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Kim YI, Chung JW. Selective or targeted gene/drug delivery for liver tumors: advantages and current status of local delivery. Expert Rev Gastroenterol Hepatol 2008; 2:791-802. [PMID: 19090739 DOI: 10.1586/17474124.2.6.791] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
There are various disorders involving the liver. They include metabolic diseases, hepatitis, liver cirrhosis and cancer, the latter of which may be the most serious. Delivery of therapeutic genes or drugs should be targeted to either one of the following cells in the liver: hepatocytes, Kupffer cells and tumor endothelial cells, or to the tumor cells themselves. To maximize the therapeutic effect and minimize systemic toxicity or nontarget injuries, the sufficient amount or dose of genes or drugs should be specifically delivered to a target, with minimal exposure in their active forms to nontarget cells. There are diverse strategies to improve selective delivery or targeting efficiency. In this article, we present potential new therapeutic strategies and clinical developments for liver cancer, with a focus on the progress in the localized delivery of therapeutic agents using image-guided procedures.
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Affiliation(s)
- Young Il Kim
- Division of Interventional Radiology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5642, USA.
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2
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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: 27] [Impact Index Per Article: 1.5] [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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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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4
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Abstract
Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is often diagnosed at an advanced stage when most potentially curative therapies such as resection, transplantation or percutaneous and transarterial interventions are of limited efficacy. The fact that HCC is resistant to conventional chemotherapy, and is rarely amenable to radiotherapy, leaves this disease with no effective therapeutic options and a very poor prognosis. Therefore, the development of more effective therapeutic tools and strategies is much needed. HCCs are phenotypically and genetically heterogeneous tumors that commonly emerge on a background of chronic liver disease. However, in spite of this heterogeneity recent insights into the biology of HCC suggest that certain signaling pathways and molecular alterations are likely to play essential roles in HCC development by promoting cell growth and survival. The identification of such mechanisms may open new avenues for the prevention and treatment of HCC through the development of targeted therapies. In this review we will describe the new potential therapeutic targets and clinical developments that have emerged from progress in the knowledge of HCC biology, In addition, recent advances in gene therapy and combined cell and gene therapy, together with new radiotherapy techniques and immunotherapy in patients with HCC will be discussed.
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Affiliation(s)
- M A Avila
- Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain
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Prieto J, Qian C, Hernandez-Alcoceba R, Gonzalez-Aseguinolaza G, Mazzolini G, Sangro B, Kramer MG. Gene therapy of liver diseases. Expert Opin Biol Ther 2005; 4:1073-91. [PMID: 15268675 DOI: 10.1517/14712598.4.7.1073] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Many liver diseases lack satisfactory treatment and alternative therapeutic options are urgently needed. Gene therapy is a new mode of treatment for both inherited and acquired diseases, based on the transfer of genetic material to the tissues. Genes are incorporated into appropriate vectors in order to facilitate their entrance and function inside the target cells. Gene therapy vectors can be constructed on the basis of viral or non-viral molecular structures. Viral vectors are frequently used, due to their higher transduction efficiency. Both the type of vector and the expression cassette determine the duration, specificity and inducibility of gene expression. A considerable number of preclinical studies indicate that a great variety of liver diseases, including inherited metabolic defects, chronic viral hepatitis, liver cirrhosis and primary and metastatic liver cancer, are amenable to gene therapy. Gene transfer to the liver can also be used to convert this organ into a factory of secreted proteins needed to treat conditions that do not affect the liver itself. Clinical trials of gene therapy for the treatment of inherited diseases and liver cancer have been initiated but human gene therapy is still in its infancy. Recent progress in vector technology and imaging techniques, allowing in vivo assessment of gene expression, will facilitate the development of clinical applications of gene therapy.
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Affiliation(s)
- Jesus Prieto
- Department of Internal Medicine, Clinica Universitaria de Navarra, Avda. Pio XII 36, 31008 Pamplona, Spain
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Shinozaki K, Ebert O, Woo SLC. Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV. Hepatology 2005; 41:196-203. [PMID: 15619242 DOI: 10.1002/hep.20536] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus with intrinsic oncolytic specificity due to attenuated antiviral responses in many tumors. We report that repeated hepatic arterial infusion of recombinant syncytia-forming VSV vector in advanced multifocal hepatocellular carcinoma (HCC)-bearing rats at a 10-fold reduced vector dose resulted in sustained tumor-selective virus replication until the onset of high-titer neutralizing antibodies in blood. No significant elevations in serum transaminases and liver pathology were noted, indicating a lack of hepatotoxicity. Substantially improved tumor response was achieved with completely necrotic tumor nodules surrounded by mononuclear phagocytic cells, followed by fibrosis and calcification of the lesions, angiogenesis, and regeneration of normal hepatic parenchyma. Survival of tumor-bearing rats treated with repeated vector infusions was not only significantly improved over that of animals after a single injection at 10 times the vector dose (P = .001), but 18% of animals in the former treatment group also achieved long-term and tumor-free survival compared with 0% of animals in the latter treatment group. In conclusion, this treatment regimen will be very useful in the future development of VSV-mediated virotherapy as a novel therapeutic modality for patients with advanced HCC.
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Affiliation(s)
- Katsunori Shinozaki
- Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029-6574, USA
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Guan XX, Chen LB, Ding GX, De W, Zhang AH. Transfection of p27 kip1 enhances radiosensitivity induced by 60Co γ-irradiation in hepatocellular carcinoma HepG 2 cell line. World J Gastroenterol 2004; 10:3103-6. [PMID: 15457552 PMCID: PMC4611250 DOI: 10.3748/wjg.v10.i21.3103] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To study the cell cycle alterations of human hepatoma cell line HepG2 in vitro after 60Co γ-irradiation and further to examine the mechanisms underlying the enhancement of radiosensitivity to γ-irradiation in HepG2 transiently transfected with wild type p27kip1.
METHODS: The proliferation of HepG2 cells was evaluated with MTT assay, and the cell cycle profile and apoptosis were assessed by cell morphology, DNA fragmentation analysis and flow cytometry. HepG2 cells were transfected with p27kip1 wild type by using Lipofectamine (LF2000), and the expression and subcellular localization of p27kip1 in HepG2 were detected by immunocytochemistry.
RESULTS: 60Co γ-irradiation inhibited the growth of HepG2 cells in a dose-dependent manner. Apoptosis of HepG2 cells was induced 48 h after γ ray exposure. Furthermore research was carried out to induce exogenous expression of p27kip1 in HepG2. The expression of p27kip1 induced G0/G1 phase arrest in HepG2 cells. The overexpression of p27kip1 enhanced 60Co γ-irradiation-induced radiosensitivity in HepG2 cells.
CONCLUSION: Overexpression of p27kip1 is a rational approach to improve conventional radiotherapy outcomes, which may be a possible strategy for human hepatoma therapy.
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Affiliation(s)
- Xiao-Xiang Guan
- Department of Oncology, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210002, Jiangsu Province, China
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Li CJ, Wang L, Tu XH, Song JX. Mechanism of thermochemotherapy with 5-fluorocytosine on human colon cancer cell line SW480 transfected cytosine deaminase gene. Shijie Huaren Xiaohua Zazhi 2004; 12:2307-2311. [DOI: 10.11569/wcjd.v12.i10.2307] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the mechanism of thermochemotherapy with 5-fluorocytosine (5-FC) on human colon cancer cell line SW480 transfected carcinoembryonic antigen (CEA) tissue-specific cytosine deaminase (CD) in vitro.
METHODS: Recombinant retroviral vector G1CEACDNa, in which the CD gene was controlled under the CEA promoter, was introduced through liposome technique to human colorectal carcinoma cell line SW480, and then the cells were selectively cultured in G418. The proliferated colonies were treated with the combined therapy of 5-FC and hyperthermia at a temperature of 43 ℃ for 30 min, 3 times. RT-PCR was performed to detect the expression of CD gene in target cells after being heated. The cell survival rate was detected by MTT method. The ultrastructures of cells were observed by electron microscopy and apoptosis was verified by flow cytometry.
RESULTS: The expression of CD genes in target cells was detected after being heated. After transfection, SW480-CEACD cells were more sensitive than their parental cells (P <0.01, t = 5.620, n = 9) to 5-FC, the killing effect of hyperthermia on SW480 cells was observed (P <0.05, t = 2.999, n = 9). Furthermore, after being treated with thermoche-motherapy of 5-FC at a temperature of 43 ℃ for 30 min, the killing effect on SW480-CEACD cells was more significant than that on SW480 cells (P <0.01, t = 4.356, n = 9). Treatment with the combination of 5-FC and hyperthermia displayed a higher anti-tumor effect than that with 5-FC alone on SW480-CEACD cells in vitro (P <0.05, t = 2.376, n = 9). Apoptotic bodies in the field of electron microscope were observed. G1 blockage was confirmed and the increased rate of apoptosis cells was verified after hyperthermia with 5-FC by flow cytometry.
CONCLUSION: The combination of 5-FC and hyperthermia will result in G1 blockage of human colorectal carcinoma cell lines SW480 transfected with the CEA tissue-specific CD genes, which will improve the outcome of the anti-tumor effect on that cell line.
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Abstract
Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, responsible for an estimated one million deaths annually. It has a poor prognosis due to its rapid infiltrating growth and complicating liver cirrhosis. Surgical resection, liver transplantation and cryosurgery are considered the best curative options, achieving a high rate of complete response, especially in patients with small HCC and good residual liver function. In nonsurgery, regional interventional therapies have led to a major breakthrough in the management of unresectable HCC, which include transarterial chemoembolization (TACE), percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), microwave coagulation therapy (MCT), laser-induced thermotherapy (LITT), etc. As a result of the technical development of locoregional approaches for HCC during the recent decades, the range of combined interventional therapies has been continuously extended. Most combined multimodal interventional therapies reveal their enormous advantages as compared with any single therapeutic regimen alone, and play more important roles in treating unresectable HCC.
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Affiliation(s)
- Jun Qian
- Department of Radiology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China.
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10
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Li CJ, Ma QJ, Lai DN, Lu JG, Wang XJ, Wang Q, Pan BR, Wu YZ, Li JM. Killing effect of CD/5-FC system on human colon cancer cell lines SW 480 and LoVo. Shijie Huaren Xiaohua Zazhi 2003; 11:535-539. [DOI: 10.11569/wcjd.v11.i5.535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the killing effect of carcinoembryonic antigen (CEA) and tissue-specific cytosine deaminase (CD)/5-fluorocytosine (5-FC) system on human colorectal carcinoma cell lines LoVo and SW480 in vitro.
METHODS Recombinant retroviral vector G1CEACDNa was constructed, in which the CD gene was controlled under the CEA promoter, and retroviral vector pCD2 were introduced through liposome technique respectively to the human colorectal carcinoma cell lines LoVo and SW480. Expression of CEA was high and low in both the cell lines respectively. The cells were selectively cultured in G418. The proliferative colonies were treated with 5-FC.
RESULTS After the transfection, LoVo-CEACD cells and LoVo-CD cells were more sensitive to 5-FC than their parental cells (P<0.01, t = 5.688, n = 9; P<0.01, t = 3.136, n = 9), and SW480-CEACD cells and SW480-CD cells were more sensitive than their parental cells as well (P<0.01, t = 3.437, n = 9; P <0.01, t = 3.516, n = 9). Furthermore, the LoVo-CEACD cells were more sensitive to 5-FC than the LoVo-CD cells (P <0.05, t =2.183, n =9) while the SW480-CEACD cells were less sensitive than SW480-CD cells.TheLoVo-CEACD cells displayed a higher anti-tumor effect than SW480-CEACD cells in vitro. The bystander effect in all cells transfected with CD gene were observed in this study.
CONCLUSION The CEA tissue-specific CD/5-FC system displays an obvious targeting anti-tumor effect on human colorectal carcinoma cell lines LoVo and SW480, but the killing effect on the LoVo-CEACD cells is higher than that on the SW480-CEACD cells in vitro.
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Affiliation(s)
- Cheng-Jin Li
- Jian-Guo Lu, Xiao-Jun Wang, Qing Wang, Yong-Zhong Wu, Jin-Mao Li, Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
| | - Qing-Jiu Ma
- Jian-Guo Lu, Xiao-Jun Wang, Qing Wang, Yong-Zhong Wu, Jin-Mao Li, Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
| | - Da-Nian Lai
- Jian-Guo Lu, Xiao-Jun Wang, Qing Wang, Yong-Zhong Wu, Jin-Mao Li, Department of General Surgery, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shaanxi Province, China
| | | | | | | | - Bo-Rong Pan
- Department of Oncolgy, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China
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11
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Abstract
Cancer gene therapy has been one of the most exciting areas of therapeutic research in the past decade. In this review, we discuss strategies to restrict transcription of transgenes to tumour cells. A range of promoters which are tissue-specific, tumour-specific, or inducible by exogenous agents are presented. Transcriptional targeting should prevent normal tissue toxicities associated with other cancer treatments, such as radiation and chemotherapy. In addition, the specificity of these strategies should provide improved targeting of metastatic tumours following systemic gene delivery. Rapid progress in the ability to specifically control transgenes will allow systemic gene delivery for cancer therapy to become a real possibility in the near future.
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Affiliation(s)
- Tracy Robson
- School of Biomedical Sciences, University of Ulster, Newtownabbey, Co. Antrim, BT37 0QB, Northern Ireland, UK
| | - David G. Hirst
- School of Biomedical Sciences, University of Ulster, Newtownabbey, Co. Antrim, BT37 0QB, Northern Ireland, UK
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Ueda K, Iwahashi M, Nakamori M, Nakamura M, Matsuura I, Ojima T, Yamaue H. Improvement of carcinoembryonic antigen-specific prodrug gene therapy for experimental colon cancer. Surgery 2003; 133:309-17. [PMID: 12660644 DOI: 10.1067/msy.2003.73] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
BACKGROUND Improvement of tumor-specific gene expression is very important for achieving successful effects in prodrug gene therapy for advanced cancer with metastatic lesions. We used the Cre/loxP system for enhancing carcinoembryonic antigen (CEA)-specific prodrug gene therapy of cytosine (CD)/5-fluorocytosine (5-FC) for the treatment of a colon cancer model accompanied with liver metastases. METHODS Orthotopic colon cancer models were developed. Seven days later, adenovirus vector (3 x 10(9) pfu)was injected into the abdominal cavity, and 5-FC was administered for the next 10 days. RESULTS In these models, the double administration of AxCEANCre expressing Cre recombinase under the control of the CEA promoter, and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre-mediated switching system, completely inhibited liver metastases, and significantly reduced primary tumor volume compared to the administration of Mock or AxCEACD (P <.001). The survival periods of the mice treated with AxCEANCre and AxCALNLCD were longer than mice treated with Mock or AxCEACD, and longer than the mice treated with AxCACD (P <.05). CONCLUSIONS The enhanced CEA-specific prodrug gene therapy using the Cre/loxP system was useful for the treatment of advanced colon cancer with liver metastases, implicating clinical application.
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Affiliation(s)
- Kentaro Ueda
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
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Abstract
Since advanced liver cancer lacks effective therapy in most cases, a considerable interest has been drawn towards gene therapy. Natural or chimerical genes can be transferred to the tumour itself, the non-tumoral liver, or even distant tissues using a variety of vectors administered by intratumoral or intravascular routes. The desired selectivity in gene expression can be achieved by increasing the specificity of gene delivery or by controlling gene expression with tumour-specific promoters, such as alpha-fetoprotein or carcinoembryonic antigen. There are two main approaches to gene therapy of liver cancer aiming at killing directly malignant cells or at improving the host's defensive systems, respectively. The former include replacing the lost function of tumour suppressor genes, inhibiting the action of activated oncogenes, sensitising tumour cells to prodrugs, or infecting the tumoral tissue with viruses that replicate selectively in cancer cells. Host defences can be improved by stimulating the antitumoral immune response, or by interfering with tumour vessel formation. Progress in gene therapy of liver cancer depends very much on information collected from well-designed clinical trials. This information includes knowledge of whether an efficient gene transfer has been achieved and what is the duration and magnitude of gene expression in the transduced tissues. Hopefully, magnetic resonance or positron emission tomography (PET) may turn out to be reliable procedures for tracing transgene expression in humans. Pre-clinical evidence and early clinical trials strongly suggest that there is a place for gene therapy of liver malignancies.
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Affiliation(s)
- Bruno Sangro
- Division of Gene Therapy, Department of Internal Medicine, Clínica Universitaria de Navarra, AP 4209, 31080, Pamplona, Spain.
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14
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Kuiper M, Sanches RM, Walford JA, Slater NKH. Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography. Biotechnol Bioeng 2002; 80:445-53. [PMID: 12325153 DOI: 10.1002/bit.10388] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.
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Affiliation(s)
- Marcel Kuiper
- Department of Chemical Engineering, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom
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15
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Reid TR, Sze DY. Developments in medical oncology and their implications for interventional radiology. Tech Vasc Interv Radiol 2002; 5:177-81. [PMID: 12524649 DOI: 10.1053/tvir.2002.36421] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Although surgery and radiation therapy have always been spatially targeted, chemotherapy as administered by oncologists has remained steadfastly committed to non-targeted systemic delivery. Decades of pharmaceutical research have yielded agents appropriate for intravenous use, but countless potentially efficacious agents have been discarded because of pharmacokinetic and toxicity profiles unsuitable for systemic delivery. With the emerging technology of biological agents comes a new series of challenges. These agents tend to be larger, less long-lived, and antigenic when compared with the agents of the past. Meanwhile, interventional radiologists have shown that targeted methods of delivery can have substantial impact on the efficacy and toxicity of agents. Laboratory scientists have developed new bullets; we interventional radiologists have developed new guns. It is time we take advantage of potential synergies.
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Affiliation(s)
- Tony R Reid
- Palo Alto Veterans Administration Medical Center and Stanford University Medical Center, Palo Alto, CA 94305-5642, USA
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16
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Harrington K, Alvarez-Vallina L, Crittenden M, Gough M, Chong H, Diaz RM, Vassaux G, Lemoine N, Vile R. Cells as vehicles for cancer gene therapy: the missing link between targeted vectors and systemic delivery? Hum Gene Ther 2002; 13:1263-80. [PMID: 12162810 DOI: 10.1089/104303402760128504] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Systemic administration of currently manufactured viral stocks has not so far achieved sufficient circulating titers to allow therapeutic targeting of metastatic disease. This is due to low initial viral titers, immune inactivation, nonspecific adhesion, and loss of particles. One way to exploit the elegant molecular manipulations that have been made to increase vector targeting is to protect these vectors until they reach the local sites of tumor growth. Various cell types home preferentially to tumors and can be loaded with the constructs required to produce targeted vectors. Here we discuss the potential of using such cell carriers to chaperone precious vectors directly to the tumors. The vectors can incorporate mechanisms to achieve tumor site-inducible expression, along with tumor cell-specific expression of the therapeutic gene and/or replicating viral genomes that would be released at the tumor. In this way, the great advances that have so far been made with the engineering of vector tropisms might be genuinely exploited and converted into clinical benefit.
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Affiliation(s)
- Kevin Harrington
- Cancer Research Campaign, Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, UK
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Mayer-Kuckuk P, Banerjee D, Kemeny N, Fong Y, Bertino JR. Molecular therapies for colorectal cancer metastatic to the liver. Mol Ther 2002; 5:492-500. [PMID: 11991739 DOI: 10.1006/mthe.2002.0596] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Colorectal cancers are the fourth most commonly diagnosed cancers and will account for over 56,000 deaths in the United States in 2002. A majority of patients with advanced colorectal cancer develop liver metastases during the course of their disease. Treatment of colorectal cancer metastatic to the liver by surgery or chemotherapy is limited and most patients succumb to their disease. Therefore, a broad spectrum of novel treatments, including innovative molecular therapies such as gene and immunotherapy or replication-competent viral therapy, is under preclinical investigation and several clinical trials are in progress. Here we review molecular therapies for colorectal cancer metastatic to the liver.
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Affiliation(s)
- Philipp Mayer-Kuckuk
- Program of Molecular Pharmacology and Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA
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18
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Shen LZ, Wu WX, Xu DH, Zheng ZC, Liu XY, Ding Q, Hua YB, Yao K. Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene. World J Gastroenterol 2002; 8:270-5. [PMID: 11925606 PMCID: PMC4658365 DOI: 10.3748/wjg.v8.i2.270] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated.
METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC50) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean ± SD of the mean. Statistical analysis was performed using ANOVA test.
RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 × 1014 pfu/L-1 after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC50 values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were > 15000, 216.5 ± 38.1 and 128.8 ± 25.4 μmol•L⁻¹, P < 0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the M.O.I of adenoviruses were enhanced (The value of IC50 of 5-FC was reduced to 27.9 ± 4.2 μmol•L-1 in 1000 M.O.I AdCEACD infected Lovo cells and 24.8 ± 7.1 μmol•L⁻¹ in 100 M.O.I AdCMVCD infected Lovo cells, P < 0.05, P < 0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC50 of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was > 15000 and 214.5 ± 31.3 μmol•L⁻¹, P < 0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30% when the proportion of transfected cells was only 10 percent.
CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.
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Affiliation(s)
- Li-Zong Shen
- Department of General Surgery, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, 210029, Jiangsu Province, China
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Abstract
For most patients with advanced or multifocal hepatocellular carcinoma (HCC) or with metastatic malignant liver disease treatment options are limited, resulting in a poor prognosis. Novel therapeutic strategies such as gene therapy are therefore urgently required. Gene therapeutic approaches use gene delivery systems (vectors) to introduce DNA constructs as therapeutic agents into living cells. Antitumour strategies include the reintroduction of tumour suppressor genes into tumour cells, the expression of foreign enzymes to render tumours susceptible to treatment with chemotherapeutic agents and the enhancement of tumour immunogenicity by expressing immunomodulatory genes or by genetic vaccination with tumour antigens. Furthermore, gene therapy may be also used for anti-angiogenesis to reduce tumour growth and metastatic potential. Other novel approaches aim at the development of genetically altered replication competent viruses, which selectively replicate in tumour cells inducing cell lysis. Although most clinical trials of antitumour gene therapy so far have failed to induce strong therapeutic effects, further improvement of antitumour gene therapy may finally result in potent clinical treatment options for patients with malignant liver tumours.
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Affiliation(s)
- Leonhard Mohr
- Department of Medicine II, University Hospital Freiburg, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
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