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Niwa H, Nakamura T, Kushiya H, Kuraya T, Inoko K, Inagaki A, Suzuki T, Sasaki K, Tsuchikawa T, Hiraoka K, Shichinohe T, Hatanaka Y, Jolly DJ, Kasahara N, Hirano S. Therapeutic activity of retroviral replicating vector-mediated gene therapy in combination with anti-PD-1 antibody in a murine pancreatic cancer model. Cancer Gene Ther 2024; 31:1390-1401. [PMID: 39039195 DOI: 10.1038/s41417-024-00810-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 05/24/2024] [Accepted: 07/11/2024] [Indexed: 07/24/2024]
Abstract
Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.
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Affiliation(s)
- Hiroki Niwa
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Toru Nakamura
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan.
| | - Hiroki Kushiya
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Tomotaka Kuraya
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Kazuho Inoko
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Akihito Inagaki
- Department of Neurological Surgery, University of California, San Francisco, CA, USA
| | - Tomohiro Suzuki
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Katsunori Sasaki
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Takahiro Tsuchikawa
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Kei Hiraoka
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
- Department of Clinical Research, NHO Hakodate National Hospital, Hakodate, Hokkaido, Japan
| | - Toshiaki Shichinohe
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
| | - Yutaka Hatanaka
- Center for Development of Advanced Diagnostics (C-DAD), Hokkaido University Hospital, Sapporo, Japan
| | - Douglas J Jolly
- Tocagen Inc., San Diego, CA, USA
- Abintus Bio Inc., San Diego, CA, USA
| | - Noriyuki Kasahara
- Department of Neurological Surgery, University of California, San Francisco, CA, USA.
- Department of Radiation Oncology, University of California, San Francisco, CA, USA.
| | - Satoshi Hirano
- Department of Gastroenterological Surgery II, Hokkaido University Faculty of Medicine, Sapporo, Hokkaido, Japan
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Wong B, Birtch R, Rezaei R, Jamieson T, Crupi MJF, Diallo JS, Ilkow CS. Optimal delivery of RNA interference by viral vectors for cancer therapy. Mol Ther 2023; 31:3127-3145. [PMID: 37735876 PMCID: PMC10638062 DOI: 10.1016/j.ymthe.2023.09.012] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 08/28/2023] [Accepted: 09/14/2023] [Indexed: 09/23/2023] Open
Abstract
In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.
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Affiliation(s)
- Boaz Wong
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Rayanna Birtch
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Reza Rezaei
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Taylor Jamieson
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Mathieu J F Crupi
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Jean-Simon Diallo
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Carolina S Ilkow
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
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Kang M, Song A, Kim J, Kang SH, Lee SJ, Kim YS. Split genome-based retroviral replicating vectors achieve efficient gene delivery and therapeutic effect in a human glioblastoma xenograft model. BMB Rep 2022; 55:615-620. [PMID: 36195571 PMCID: PMC9813426] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2022] [Indexed: 12/29/2022] Open
Abstract
The murine leukemia virus-based semi-retroviral replicating vectors (MuLV-based sRRV) had been developed to improve safety and transgene capacity for cancer gene therapy. However, despite the apparent advantages of the sRRV, improvements in the in vivo transduction efficiency are still required to deliver therapeutic genes efficiently for clinical use. In this study, we established a gibbon ape leukemia virus (GaLV) envelopepseudotyped semi-replication-competent retrovirus vector system (spRRV) which is composed of two transcomplementing replication-defective retroviral vectors termed MuLV-Gag-Pol and GaLV-Env. We found that the spRRV shows considerable improvement in efficiencies of gene transfer and spreading in both human glioblastoma cells and pre-established human glioblastoma mouse model compared with an sRRV system. When treated with ganciclovir after intratumoral injection of each vector system into pre-established U-87 MG glioblastomas, the group of mice injected with spRRV expressing the herpes simplex virus type 1-thymidine kinase (HSV1-tk) gene showed a survival rate of 100% for more than 150 days, but all control groups of mice (HSV1-tk/PBS-treated and GFP/GCV-treated groups) died within 45 days after tumor injection. In conclusion, these findings sug-gest that intratumoral delivery of the HSV1-tk gene by the spRRV system is worthy of development in clinical trials for the treatment of malignant solid tumors. [BMB Reports 2022; 55(12): 615-620].
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Affiliation(s)
- Moonkyung Kang
- Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Korea
| | - Ayoung Song
- Institue of Molecular Biology, Inje University, Seoul 04551, Korea
| | - Jiyoung Kim
- Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Korea
| | - Se Hun Kang
- Research Institute and Hospital, National Cancer Center of Korea, Goyang 10408, Korea
| | - Sang-Jin Lee
- Research Institute and Hospital, National Cancer Center of Korea, Goyang 10408, Korea
| | - Yeon-Soo Kim
- Graduate School of New Drug Discovery and Development, Chungnam National University, Daejeon 34134, Korea,Corresponding author. Tel: +82-42-821-8631; Fax: +82-42-821-8923; E-mail:
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Retroviral Replicating Vector Toca 511 ( Vocimagene Amiretrorepvec) for Prodrug Activator Gene Therapy of Lung Cancer. Cancers (Basel) 2022; 14:cancers14235820. [PMID: 36497300 PMCID: PMC9736610 DOI: 10.3390/cancers14235820] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2022] [Revised: 11/23/2022] [Accepted: 11/24/2022] [Indexed: 11/29/2022] Open
Abstract
Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.
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Evaluation of the stability and intratumoral delivery of foreign transgenes encoded by an oncolytic Foamy Virus vector. Cancer Gene Ther 2022; 29:1240-1251. [PMID: 35145270 PMCID: PMC9363555 DOI: 10.1038/s41417-022-00431-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2021] [Revised: 12/21/2021] [Accepted: 01/25/2022] [Indexed: 11/12/2022]
Abstract
Foamy Viruses are cell cycle-dependent retroviruses capable of persisting unintegrated in quiescent cells until cell division occurs. This unique ability allows them to target slowly dividing human tumor cells which remains an unmet need in oncolytic virotherapy. We have previously reported the generation of oncolytic Foamy Virus (oFV) vector system and demonstrated its superiority over oncolytic Murine Leukemia Virus vectors in infecting slowly dividing cancer cells. In the present study we evaluated (i) the ability of oFV to carry foreign transgenes and (ii) the genetic stability of these vectors upon serial passage. The thymidine kinase (TK) and inducible caspase 9 (iCasp9) cDNAs could be detected in the oFV backbone for up to 3 in vitro passages. In vivo, GFP-, TK- and iCasp9- carrying oFV vectors propagated efficiently in subcutaneous xenograft glioblastoma tumors and drove transgene expression for up to 66 days. However, in vivo oFV vector spread eventually resulted in complete loss of the iCasp9 cDNA, minor loss of the TK cDNA and negligible loss of the GFP. Our results suggest that oFV is a promising gene delivery platform and that transgenes smaller than 1 kb might be most suitable for oFV arming.
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Jeon YH, Jung YT. Production of a replicating retroviral vector expressing Reovirus fast protein for cancer gene therapy. J Virol Methods 2021; 299:114332. [PMID: 34655690 DOI: 10.1016/j.jviromet.2021.114332] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Revised: 10/12/2021] [Accepted: 10/12/2021] [Indexed: 01/09/2023]
Abstract
Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.
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Affiliation(s)
- Young Hyun Jeon
- Department of Microbiology, Dankook University, Cheonan, 330-714, Republic of Korea
| | - Yong-Tae Jung
- Department of Microbiology, Dankook University, Cheonan, 330-714, Republic of Korea.
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Collins SA, Shah AH, Ostertag D, Kasahara N, Jolly DJ. Clinical development of retroviral replicating vector Toca 511 for gene therapy of cancer. Expert Opin Biol Ther 2021; 21:1199-1214. [PMID: 33724117 PMCID: PMC8429069 DOI: 10.1080/14712598.2021.1902982] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Accepted: 03/10/2021] [Indexed: 01/23/2023]
Abstract
INTRODUCTION The use of tumor-selectively replicating viruses is a rapidly expanding field that is showing considerable promise for cancer treatment. Retroviral replicating vectors (RRV) are unique among the various replication-competent viruses currently being investigated for potential clinical utility, because they permanently integrate into the cancer cell genome and are capable of long-term persistence within tumors. RRV can mediate efficient tumor-specific delivery of prodrug activator genes, and subsequent prodrug treatment leads to synchronized cell killing of infected cancer cells, as well as activation of antitumor immune responses. AREAS COVERED Here we review preclinical studies supporting bench-to-bedside translation of Toca 511, an optimized RRV for prodrug activator gene therapy, the results from Phase I through III clinical trials to date, and potential future directions for this therapy as well as other clinical candidate RRV. EXPERT OPINION Toca 511 has shown highly promising results in early-stage clinical trials. This vector progressed to a registrational Phase III trial, but the results announced in late 2019 appeared negative overall. However, the median prodrug dosing schedule was not optimal, and promising possible efficacy was observed in some prespecified subgroups. Further clinical investigation, as well as development of RRV with other transgene payloads, is merited.
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Affiliation(s)
- Sara A Collins
- Department of Neurological Surgery, University of California, San Francisco (UCSF), San Francisco, California, United States of America
| | - Ashish H Shah
- Department of Neurological Surgery, Miller School of Medicine, University of Miami, Florida, United States of America
| | - Derek Ostertag
- Tocagen, Inc., San Diego, California, United States of America
| | - Noriyuki Kasahara
- Department of Neurological Surgery, University of California, San Francisco (UCSF), San Francisco, California, United States of America
- Department of Radiation Oncology, University of California, San Francisco (UCSF), California, United States of America
| | - Douglas J Jolly
- Tocagen, Inc., San Diego, California, United States of America
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Oncolytic Foamy Virus - generation and properties of a nonpathogenic replicating retroviral vector system that targets chronically proliferating cancer cells. J Virol 2021; 95:JVI.00015-21. [PMID: 33692205 PMCID: PMC8139661 DOI: 10.1128/jvi.00015-21] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Nonpathogenic retroviruses of the Spumaretrovirinae subfamily can persist long-term in the cytoplasm of infected cells, completing their lifecycle only after the nuclear membrane dissolves at the time of cell division. Since the targeting of slowly dividing cancer cells remains an unmet need in oncolytic virotherapy we constructed a replication competent Foamy Virus vector (oFV) from the genomes of two chimpanzee Simian Foamy Viruses (PAN1 and PAN2) and inserted a GFP transgene in place of the bel-2 open reading frame. oFV-GFP infected and propagated with slow kinetics in multiple human tumor cell lines, inducing a syncytial cytopathic effect. Infection of growth arrested MRC5 cells was not productive, but oFV genomes persisted in the cytoplasm and the productive viral lifecycle resumed when cell division was later restored. In vivo, the virus propagated extensively in intraperitoneal ovarian cancer xenografts, slowing tumor growth, significantly prolonging survival of the treated mice and sustaining GFP transgene expression for at least 45 days. Our data indicate that oFV is a promising new replication-competent viral and gene delivery platform for efficient targeting of the most fundamental trait of cancer cells, their ability to sustain chronic proliferation.Significance:The infectivity of certain retroviruses is limited to dividing cells, which makes them attractive tools for targeting cancer cell proliferation. Previously developed replication-competent gammaretroviral vectors spread efficiently in rapidly dividing cancer cells, but not in cancer cells that divide more slowly. In contrast to rapidly proliferating transplantable mouse tumors, slow proliferation is a hallmark of human cancers and may have contributed to the clinical failure of the preclinically promising Murine Leukemia Virus vector Toca511 which failed to show efficacy in a phase 3 clinical trial in patients with glioblastoma. The studies presented in our manuscript show that oncolytic Foamy Virus (oFV) vectors are capable of persisting unintegrated in quiescent cells and resuming their life cycle once the cells start dividing again. This property of oFVs, together with their lack of pathogenicity and their ability to catalyze the fusion of infected cancer cells, makes them an attractive platform for further investigation.
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Voropaev H, Gimmelshein Vatkin M, Shneor D, Luski S, Honigman A, Frenkel S. Infectious Knockdown of CREB and HIF-1 for the Treatment of Metastatic Uveal Melanoma. Cancers (Basel) 2019; 11:cancers11081056. [PMID: 31357444 PMCID: PMC6721386 DOI: 10.3390/cancers11081056] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Revised: 07/17/2019] [Accepted: 07/17/2019] [Indexed: 12/12/2022] Open
Abstract
Uveal melanoma (UM) is the most prevalent primary intraocular cancer in adults. Up to half the patients develop metastases that are currently incurable, and most patients die within two years following the diagnosis of metastases. Therefore, novel therapeutic approaches are required. It has been established that tumor cells are more resistant to the hypoxia cue than non-malignant cells and can remain viable in hypoxia. Oxygen absence in hypoxic tumor areas means the absence of chemotherapeutics and the absence of the effector for radiotherapy (free oxygen radicals). To overcome this treatment resistance, we constructed MuLV-based replication-competent retroviral (RCR) vectors expressing shRNA targeting the hypoxia-response regulating genes CREB and HIF-1. These RCRs express shRNAs either against a single exon or against an exon and the poly-A signal to minimize the point-mutation resistance. These RCRs that only infect replicating cells will preferentially infect tumor cells. Pre-infected Mel270 UM subcutaneous xenografts in SCID mice were monitored weekly in vivo via bioluminescence. Here, we demonstrate that the knockdown of CREB or HIF-1 in UM cells dramatically decreases UM tumor progression. The reduction of the expression of Glut-1, which is a major glucose transporter in cancer cells, within tumors that are infected with the armed viruses may indicate UM's dependence on glycolysis for tumor progression.
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Affiliation(s)
- Hanna Voropaev
- Division of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
| | - Maria Gimmelshein Vatkin
- Division of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
| | - Dudi Shneor
- Division of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
| | - Shahar Luski
- Division of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
| | - Alik Honigman
- Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
- Department of Biotechnology, Hadassah Academic College, Jerusalem 9101001, Israel
| | - Shahar Frenkel
- Division of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel.
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Dual-vector prodrug activator gene therapy using retroviral replicating vectors. Cancer Gene Ther 2018; 26:128-135. [PMID: 30348946 PMCID: PMC6760537 DOI: 10.1038/s41417-018-0051-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Revised: 09/24/2018] [Accepted: 09/29/2018] [Indexed: 01/08/2023]
Abstract
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.
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A novel combinatorial treatment option for metastatic uveal melanoma. Oncotarget 2018; 9:26096-26108. [PMID: 29899845 PMCID: PMC5995237 DOI: 10.18632/oncotarget.25445] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Accepted: 04/28/2018] [Indexed: 01/01/2023] Open
Abstract
Uveal melanoma (UM) is the most frequent intraocular tumor in adult patients. When metastases occur, systemic therapy with alkylating agents (fotemustine or dacarbazine (DTIC)) has shown only modest efficacy. The common chemotherapeutic drug doxorubicin (DOX) is not used to treat metastatic UM (mUM). To expand the chemotherapeutic arsenal for mUM, we tested the effect of DOX on UM cell mortality. We have previously shown that CREB knockdown enhances sensitivity to DOX. UM cells infected with recombinant MuLV-based replicative competent retroviruses (RCR) expressing shRNA targeting CREB were co-treated with either DTIC or DOX. We found that CREB knockdown increases the sensitivity of these cells to both DOX and DTIC in normoxia and more so in hypoxia as measured by cell survival and Caspase 3 activation. The ability to combine CREB knockdown by infection with the RCR recombinant virus which preferentially infects replicating tumor cells and chemotherapy to achieve the same amount of cell death in lower concentrations may result in fewer side effects of the drugs. This combination is a possible new treatment for mUM.
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Hofacre A, Yagiz K, Mendoza D, Lopez Espinoza F, Munday AW, Burrascano C, Singer O, Gruber HE, Jolly DJ, Lin AH. Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models. Hum Gene Ther 2018; 29:437-451. [PMID: 29216761 DOI: 10.1089/hum.2017.205] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (∼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - Amy H Lin
- Tocagen, Inc. , San Diego, California
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Shneor D, Folberg R, Pe'er J, Honigman A, Frenkel S. Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma. Cancer Gene Ther 2016; 24:64-74. [PMID: 27934882 PMCID: PMC5339434 DOI: 10.1038/cgt.2016.68] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2016] [Revised: 09/05/2016] [Accepted: 09/07/2016] [Indexed: 12/19/2022]
Abstract
The fast proliferation of tumor cells develops faster than the vasculature, resulting, in most malignant tumors, in generation of hypoxic regions. Hypoxia renders solid tumors resistant to radiation and chemotherapeutics while providing opportunities for tumor-selective therapies targeting tumor hypoxia. Here we exploit two properties of tumors: propagation of tumor cells and ongoing generation of hypoxic regions to construct a system that preferentially leads to the death of tumor cells and thus hinders tumor growth. We constructed murine leukemia virus replication-competent (RCR) viruses that infect only propagating cells. These viruses express small hairpin RNAs (shRNAs) targeting cyclic AMP-response-element binding protein (CREB), hypoxia-inducible factors 1 (HIF)-1 or HIF-2 individually or all three together (X3). These viruses efficiently infected in vitro human hepatocellular carcinoma (HepG2 and FLC4) cells and established persistence of the virus and knocked down the expression of the regulators of the hypoxia-responding genes. Knockdown of either HIF-1 or CREB or both in hypoxia reduced the expression of hypoxia-response elements- and CRE-mediated gene expression, diminished cell proliferation and increased caspase-3 activity. We did not detect any significant effect of the efficiently knocked down HIF-2 on any of the functions tested in vitro. Moreover, severe combined immunodeficiency mice implanted subcutaneously with HepG2 stably infected with recombinant RCRs showed reduction of tumor growth and vascular endothelial growth factor expression, and no hypoxia-guided neovascularization. Combined treatment (RCRs+doxorubicin) improved efficacy in the context of in vitro hypoxia and in vivo (with either vACE-CREB or vACE-X3). This synergistic effect may lead to an improved efficacy and safety profile of the treatment that may result in fewer side effects.
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Affiliation(s)
- D Shneor
- Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.,Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
| | - R Folberg
- Departments of Pathology, Ophthalmology and Biomedical Sciences, Oakland University William Beaumont School of Medicine, Rochester, MI, USA
| | - J Pe'er
- Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
| | - A Honigman
- Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.,Department of Biotechnology, Hadassah Academic College, Jerusalem, Israel
| | - S Frenkel
- Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel
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Twitty CG, Diago OR, Hogan DJ, Burrascano C, Ibanez CE, Jolly DJ, Ostertag D. Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types. Hum Gene Ther Methods 2015; 27:17-31. [PMID: 26467507 DOI: 10.1089/hgtb.2015.106] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6 μg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals.
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15
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Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors. Gene Ther 2014; 22:155-62. [PMID: 25354682 DOI: 10.1038/gt.2014.98] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2014] [Revised: 09/12/2014] [Accepted: 09/17/2014] [Indexed: 12/21/2022]
Abstract
Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a major concern. To design a tumor-specific retroviral replicating vector (RRV), we replaced the U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the hepatitis B virus enhancer II (EII) and human α-fetoprotein (AFP) core promoter to produce ACE-GFP-EIIAFP, a hepatocellular carcinoma (HCC)-targeting RRV. Similar to ACE-GFP, ACE-GFP-EIIAFP exhibited robust green fluorescent protein (GFP) expression in HCC cells and, most importantly, it exhibited HCC-specific replication and did not replicate in non-HCC tumor cells or normal liver cells. We sequenced the promoter region of ACE-GFP-EIIAFP collected from serial infection cycles to examine the genomic stability of the vector during its replicative spread, and found that the vector could retain the hybrid promoter in the genome for at least six infection cycles. In vitro studies revealed that ACE-CD-EIIAFP and ACE-PNP-EIIAFP, which express the yeast cytosine deaminase and Escherichia coli purine nucleoside phosphorylase, respectively, exert a highly potent cytotoxic effect on HCC cells in the presence of their respective prodrugs. In vivo, ACE-CD-EIIAFP-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signal was observed in extratumoral tissues. These results suggest that the tumor-specific, suicide-gene-encoding RRV may fulfill the promise of retroviral gene therapy for cancer.
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16
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Takahashi M, Valdes G, Hiraoka K, Inagaki A, Kamijima S, Micewicz E, Gruber HE, Robbins JM, Jolly DJ, McBride WH, Iwamoto KS, Kasahara N. Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector. Cancer Gene Ther 2014; 21:405-410. [PMID: 25301172 PMCID: PMC4246057 DOI: 10.1038/cgt.2014.38] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2014] [Revised: 06/17/2014] [Accepted: 06/17/2014] [Indexed: 12/28/2022]
Abstract
A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794 and NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.
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Affiliation(s)
- Masamichi Takahashi
- Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Gilmer Valdes
- Department of Radiation Oncology, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Kei Hiraoka
- Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Akihito Inagaki
- Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Shuichi Kamijima
- Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Ewa Micewicz
- Department of Radiation Oncology, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | | | | | | | - William H McBride
- Department of Radiation Oncology, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Keisuke S Iwamoto
- Department of Radiation Oncology, University of California Los Angeles (UCLA), Los Angeles, California, USA
| | - Noriyuki Kasahara
- Department of Medicine, University of California Los Angeles (UCLA), Los Angeles, California, USA
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17
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Kubo S, Takagi-Kimura M, Logg CR, Kasahara N. Highly efficient tumor transduction and antitumor efficacy in experimental human malignant mesothelioma using replicating gibbon ape leukemia virus. Cancer Gene Ther 2013; 20:671-7. [PMID: 24201868 DOI: 10.1038/cgt.2013.67] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2013] [Accepted: 10/17/2013] [Indexed: 11/10/2022]
Abstract
Retroviral replicating vectors (RRVs) have been shown to achieve 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), in human malignant mesothelioma cells. In vitro, both RRVs expressing the green fluorescent protein gene efficiently replicated in most mesothelioma cell lines tested, but not in normal mesothelial cells. Notably, in ACC-MESO-1 mesothelioma cells that were not permissive for AMLV-RRV, the GALV-RRV could spread efficiently in culture and in mice with subcutaneous xenografts by in vivo fluorescence imaging. Next, GALV-RRV expressing the cytosine deaminase prodrug activator gene showed efficient killing of ACC-MESO-1 cells in a prodrug 5-fluorocytosine dose-dependent manner, compared with AMLV-RRV. GALV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous ACC-MESO-1 tumor growth in nude mice. Quantitative reverse transcription PCR demonstrated that ACC-MESO-1 cells express higher PiT-1 (GALV receptor) and lower PiT-2 (AMLV receptor) compared with normal mesothelial cells and other mesothelioma cells, presumably accounting for the distinctive finding that GALV-RRV replicates much more robustly than AMLV-RRV in these cells. These data indicate the potential utility of GALV-RRV-mediated prodrug activator gene therapy in the treatment of mesothelioma.
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Affiliation(s)
- S Kubo
- Department of Genetics, Hyogo College of Medicine, Nishinomiya, Japan
| | - M Takagi-Kimura
- Department of Genetics, Hyogo College of Medicine, Nishinomiya, Japan
| | - C R Logg
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
| | - N Kasahara
- Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA
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18
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Hickey MJ, Malone CC, Erickson KL, Lin A, Soto H, Ha ET, Kamijima S, Inagaki A, Takahashi M, Kato Y, Kasahara N, Mueller BM, Kruse CA. Combined alloreactive CTL cellular therapy with prodrug activator gene therapy in a model of breast cancer metastatic to the brain. Clin Cancer Res 2013; 19:4137-48. [PMID: 23780889 DOI: 10.1158/1078-0432.ccr-12-3735] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. EXPERIMENTAL DESIGN AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. RESULTS AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy-treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50-83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. CONCLUSION The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain.
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Affiliation(s)
- Michelle J Hickey
- Department of Neurosurgery, University of California Los Angeles, Los Angeles, California, USA
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19
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Perez OD, Logg CR, Hiraoka K, Diago O, Burnett R, Inagaki A, Jolson D, Amundson K, Buckley T, Lohse D, Lin A, Burrascano C, Ibanez C, Kasahara N, Gruber HE, Jolly DJ. Design and selection of Toca 511 for clinical use: modified retroviral replicating vector with improved stability and gene expression. Mol Ther 2012; 20:1689-98. [PMID: 22547150 DOI: 10.1038/mt.2012.83] [Citation(s) in RCA: 107] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.
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20
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Logg CR, Robbins JM, Jolly DJ, Gruber HE, Kasahara N. Retroviral replicating vectors in cancer. Methods Enzymol 2012; 507:199-228. [PMID: 22365776 DOI: 10.1016/b978-0-12-386509-0.00011-9] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The use of replication-competent viruses for the treatment of cancer is an emerging technology that shows significant promise. Among the various different types of viruses currently being developed as oncolytic agents, retroviral replicating vectors (RRVs) possess unique characteristics that allow highly efficient, non-lytic, and tumor-selective gene transfer. By retaining all of the elements necessary for viral replication, RRVs are capable of transmitting genes via exponential in situ amplification. Their replication-competence also provides a powerful means whereby novel and useful RRV variants can be generated using natural selection. Their stringent requirement for cell division in order to achieve productive infection, and their preferential replication in cells with defective innate immunity, confer a considerable degree of natural specificity for tumors. Furthermore, their ability to integrate stably into the genome of cancer cells, without immediate cytolysis, contributes to long-lasting therapeutic efficacy. Thus, RRVs show much promise as therapeutic agents for cancer and are currently being tested in the clinic. Here we describe experimental methods for their production and quantitation, for adaptive evolution and natural selection to develop novel or improved RRV, and for in vitro and in vivo assessment of the therapeutic efficacy of RRVs carrying prodrug activator genes for treatment of cancer.
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Affiliation(s)
- Christopher R Logg
- Department of Medicine, University of California, Los Angeles, California, USA
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21
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Virotherapy induces massive infiltration of neutrophils in a subset of tumors defined by a strong endogenous interferon response activity. Cancer Gene Ther 2011; 18:785-94. [PMID: 21869820 PMCID: PMC3196785 DOI: 10.1038/cgt.2011.46] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Oncolytic virotherapy has shown substantial promises as an alternative therapeutic modality for solid tumors in both preclinical studies and clinical trials. The main therapeutic activity of virotherapy derives from the direct lytic effect associated with virus replication and the induction of host immune responses to the infected tumor cells. Here we show that some human and murine tumor cell lines are highly resistant to the lytic effect of a type II herpes simplex virus-derived oncolytic virus, FusOn-H2, which was constructed by deleting the N-terminal region of the ICP10 gene. However, these tumor cells still respond exceptionally well to FusOn-H2 virotherapy in vivo. Histological examination of the treated tumors revealed that, in contrast to tumors supporting FusOn-H2 replication, implants of these highly resistant lines showed massive infiltration of neutrophils after virotherapy. Further analysis showed that there is a correlation between an intrinsically strong interferon response activity and the recruitment of neutrophils in these tumors. These results suggest that an innate immune response mainly represented by neutrophils may be part of the virotherapy-mediated antitumor mechanism in these tumors.
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22
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Kawasaki Y, Tamamoto A, Takagi-Kimura M, Maeyama Y, Yamaoka N, Terada N, Okamura H, Kasahara N, Kubo S. Replication-competent retrovirus vector-mediated prodrug activator gene therapy in experimental models of human malignant mesothelioma. Cancer Gene Ther 2011; 18:571-8. [PMID: 21660062 PMCID: PMC3159547 DOI: 10.1038/cgt.2011.25] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Replication-competent retrovirus (RCR) vectors have been shown to achieve significantly enhanced tumor transduction efficiency and therapeutic efficacy in various cancer models. In the present study, we investigated RCR vector-mediated prodrug activator gene therapy for the treatment of malignant mesothelioma, a highly aggressive tumor with poor prognosis. RCR-GFP vector expressing the green fluorescent protein marker gene successfully infected and efficiently replicated in human malignant mesothelioma cell lines, as compared with non-malignant mesothelial cells in vitro. In mice with pre-established subcutaneous tumor xenografts, RCR-GFP vector showed robust spread throughout entire tumor masses after intratumoral administration. Next, RCR-cytosine deaminase (RCR-CD), expressing the yeast CD prodrug activator gene, showed efficient transmission of the prodrug activator gene associated with replicative spread of the virus, resulting in efficient killing of malignant mesothelioma cells in a prodrug 5-fluorocytosine (5FC)-dose dependent manner in vitro. After a single intratumoral injection of RCR-CD followed by intraperitoneal administration of 5FC, RCR vector-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous tumor growth, and significantly prolonged survival in the disseminated peritoneal model of malignant mesothelioma. These data indicate the potential utility of RCR vector-mediated prodrug activator gene therapy in the treatment of malignant mesothelioma.
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Affiliation(s)
- Y Kawasaki
- Laboratory of Host Defenses, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Japan
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23
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Koppers-Lalic D, Hoeben RC. Non-human viruses developed as therapeutic agent for use in humans. Rev Med Virol 2011; 21:227-39. [PMID: 21560181 PMCID: PMC7169051 DOI: 10.1002/rmv.694] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2011] [Revised: 03/28/2011] [Accepted: 03/29/2011] [Indexed: 11/08/2022]
Abstract
Viruses usually infect a restricted set of host species, and only in rare cases does productive infection occur outside the natural host range. Infection of a new host species can manifest as a distinct disease. In this respect, the use of non‐human viruses in clinical therapy may be a cause for concern. It could provide the opportunity for the viruses to adapt to the new host and be transferred to the recipient's relatives or medical caretakers, or even to the normal host species. Such environmental impact is evidently undesirable. To forecast future clinical use of non‐human viruses, a literature study was performed to identify the viruses that are being considered for application as therapeutic agents for use in humans. Twenty‐seven non‐human virus species were identified that are in (pre)clinical development, mainly as oncolytic agents. For risk management, it is essential that the potential environmental consequences are assessed before initiating clinical use, even if the virus is not formally classified as a genetically modified organism. To aid such assessment, each of these viruses was classified in one of five relative environmental risk categories, ranging from “Negligible” to “Very High”. Canary pox virus and the Autographa californica baculovirus were assigned a “Negligible” classification, and Seneca Valley virus, murine leukemia virus, and Maraba virus to the “High” category. A complicating factor in the classification is the scarcity of publicly available information on key aspects of virus biology in some species. In such cases the relative environmental risk score was increased as a precaution. Copyright © 2011 John Wiley & Sons, Ltd.
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24
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The rapid production of high-titer porcine endogenous retrovirus(PERV)-B env pseudotype and construction of an EGFP-expressing replication competent PERV-A vector. J Virol Methods 2010; 171:61-6. [PMID: 20933542 DOI: 10.1016/j.jviromet.2010.09.030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2010] [Revised: 09/29/2010] [Accepted: 09/30/2010] [Indexed: 11/22/2022]
Abstract
Porcine endogenous retroviruses (PERVs) present a unique concern associated with xenotransplantation because they have been shown to infect certain human cells in vitro and it is also difficult to generate herds of pigs free of PERVs. A simple system for the production of high-titer MoMLV-PERV pseudotypes is reported; an EGFP-expressing replication-competent molecular clone that allows direct measurement of titer was also constructed. To improve the MLV-based retroviral vector system, a 2.1-kb PERV-B env product was amplified from PK-15 genomic DNA and cloned into the pCL-Eco retroviral vector. The titer of lacZ (PERV-B) from the 293 cells was about 1.0×10(4) CFU/ml. In contrast, the titer of lacZ (PERV-B) from a conventional murine retroviral vector (split genome) was found to be 1.2×10(2) CFU/ml when the PERV-B env expression vector was transfected into TELCeB6 cells, which harbor MFGnlslacZ and the gag-pol-expressing vector. In addition, an infectious PERV-A clone containing enhanced GFP (EGFP) by using a PCR-based method was developed. This EGFP-expressing PERV-A-IRES-EGFP molecular clone was found to be stable genetically on transfection in 293 cells.
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25
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Tai CK, Wang W, Lai YH, Logg CR, Parker WB, Li YF, Hong JS, Sorscher EJ, Chen TC, Kasahara N. Enhanced efficiency of prodrug activation therapy by tumor-selective replicating retrovirus vectors armed with the Escherichia coli purine nucleoside phosphorylase gene. Cancer Gene Ther 2010; 17:614-23. [PMID: 20467451 PMCID: PMC2938767 DOI: 10.1038/cgt.2010.17] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2009] [Revised: 11/22/2009] [Accepted: 12/27/2009] [Indexed: 11/08/2022]
Abstract
Gene transfer of the Escherichia coli purine nucleoside phosphorylase (PNP) results in potent cytotoxicity after administration of the prodrug fludarabine phosphate (F-araAMP). Here, we have tested whether application of this strategy in the context of replication-competent retrovirus (RCR) vectors, which can achieve highly efficient tumor-restricted transduction as well as persistent expression of transgenes, would result in effective tumor inhibition, or, alternatively, would adversely affect viral replication. We found that RCR vectors could achieve high levels of PNP expression concomitant with the efficiency of their replicative spread, with significant cell killing activity in vitro and potent therapeutic effects in vivo. In U-87 xenograft models, replicative spread of the vector resulted in progressive transmission of the PNP transgene, as evidenced by increasing PNP enzyme activity with time after vector inoculation. On F-araAMP administration, high efficiency gene transfer of PNP by the RCR vector resulted in significant suppression of tumor growth and extended survival time. As the RCR mediates stable integration of the PNP gene and continuous expression, an additional round of F-araAMP administration resulted in further survival benefit. RCR-mediated PNP suicide gene therapy thus represents a highly efficient form of intracellular chemotherapy, and may achieve effective antitumor activity with less systemic toxicity.
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Affiliation(s)
- C-K Tai
- Department of Life Science, National Chung Cheng University, Min-Hsiung, Chia-Yi, Taiwan
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26
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Adenovirus-retrovirus hybrid vectors achieve highly enhanced tumor transduction and antitumor efficacy in vivo. Mol Ther 2010; 19:76-82. [PMID: 20808291 DOI: 10.1038/mt.2010.182] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors have been shown to mediate efficient, selective, and persistent tumor transduction, thereby achieving significant therapeutic benefit in a wide variety of cancer models. To further augment the efficiency of this strategy, we have developed a delivery method employing a gutted adenovirus encoding an RCR vector (AdRCR); thus, tumor cells transduced with the adenoviral vector transiently become RCR vector producer cells in situ. As expected, high-titer AdRCR achieved significantly higher initial transduction levels in human cancer cells both in vitro and in vivo, as compared to the original RCR vector itself. Notably, even at equivalent initial transduction levels, more secondary RCR progeny were produced from AdRCR-transduced cells as compared to RCR-transduced cells, resulting in further acceleration of subsequent RCR replication kinetics. In pre-established tumor models in vivo, prodrug activator gene therapy with high-titer AdRCR could achieve enhanced efficacy compared to RCR alone, in a dose-dependent manner. Thus, AdRCR hybrid vectors offer the advantages of high production titers characteristic of adenovirus and secondary production of RCR in situ, which not only accelerates subsequent vector spread and progressive tumor transduction, but can also significantly enhance the therapeutic efficacy of RCR-mediated prodrug activator gene therapy.
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27
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Kimura T, Hiraoka K, Kasahara N, Logg CR. Optimization of enzyme-substrate pairing for bioluminescence imaging of gene transfer using Renilla and Gaussia luciferases. J Gene Med 2010; 12:528-37. [PMID: 20527045 PMCID: PMC2989363 DOI: 10.1002/jgm.1463] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Bioluminescence imaging (BLI) permits the non-invasive quantification and localization of transduction and expression by gene transfer vectors. The tendency of tissue to attenuate light in the optical region, however, limits the sensitivity of BLI. Improvements in light output from bioluminescent reporter systems would allow the detection of lower levels of expression, smaller numbers of cells and expression from deeper and more attenuating tissues within an animal. METHODS With the goal of identifying substrates that allow improved sensitivity with Renilla luciferase (RLuc) and Gaussia luciferase (GLuc) reporter genes, we evaluated native coelenterazine and three of its most promising derivatives in BLI of cultured cells transduced with retroviral vectors encoding these reporters. Of the eight enzyme-substrate pairs tested, the two that performed best were further evaluated in mice to compare their effectiveness for imaging vector-modified cells in live animals. RESULTS In cell culture, we observed striking differences in luminescence levels from the various enzyme-substrate combinations and found that the two luciferases exhibited markedly distinct abilities to generate light with the substrates. The most effective pairs were RLuc with the synthetic coelenterazine derivative ViviRen, and GLuc with native coelenterazine. In animals, these two pairs allowed similar detection sensitivities, which were eight- to 15-fold higher than that of the prototypical RLuc-native coelenterazine combination. CONCLUSIONS Substrate selection can dramatically influence the detection sensitivity of RLuc and GLuc and appropriate choice of substrate can greatly improve the performance of reporter genes encoding these enzymes for monitoring gene transfer by BLI.
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Affiliation(s)
- Takahiro Kimura
- Department of Medicine, University of California, Los Angeles
- Department of Urology, Jikei University School of Medicine, Tokyo, Japan
| | - Kei Hiraoka
- Department of Medicine, University of California, Los Angeles
| | - Noriyuki Kasahara
- Department of Medicine, University of California, Los Angeles
- Department of Molecular and Medical Pharmacology, University of California, Los Angeles
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Paar M, Klein D, Salmons B, Günzburg WH, Renner M, Portsmouth D. Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors. BMC Mol Biol 2009; 10:8. [PMID: 19203366 PMCID: PMC2645402 DOI: 10.1186/1471-2199-10-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2008] [Accepted: 02/09/2009] [Indexed: 12/22/2022] Open
Abstract
Background The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design. Results In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed. Conclusion Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.
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Affiliation(s)
- Matthias Paar
- Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, Vienna, Austria.
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29
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Guinn B, Casey G, Collins S, O'Brien T, Alexander MY, Tangney M. Tripartite Meeting in Gene and Cell Therapy, 2008: Irish Society for Gene and Cell Therapy, British Society for Gene Therapy, and International Society for Cell and Gene Therapy of Cancer. Hum Gene Ther 2008; 19:967-78. [DOI: 10.1089/hum.2008.085] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Barbara Guinn
- Department of Haematological Medicine, King's College London School of Medicine, London SE5 9NU, United Kingdom
| | - Garrett Casey
- Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jr. Laboratory, University College Cork, Cork, Ireland
| | - Sara Collins
- Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jr. Laboratory, University College Cork, Cork, Ireland
| | - Tim O'Brien
- Regenerative Medicine Institute, National University of Ireland, Galway, Ireland
| | - M. Yvonne Alexander
- Cardiovascular Group, School of Medicine, University of Manchester, Manchester M13 9NT, United Kingdom
| | - Mark Tangney
- Cork Cancer Research Centre, Mercy University Hospital and Leslie C. Quick Jr. Laboratory, University College Cork, Cork, Ireland
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30
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Duerner LJ, Schwantes A, Schneider IC, Cichutek K, Buchholz CJ. Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue. Gene Ther 2008; 15:1500-10. [PMID: 18509380 DOI: 10.1038/gt.2008.92] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Virotherapy is currently being developed for many different types of viruses including replication-competent murine leukaemia virus (MLV) as a novel tool in cancer therapy. However, there is the risk of insertional mutagenesis associated with this virus, making careful preclinical studies necessary before its first application in man. We have previously generated conditionally replication-competent MLV variants that require activation by tumour-associated proteases to become infectious. Here we analysed in a comparative study the spreading of non-targeted and of such tumour-targeted MLV variants to tumour and extratumoural organs in immunodeficient mice. Both virus types were able to efficiently infect tumour cells after systemic administration. The non-targeted virus, however, also infected extratumoural organs like bone marrow, spleen and liver efficiently. In contrast, the targeted viruses revealed in a quantitative analysis of virus spreading an up to 500-fold more selective infection of tumour tissue than the non-targeted virus. The data raise serious doubts about a safe clinical use of non-targeted MLV. Engineering the virus to become activatable by tumour-associated proteases can significantly improve the safety of MLV.
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Affiliation(s)
- L J Duerner
- Abteilung Medizinische Biotechnologie, Paul-Ehrlich-Institut, Langen, Germany
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31
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Hiraoka K, Kimura T, Logg CR, Kasahara N. Tumor-selective gene expression in a hepatic metastasis model after locoregional delivery of a replication-competent retrovirus vector. Clin Cancer Res 2007; 12:7108-16. [PMID: 17145835 PMCID: PMC8207453 DOI: 10.1158/1078-0432.ccr-06-1452] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Replication-competent retrovirus (RCR) vectors have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. In this setting, the intrinsic inability of retroviruses to infect postmitotic normal cells, combined with their unique ability to persist through stable integration, allow further transduction of ectopic tumor foci as the infected cancer cells migrate. However, i.v. delivery of RCR vectors has never been tested previously, particularly in an immunocompetent tumor model. EXPERIMENTAL DESIGN We combined optical imaging, flow cytometry, and molecular analysis to monitor RCR vector spread after administration via locoregional infusion in a hepatic metastasis model of colorectal cancer. RESULTS Robust RCR replication was first confirmed in both human WiDr and murine CT26 colorectal cancer cells in vitro, with transduction levels reaching >90% in <12 days after virus inoculation at multiplicities of infection of 0.01 to 0.1. In vivo, infusion of RCR supernatant into the portal circulation resulted in progressive and significant transduction of multifocal intrahepatic CT26 tumors in syngeneic mice, averaging about 30% but with up to 60% transduction in some tumors within 4 weeks. However, immunohistochemistry and quantitative PCR analysis showed no evidence of RCR spread to adjacent normal liver or to any other normal tissues. CONCLUSIONS Our results thus show that locoregional infusion of RCR vectors can be used to deliver therapeutic genes selectively to tumor cells in the liver while sparing normal hepatocytes and without dissemination to extrahepatic normal tissues.
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Affiliation(s)
- Kei Hiraoka
- Department of Medicine, University of California at Los Angeles, California 90095, USA
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32
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Kikuchi E, Menendez S, Ozu C, Ohori M, Cordon-Cardo C, Logg CR, Kasahara N, Bochner BH. Highly Efficient Gene Delivery for Bladder Cancers by Intravesically Administered Replication-Competent Retroviral Vectors. Clin Cancer Res 2007; 13:4511-8. [PMID: 17671137 DOI: 10.1158/1078-0432.ccr-07-0151] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE In an attempt to improve viral delivery of potentially therapeutic genes via an intravesical route, we have recently developed murine leukemia virus-based replication-competent retrovirus (RCR) vectors. EXPERIMENTAL DESIGN We evaluated the transduction efficiency of intravesically administered RCR vectors to bladder tumor using orthotopic animal models to determine their potential as delivery vectors for bladder cancer. RESULTS The RCR vector containing green fluorescent protein (GFP) marker gene achieved efficient in vitro transmission of the GFP transgene. Murine bladder tumor-2 mouse bladder tumors exposed to intravesically administered RCR vectors exhibited 0%, 9.2 +/- 2.9%, and 30.0 +/- 6.2% of GFP expression at 9, 18, and 27 days after exposure in the orthotopic model, respectively. Orthotopic KU-19-19 human bladder tumors exposed to intravesically administered RCR vectors exhibited 3%, 85 +/- 1.0%, and 100% of GFP expression at 7, 21, and 35 days after exposure, respectively. GFP staining was observed only in the tumor cells in the bladder. No detectable PCR products of GFP gene could be observed in distant organs. Treatment with RCR vectors containing yeast cytosine deaminase (CD) gene plus 5-fluorocytosine (5-FC) dramatically inhibited the growth of preestablished murine bladder tumor-2 tumors. A single course of 5-FC treatment resulted in a 50% animal survival in mice exposed to RCR-CD compared with a 0% survival in all controls over a 70-day follow-up period. CONCLUSIONS Intravesically administered RCR vectors can efficiently deliver genes to orthotopic bladder tumor without viral spread in distant organs. RCR-CD/5-FC suicide gene therapy promises to be a novel and potentially therapeutic modality for bladder cancer.
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Affiliation(s)
- Eiji Kikuchi
- Departments of Urology and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA
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33
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Hiraoka K, Kimura T, Logg CR, Tai CK, Haga K, Lawson GW, Kasahara N. Therapeutic efficacy of replication-competent retrovirus vector-mediated suicide gene therapy in a multifocal colorectal cancer metastasis model. Cancer Res 2007; 67:5345-53. [PMID: 17545615 PMCID: PMC8207455 DOI: 10.1158/0008-5472.can-06-4673] [Citation(s) in RCA: 50] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.
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Affiliation(s)
- Kei Hiraoka
- Department of Medicine, University of California Los Angeles, Los Angeles, California 90095, USA
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34
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Logg CR, Baranick BT, Lemp NA, Kasahara N. Adaptive evolution of a tagged chimeric gammaretrovirus: identification of novel cis-acting elements that modulate splicing. J Mol Biol 2007; 369:1214-29. [PMID: 17498744 PMCID: PMC2938735 DOI: 10.1016/j.jmb.2007.04.026] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2006] [Revised: 04/05/2007] [Accepted: 04/10/2007] [Indexed: 11/19/2022]
Abstract
Retroviruses are well known for their ability to incorporate envelope (Env) proteins from other retroviral strains and genera, and even from other virus families. This characteristic has been widely exploited for the generation of replication-defective retroviral vectors, including those derived from murine leukemia virus (MLV), bearing heterologous Env proteins. We investigated the possibility of "genetically pseudotyping" replication-competent MLV by replacing the native env gene in a full-length viral genome with that of another gammaretrovirus. Earlier, we developed replication-competent versions of MLV that stably transmit and express transgenes inserted into the 3' untranslated region of the viral genome. In one such tagged MLV expressing green fluorescent protein, we replaced the native env sequence with that of gibbon ape leukemia virus (GALV). Although the GALV Env protein is commonly used to make high-titer pseudotypes of MLV vectors, we found that the env replacement greatly attenuated viral replication. However, extended cultivation of cells exposed to the chimeric virus resulted in selection of mutants exhibiting rapid replication kinetics and different variants arose in different infections. Two of these variants had acquired mutations at or adjacent to the splice acceptor site, and three others had acquired dual mutations within the long terminal repeat. Analysis of the levels of unspliced and spliced viral RNA produced by the parental and adapted viruses showed that the mutations gained by each of these variants functioned to reverse an imbalance in splicing caused by the env gene substitution. Our results reveal the presence of previously unknown cis-acting sequences in MLV that modulate splicing of the viral transcript and demonstrate that tagging of the retroviral genome with an easily assayed transgene can be combined with in vitro evolution as an approach to efficiently generating and screening for replicating mutants of replication-impaired recombinant viruses.
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Affiliation(s)
- Christopher R Logg
- Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
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35
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Paar M, Schwab S, Rosenfellner D, Salmons B, Günzburg WH, Renner M, Portsmouth D. Effects of viral strain, transgene position, and target cell type on replication kinetics, genomic stability, and transgene expression of replication-competent murine leukemia virus-based vectors. J Virol 2007; 81:6973-83. [PMID: 17442710 PMCID: PMC1933297 DOI: 10.1128/jvi.02470-06] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3'-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.
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Affiliation(s)
- Matthias Paar
- Research Institute for Virology and Biomedicine, University of Veterinary Medicine, Vienna, Austria
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36
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Dalba C, Bellier B, Kasahara N, Klatzmann D. Replication-competent Vectors and Empty Virus-like Particles: New Retroviral Vector Designs for Cancer Gene Therapy or Vaccines. Mol Ther 2007; 15:457-66. [PMID: 17245356 DOI: 10.1038/sj.mt.6300054] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Replication-defective vectors based on murine oncoretroviruses were the first gene transfer vectors to be used in successful gene therapies. Despite this achievement, they have two major drawbacks: insufficient efficacy for in vivo gene transfer and insertional mutagenesis. Attempts to overcome these problems have led to two retroviral vector designs of principally opposite character: replication-competent vectors transducing largely intact genomes and genome-free vectors. Replication-competent retroviral vectors have achieved dramatically improved efficacy for in vivo cancer gene therapy and genome-free retroviral vectors expressing different kinds of antigens have proven excellent as immunogens. Current developments aim to improve the safety of the replication-competent vectors and to augment the production efficiency of the genome-free vectors by expression from heterologous viral or non-viral vectors. Together with the continuous advances of classical defective retroviral vectors for ex vivo gene therapy, these developments illustrate that, due to their tremendous design versatility, retroviral vectors remain important vectors for gene therapy applications.
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Affiliation(s)
- Charlotte Dalba
- Biologie et Thérapeutique des Pathologies Immunitaires, CNRS, Université Pierre et Marie Curie-Paris, UMR 7087, Paris, France.
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37
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Kikuchi E, Menendez S, Ozu C, Ohori M, Cordon-Cardo C, Logg CR, Kasahara N, Bochner BH. Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts. Cancer Gene Ther 2007; 14:279-86. [PMID: 17218950 DOI: 10.1038/sj.cgt.7701013] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3+/-16.1, 46.6+/-5.8 and 93.7+/-7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.
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Affiliation(s)
- E Kikuchi
- Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
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Abstract
Since the relationship between angiogenesis and tumor growth was established by Folkman in 1971, scientists have made efforts exploring the possibilities in treating cancer by targeting angiogenesis. Inhibition of angiogenesis growth factors and administration of angiogenesis inhibitors are the basics of anti-angiogenesis therapy. Transfer of anti-angiogenesis genes has received attention recently not only because of the advancement of recombinant vectors, but also because of the localized and sustained expression of therapeutic gene product inside the tumor after gene transfer. This review provides the up-to-date information about the strategies and the vectors studied in the field of anti-angiogenesis cancer gene therapy.
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Affiliation(s)
- Ching-Chiu Liu
- Institute of Molecular Technology for Drug Discovery and Synthesis, Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, China
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Metzl C, Mischek D, Salmons B, Günzburg WH, Renner M, Portsmouth D. Tissue- and tumor-specific targeting of murine leukemia virus-based replication-competent retroviral vectors. J Virol 2006; 80:7070-8. [PMID: 16809312 PMCID: PMC1489065 DOI: 10.1128/jvi.00020-06] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human alpha1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and beta-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.
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Affiliation(s)
- Christian Metzl
- Research Institute for Virology and Biomedicine, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria
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40
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Qiao J, Moreno J, Sanchez-Perez L, Kottke T, Thompson J, Caruso M, Diaz RM, Vile R. VSV-G pseudotyped, MuLV-based, semi-replication-competent retrovirus for cancer treatment. Gene Ther 2006; 13:1457-70. [PMID: 16724095 DOI: 10.1038/sj.gt.3302782] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
Low levels of gene delivery in vivo using replication-defective retroviral vectors have severely limited their application for clinical protocols. To overcome this problem, we describe here a semi-replication-competent retrovirus (s-RCR) in which the gag-pol and envelope (VSV-G, vesicular stomatitis virus G protein) genes were split into two vectors. This system offers potential advantages over both replication-defective vectors, in terms of efficiency of in vivo spread through a tumor, and all-in-one replication-competent vectors in terms of the payload of therapeutic genes that can be carried. We achieved a viral titer of s-RCR viruses approximately 70-fold higher than VSV-G pseudotyped, replication-defective vectors. In addition, s-RCR vectors induced tumor killing by the cytotoxicity of VSV-G during viral spread. Inclusion of the herpes simplex virus thymidine kinase (HSVtk30) gene into vectors significantly improved tumor killing activity followed by ganciclovir (GCV) treatment in vitro under conditions of low-level viral replication. However, at high levels of viral spread, VSV-G-mediated cytotoxicity predominated. Xenografts of human fibrosarcoma HT1080 cells, preinfected by semi-replicative green fluorescent protein vectors (semi-GFP), were completely non-tumorigenic in nude mice. Implantation of cells preinfected by semi-replicative TK30 vectors (semi-TK30) mixed with parental HT1080 cells at a ratio of 1:1 efficiently prevented tumor growth in mice treated by GCV. Direct intratumoral injection of HT1080 tumors growing in nude mice, or B16 murine melanoma in immunocompetent mice, with semi-TK30 viruses significantly prolonged survival. Injection of autologous cells (B16) producing semi-TK30 vector into B16 tumors prolonged survival only in mice treated with GCV but not with phosphate-buffered saline (PBS). In contrast, when xenogeneic cells (293T) producing semi-TK30 vectors were injected into B16 tumors, an optimal survival advantage was obtained in mice treated with PBS rather than GCV. These data indicate that complex interactions exist between direct cytotoxicity of VSV-G and HSVtk expression when placed in the context of additional immune parameters, which combine to determine the efficacy of the therapy. Taken together, our data suggest that s-RCR vectors have some potential advantages for development to deliver genes into tumors for cancer treatment but that a combination of factors will impact on the decision as to whether the s-RCR strategy is worth developing to full clinical trials.
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Affiliation(s)
- J Qiao
- Molecular Medicine Program, Mayo Clinic, Rochester, MN 55902, USA
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Wang W, Tai CK, Kershaw AD, Solly SK, Klatzmann D, Kasahara N, Chen TC. Use of replication-competent retroviral vectors in an immunocompetent intracranial glioma model. Neurosurg Focus 2006; 20:E25. [PMID: 16709031 PMCID: PMC8295718 DOI: 10.3171/foc.2006.20.4.1] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
OBJECT The authors had previously reported on a replication-competent retrovirus (RCR) that has been demonstrated to be stable, capable of effective transduction, and able to prolong survival in an intracranial tumor model in nude mice. The purpose of this study was further investigation of this gene therapy option. METHODS The transduction efficiency of RCR in RG2, an immunocompetent intracranial tumor model, was tested in Fischer 344 rats. The immune response to the RCR vector was expressed by the quantification of CD4, CD8, and CD11/b in tumors. The pharmaceutical efficacy of the suicide gene CD in converting prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) was measured using fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy. Animal survival data were plotted on Kaplan-Meier survival curves. Finally, the biodistribution of RCR was determined using quantitative real-time polymerase chain reaction (RT-PCR) for the detection of retroviral env gene. There was no evidence of viral transduction in normal brain cells. Neither severe inflammation nor immunoreaction occurred after intracranial injection of RCR-green fluorescent protein compared with phosphate-buffered saline (PBS). The 19F-NMR spectroscopy studies demonstrated that RCR-CD was able to convert 5-FC to 5-FU effectively in vitro. The infection of RG2 brain tumors with RCR-CD and their subsequent treatment with 5-FC significantly prolonged survival compared with that in animals with RG2 transduced tumors treated with PBS. In contrast to the nude mouse model, evidence of virus dissemination to the systemic organs after intracranial injection was not detected using RT-PCR. CONCLUSIONS The RCR-mediated suicide gene therapy described in this paper effectively transduced malignant gliomas in an immunocompetent in vivo rodent model, prolonging survival, without evidence of severe intracranial inflammation, and without local transduction of normal brain cells or systemic organs.
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Affiliation(s)
- Weijun Wang
- Department of Neurosurgery, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA
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42
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Tai CK, Wang WJ, Chen TC, Kasahara N. Single-shot, multicycle suicide gene therapy by replication-competent retrovirus vectors achieves long-term survival benefit in experimental glioma. Mol Ther 2006; 12:842-51. [PMID: 16257382 PMCID: PMC8185609 DOI: 10.1016/j.ymthe.2005.03.017] [Citation(s) in RCA: 86] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2005] [Revised: 03/16/2005] [Accepted: 03/17/2005] [Indexed: 10/25/2022] Open
Abstract
Achieving therapeutically efficacious levels of gene transfer in tumors has been a major obstacle for cancer gene therapy using replication-defective virus vectors. Recently, replicating viruses have emerged as attractive tools for cancer therapy, but generally achieve only transitory tumor regression. In contrast to other replicating virus systems, transduction by replication-competent retrovirus (RCR) vectors is efficient, tumor-selective, and persistent. Correlating with its efficient replicative spread, RCR vector expressing the yeast cytosine deaminase suicide gene exhibited remarkably enhanced cytotoxicity in vitro after administration of the prodrug 5-fluorocytosine. In vivo, RCR vectors replicated throughout preestablished primary gliomas without spread to adjacent normal brain, resulting in profound tumor inhibition after a single injection of virus and single cycle of prodrug administration. Furthermore, stable integration of the replicating vector resulted in persistent infection that achieved complete transduction of ectopic glioma foci that had migrated away from the primary tumor site. Thus, efficient and stable integration of suicide genes represents a unique property of the RCR vector that achieved multiple cycles of synchronous cell killing upon repeated prodrug administration, resulting in chronic suppression of tumor growth and prolonged survival.
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Affiliation(s)
- Chien-Kuo Tai
- Department of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Wei Jun Wang
- Department of Neurological Surgery, University of Southern California, Los Angeles, CA 90033, USA
| | - Thomas C. Chen
- Department of Neurological Surgery, University of Southern California, Los Angeles, CA 90033, USA
| | - Noriyuki Kasahara
- Department of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA
- To whom correspondence and reprint request should be addressed at the UCLA Geffen School of Medicine, MRL-1551, 675 Charles E. Young Drive South, Los Angeles, CA 90095, USA. Fax: +1 (310) 825 5204.
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43
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Trajcevski S, Solly SK, Frisén C, Trenado A, Cosset FL, Klatzmann D. Characterization of a semi-replicative gene delivery system allowing propagation of complementary defective retroviral vectors. J Gene Med 2005; 7:276-87. [PMID: 15515136 DOI: 10.1002/jgm.663] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND Recently, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. METHODS The semi-replication-competent retroviral vector system is based on two transcomplementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumors in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. RESULTS In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. CONCLUSIONS This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumors in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in in vitro studies and allows envisioning its further development for cancer gene therapy.
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Affiliation(s)
- Stéphane Trajcevski
- Laboratoire de biologie et thérapeutiques des pathologies immunitaires, CNRS UMR7087, Université Pierre et Marie Curie, Groupe hospitalier Pitié-Salpêtrière, 83 boulevard de l'hôpital, 75651 Paris cedex 13, France
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44
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Sun Y, Finger C, Alvarez-Vallina L, Cichutek K, Buchholz CJ. Chronic gene delivery of interferon-inducible protein 10 through replication-competent retrovirus vectors suppresses tumor growth. Cancer Gene Ther 2005; 12:900-12. [PMID: 15905858 DOI: 10.1038/sj.cgt.7700854] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Sustained maintenance of therapeutic levels of angiostatic proteins in tumor tissues continues to represent a major challenge to antiangiogenesis therapy of cancer. In this study, we tested the hypothesis of utilizing gene transfer via replication-competent retroviral (RCR) vectors for chronic protein delivery. We now show that bioactive human interferon-inducible protein-10 (IP10) can be secreted from a variety of mammalian cells upon transduction with RCR vectors carrying the human IP10 gene. The production of IP10 from RCR-transduced cells could be maintained for at least three months in culture. The level and duration of IP10 expression in vivo was sufficient to inhibit growth of subcutaneous (s.c.) tumors as well as metastatic lesions in mice. This tumor inhibition was correlated to a marked reduction in tumor vascularization and mitotic activity. By conducting immunohistological studies, we have been able to show that IP10 vector-affected tumors evidenced elevated levels of IL-12p35 mRNA, with no sign of changes in the local inflammatory response, however, as determined by macrophage infiltration and the expression of proinflammatory cytokines. We are addressing the feasibility of using RCR vector-based gene therapy as a more convenient alternative tool to chronically deliver antiangiogenic proteins for cancer therapy.
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Affiliation(s)
- Yuansheng Sun
- Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany
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45
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Sun Y, Stevanovic S, Song M, Schwantes A, Kirkpatrick CJ, Schadendorf D, Cichutek K. The kinase insert domain-containing receptor is an angiogenesis-associated antigen recognized by human cytotoxic T lymphocytes. Blood 2005; 107:1476-83. [PMID: 16234362 DOI: 10.1182/blood-2005-05-1912] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mononuclear cells (PBMCs) of 3 HLA-A*0201 donors. The decapeptide KDR288-297 was efficient in sensitizing target cells for recognition by a CTL clone at a concentration of 10 nM. More important, KDR288-297-specific CTLs lysed target cells transfected with HLA-A2/KDR cDNAs and a range of HLA-matched KDR+ angiogenic endothelial cells (aECs) and also recognized CD34+ endothelial progenitor cells. The specificity of CTLs was further confirmed by tetramer assay and cold-target inhibition assay. In addition, ex vivo exposure of aECs to the inflammatory cytokines enhanced CTL reactivity, which is in keeping with up-regulated KDR and HLA class 1 expression. In Matrigel assays, recognition of aECs by specific CTLs triggered an antivascular effect. These findings provide the first proof of the antigenic property of KDR protein and may be useful for devising new immunotherapeutic approaches to human cancers.
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Affiliation(s)
- Yuansheng Sun
- Division of Medical Biotechnology, Paul-Ehrlich-Institute, Paul-Ehrlich-Str 51-59, D-63225 Langen, Germany.
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46
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Hartl I, Schneider RM, Sun Y, Medvedovska J, Chadwick MP, Russell SJ, Cichutek K, Buchholz CJ. Library-based selection of retroviruses selectively spreading through matrix metalloprotease-positive cells. Gene Ther 2005; 12:918-26. [PMID: 15716977 DOI: 10.1038/sj.gt.3302467] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Viruses conditionally replicating in cancer cells form an attractive novel class of antitumoral agents. To engineer such viruses infectivity can be coupled with proteolytic activity of the target cell by modifying the envelope (Env) protein of murine leukaemia virus (MLV) with blocking domains that prevent cell entry unless they are cleaved off by tumour-associated proteases like the matrix metalloproteases (MMP). Here we show that MLV variants selectively spreading through MMP-positive cells can be evolved from virus libraries, in which a standard MMP-2 substrate peptide connecting the blocking domain CD40L with the Env protein was diversified. Passaging the virus library on human fibrosarcoma or glioma cell lines resulted in the selection of about 10 virus clones, of which the three most frequent ones were shown to become activated by MMPs and to be replication competent on MMP-positive cells only. On these cells, the selected linker peptides improved the spreading by several orders of magnitude in vitro, as well as in tumour xenografts in vivo, approaching the kinetic of the unmodified wild-type virus. The data suggest that retroviral protease substrate libraries form a potent tool for the engineering of viruses conditionally replicating in a given cancer cell type of interest.
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Affiliation(s)
- I Hartl
- Medizinische Biotechnologie, Paul-Ehrlich-Institut, Langen, Germany
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Finger C, Sun Y, Sanz L, Alvarez-Vallina L, Buchholz CJ, Cichutek K. Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells. Cancer Gene Ther 2005; 12:464-74. [PMID: 15692609 DOI: 10.1038/sj.cgt.7700805] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3-4 mug/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.
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Affiliation(s)
- Carsten Finger
- Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany
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Sliva K, Erlwein O, Bittner A, Schnierle BS. Murine leukemia virus (MLV) replication monitored with fluorescent proteins. Virol J 2004; 1:14. [PMID: 15610559 PMCID: PMC544597 DOI: 10.1186/1743-422x-1-14] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2004] [Accepted: 12/20/2004] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Cancer gene therapy will benefit from vectors that are able to replicate in tumor tissue and cause a bystander effect. Replication-competent murine leukemia virus (MLV) has been described to have potential as cancer therapeutics, however, MLV infection does not cause a cytopathic effect in the infected cell and viral replication can only be studied by immunostaining or measurement of reverse transcriptase activity. RESULTS We inserted the coding sequences for green fluorescent protein (GFP) into the proline-rich region (PRR) of the ecotropic envelope protein (Env) and were able to fluorescently label MLV. This allowed us to directly monitor viral replication and attachment to target cells by flow cytometry. We used this method to study viral replication of recombinant MLVs and split viral genomes, which were generated by replacement of the MLV env gene with the red fluorescent protein (RFP) and separately cloning GFP-Env into a retroviral vector. Co-transfection of both plasmids into target cells resulted in the generation of semi-replicative vectors, and the two color labeling allowed to determine the distribution of the individual genomes in the target cells and was indicative for the occurrence of recombination events. CONCLUSIONS Fluorescently labeled MLVs are excellent tools for the study of factors that influence viral replication and can be used to optimize MLV-based replication-competent viruses or vectors for gene therapy.
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Affiliation(s)
- Katja Sliva
- Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt/Main, Germany
| | - Otto Erlwein
- Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt/Main, Germany
| | - Alexandra Bittner
- Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt/Main, Germany
| | - Barbara S Schnierle
- Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt/Main, Germany
- Paul-Ehrlich-Institute, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany
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Duch M, Carrasco ML, Jespersen T, Hansen BD, Pedersen FS. Transgene stability for three replication-competent murine leukemia virus vectors. Gene 2004; 329:61-9. [PMID: 15033529 DOI: 10.1016/j.gene.2003.12.032] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2003] [Revised: 12/01/2003] [Accepted: 12/23/2003] [Indexed: 10/26/2022]
Abstract
Retroviral vectors that are able to sustain multiple rounds of replication may find many applications. However, one critical feature of such vectors is the ability to maintain an intact transgene cassette during repeated rounds of replication. We here report on the stability of a translational cassette consisting of an internal ribosome entry site followed by the enhanced green fluorescent protein coding sequence inserted in different configurations into murine leukemia virus genomes. In two of the constructs, the insert was located in the upstream part of the U3 region while in the third construct it was inserted in the 3' untranslated region of the viral genome. Furthermore, in two of the constructs, the translational cassette was flanked by a direct repeat, while no such structure flanked the third construct. Our results show that deletion of the heterologous translational cassette is observed for all constructs upon extended cell culture and that the number of replication rounds before revertants are detected can be postponed by decreasing the length of the repeat flanking the translational cassette.
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Affiliation(s)
- Mogens Duch
- Department of Molecular Biology, University of Aarhus, DK8000, Aarhus, Denmark
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50
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Tai CK, Logg CR, Park JM, Anderson WF, Press MF, Kasahara N. Antibody-mediated targeting of replication-competent retroviral vectors. Hum Gene Ther 2003; 14:789-802. [PMID: 12804141 DOI: 10.1089/104303403765255174] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.
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MESH Headings
- Animals
- Antibodies, Monoclonal/immunology
- Antibodies, Monoclonal/metabolism
- Breast Neoplasms/metabolism
- Cell Line
- Cell Line, Tumor
- DNA, Viral/analysis
- Female
- Flow Cytometry
- Genetic Vectors/metabolism
- Green Fluorescent Proteins
- Humans
- Kinetics
- Leukemia Virus, Murine/genetics
- Leukemia Virus, Murine/physiology
- Luminescent Proteins/analysis
- Luminescent Proteins/genetics
- Mice
- NIH 3T3 Cells
- Plasmids
- Protein Structure, Tertiary
- Receptor, ErbB-2/antagonists & inhibitors
- Receptor, ErbB-2/genetics
- Receptor, ErbB-2/immunology
- Recombinant Fusion Proteins/genetics
- Staphylococcal Protein A/chemistry
- Transduction, Genetic
- Viral Envelope Proteins/genetics
- Virion/chemistry
- Virus Replication
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Affiliation(s)
- Chien-Kuo Tai
- Institute for Genetic Medicine, Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA
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