction with RRV derived from a different strain, indicating that this approach could overcome receptor interference. In order to investigate the effect of combined prodrug-dependent cell killing in vitro, cells were then transduced with MLV- or GALV-based RRV expressing the yeast cytosine deaminase suicide gene (MLV-CD, GALV-CD), which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil (5-FU), or with RRV expressing the Herpes thymidine kinase gene (MLV-TK, GALV-TK) which converts Ganciclovir (GCV) to GCV-monophosphate, either individually or in combination. In vitro cytocidal effects obtained by combining different prodrug activator genes were significantly greater when these genes were delivered with RRV derived from two different strains. These data indicate the potential utility of using RRV pseudotyped with envelopes derived from different viral strains to overcome receptor interference leading to superinfection resistance, and achieve effective combined prodrug activator gene therapy. Citation Format: James Grosso, Sara Collins, Akihito Inagaki, Suzanne Matsuura, Brian Slomovitz, Tan Ince, Noriyuki Kasahara. Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A42.

Highly Efficient Transfer of Chromosomes to a Broad Range of Target Cells Using Chinese Hamster Ovary Cells Expressing Murine Leukemia Virus-Derived Envelope Proteins.

Microcell-mediated chromosome transfer (MMCT) is an essential step for introducing chromosomes from donor cells to recipient cells. MMCT allows not only for genetic/epigenetic analysis of specific chromosomes, but also for utilization of human and mouse artificial chromosomes (HACs/MACs) as gene delivery vectors. Although the scientific demand for genome scale analyses is increasing, the poor transfer efficiency of the current method has hampered the application of chromosome engineering technology. Here, we developed a highly efficient chromosome transfer method, called retro-MMCT, which is based on Chinese hamster ovary cells expressing envelope proteins derived from ecotropic or amphotropic murine leukemia viruses. Using this method, we transferred MACs to NIH3T3 cells with 26.5 times greater efficiency than that obtained using the conventional MMCT method. Retro-MMCT was applicable to a variety of recipient cells, including embryonic stem cells. Moreover, retro-MMCT enabled efficient transfer of MAC to recipient cells derived from humans, monkeys, mice, rats, and rabbits. These results demonstrate the utility of retro-MMCT for the efficient transfer of chromosomes to various types of target cell.

Small synthetic ligands for the enrichment of viral particles pseudotyped with amphotropic murine leukemia virus envelope

Retroviral vectors gained popularity toward other viral vectors as they integrate their genome into hosts’ genome, a characteristic required for the modification of stem cells. However, the production of viable particles for gene therapy is hampered by the low ratio of infectious to non-infectious viral particles after purification, low titers and limited number of competent viral receptors. We have developed de novo two fully synthetic triazine-based ligands that can selectively bind retroviral particles pseudotyped with amphotropic murine leukemia virus envelope (AMPHO4070A). A 78-membered library of triazine-based ligands was designed in silico and was virtually screened against the modeled structure of the AMPHO4070A protein. Ligands displaying the highest energy of binding were synthesized on cross-linked agarose and experimentally tested. Adsorbents containing ligands A5A10 and A10A11 showed selectivity toward viral particles containing the target protein (VLP-AMPHO), binding 19±5μg/g support and 47±13μg/g support, respectively. The elution conditions for both ligands were mild and with high recovery yields (80–100%), in comparison with common purification practices. These results were based on a lab-scale experimental setting with VLP integrity being confirmed through TEM. In particular, the elution buffer containing 12mM imidazole allowed the recovery of intact amphotropic viral particles.

536. Dual-Vector Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors Derived from Amphotropic Murine Leukemia Virus and Gibbon Ape Leukemia Virus

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. An amphotropic murine leukemia virus (AMLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is currently being investigated in combination with Toca FC (extended-release 5-FC) in multi-center clinical trials in the United States for patients with recurrent high-grade glioma (http://www.clinicaltrials.gov NCT01156584, NCT01985256, NCT01470794). This treatment has been safe and well tolerated and median overall survival exceed historical benchmarks. RRV-mediated prodrug activator gene therapy represents the ultimate form of ‘intracellular’ chemotherapy, generated selectively and directly from within the infected cancer cells themselves, without incurring systemic toxicity. Moreover, preclinical data support subsequent activation of the immune system selectively against the cancer. However, some patients may not respond to this treatment, therefore, combination with additional therapeutic agents may be desirable to optimize treatment outcomes. Here we evaluated two different RRVs derived from AMLV and gibbon ape leukemia virus (GALV), encoding two different prodrug activator genes, CD and herpes simplex virus thymidine kinase (TK) in Hep3B human hepatocellular carcinoma cells.Both RRVs expressing the green fluorescent protein gene (AMLV-GFP and GALV-GFP) efficiently replicated in Hep3B cells and spread in culture. Additionally, AMLV-GFP can spread in GALV-mCherry pretransduced Hep3B cells but not in AMLV-mCherry pretransduced cells. Similarly, GALV-GFP can spread in AMLV-mCherry pretransduced cells but not in GALV-mCherry pretransduced cells. This mutually exclusive infection pattern is likely due to receptor interference resulting in superinfection resistance when the same viral strain is used. Notably, however, replication and spread of either RRV in culture was not affected by pretransduction with the counterpart RRV encoated with the other envelope.In order to investigate the effect of combined prodrug-dependent cell killing vs. multiple vector copy transduction in vitro, Hep3B cells were transduced with the AMLV-CD (Ca), GALV-CD (Cg), AMLV-TK (Ta), GALV-TK (Tg), respectively or in combination (Ca/Cg, Ta/Tg, Ca/Tg, Cg/Ta). The resultant cells were used to evaluate the cytotoxic effect of RRV-mediated prodrug activator gene therapy with CD and TK in the presence of their respective prodrugs, 5-fluorocytosine (5-FC) and Ganciclovir (GCV). In vitro cytocidal effects obtained by combining different prodrug activator genes (Ca/Tg and Cg/Ta) were significantly greater than when the same prodrug activator genes (Ca/Cg and Ta/Tg) were delivered with two different vectors. These data indicate the potential utility of dual-vector prodrug activator gene therapy using two different RRVs carrying different prodrug activator genes.

A Sorting Signal Suppresses IFITM1 Restriction of Viral Entry

The interferon-induced transmembrane proteins (IFITMs) broadly inhibit virus infections, particularly at the viral entry level. However, despite this shared ability to inhibit fusion, IFITMs differ in the potency and breadth of viruses restricted, an anomaly that is not fully understood. Here, we show that differences in the range of viruses restricted by IFITM1 are regulated by a C-terminal non-canonical dibasic sorting signal KRXX that suppresses restriction of some viruses by governing its intracellular distribution. Replacing the two basic residues with alanine (KR/AA) increased restriction of jaagsiekte sheep retrovirus and 10A1 amphotropic murine leukemia virus. Deconvolution microscopy revealed an altered subcellular distribution for KR/AA, with fewer molecules in LAMP1-positive lysosomes balanced by increased levels in CD63-positive multivesicular bodies, where jaagsiekte sheep retrovirus pseudovirions are colocalized. IFITM1 binds to cellular adaptor protein complex 3 (AP-3), an association that is lost when the dibasic motif is altered. Although knockdown of AP-3 itself decreases some virus entry, expression of parental IFITM1, but not its KR/AA mutant, potentiates inhibition of viral infections in AP-3 knockdown cells. By using the substituted cysteine accessibility method, we provide evidence that IFITM1 adopts more than one membrane topology co-existing in cellular membranes. Because the C-terminal dibasic sorting signal is unique to human IFITM1, our results provide novel insight into understanding the species- and virus-specific antiviral effect of IFITMs.

C3H strain of mouse mammary tumour virus, like GR strain, infects human mammary epithelial cells, albeit less efficiently than murine mammary epithelial cells.

Mouse mammary tumour virus (MMTV) is a member of the genus Betaretrovirus, infects rodent cells and uses mouse tranferrin receptor 1 for cell entry. Several MMTV strains have been shown to productively infect, in addition to murine cells, various heterologous cell lines including those of human origin, albeit less efficiently than murine cells. Here, we analysed whether MMTV from C3H mice [MMTV(C3H)], reported previously to be incapable of infecting human cells, could productively infect human cells. Using a recently described high-titre MMTV-based vector carrying MMTV(C3H) envelope protein (Env), we successfully transduced cells of human origin. Furthermore, WT MMTV(C3H) was able to infect human cells, albeit less efficiently than mouse cells. The established infection was, however, sufficient to enable virus spread to every cell in culture. The infectivity of WT MMTV(C3H) and MMTV-based vectors carrying MMTV(C3H)Env was blocked by heat inactivation, an inhibitor of reverse transcription (3'-azido-3'-deoxythymidine) and pre-incubation with neutralizing anti-MMTV antibodies that did not neutralize vectors pseudotyped with amphotropic murine leukemia virus Env, providing evidence for an authentic, receptor-mediated and reverse transcriptase-dependent infection process. Persistently infected human Hs578T cells produced infectious virions capable of infecting naïve human breast cells in culture, the infectivity of which could also be blocked by neutralizing anti-MMTV antibodies, demonstrating that virus particles released by the persistently infected Hs578T cells were related antigenically to the virus produced from murine cells. Taken together, our results show that MMTV(C3H), like MMTV(GR) and MMTV(RIII), is able not only to infect but also to replicate in cultured human breast cells.

Macropinocytosis is the entry mechanism of amphotropic murine leukemia virus.

The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the infection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that A-MLV use macropinocytosis as the predominant entry portal into cells. Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutic schemes and methods of drug delivery. We show here that amphotropic murine leukemia virus (A-MLV) pseudotyped with the amphotropic envelope protein (which expands the host range to many mammalian cells) gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 μm) following the collapse of cell surface protrusions and membrane scission. We used drugs or the introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection are correlated, and we provide both light- and electron-microscopic evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals, including caveolae, previously suggested to internalize A-MLV.