Combined therapy of suicide gene and cytokine gene
1Changan University, 2Department of Ultrasonic Diagnosis, Outpatient Clinic, Henan Provincial Military Command, Zhengzhou 450003, China, 3Department of Biomedical Engineering, School of Life Science & Technology, Xian Jiaotong University, Xian 710049, China, 4Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xian 710033, China
【Keywords】 suicide gene; gene therapy; neoplasms
INTRODUCTION
The transfer of suicide genes into tumor cells is currently being used in a variety of clinical gene therapy trials for the treatment of cancer, and suicide gene therapy is the transduction of a gene that transforms a nontoxic into a toxic substance[1]. Herpes simplex virus thymidine kinase gene (HSVtk) + ganciclovir (GCV), HSVtk phosphorylates GCV inhibit the synthesis of DNA, and this combination produces a bystander effect and has been demonstrated to be of certain effect for the treatment of tumors[2-6]. But the systemic antitumoral immune response, however, is not developed fully to eliminate tumor. The unpredictability of the bystander effect and the difficulties in transduction have kept cure rates low. The use of tissuespecific vectors to deliver the genes and the combination of the strategy with cytokines may improve the efficacy of the approach in time. Gene therapy of suicide gene combined with cytokine gene has become a new insight for the treatment of malignancies because of their complementary effect. Some data[7-12] indicated that cytokine has the ability to improve the efficiency of HSVtk/GCV therapy in the treatment of cancer and suggested that the targeting of different pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.
SUICIDE GENE THERAPY THEORY
HSVtk + ganciclovir (GCV), or HSVtk phosphorylates GCV can inhibit the synthesis of DNA[2-6]. HSVtk itself also causes a bystander effect. A bystander effect is described when nontransduced or genetically unmodified cells are killed during the death of genetically modified tumor cells transduced with a suicide gene. The bystander effect greatly enhances the efficacy of HSVtk/GCV gene therapy for cancer. Despite promising results in vitro and in vivo, the antitumor effect in clinical trials remains poor, due to very low transfection efficiency. However, high percentages of transfected cells are not mandatory for complete eradication of a tumor in vivo. Transfected tumor cells appear to be capable of inducing the death of neighboring untransfected cells. A substantial bystander effect can overcome the limitations of low transfection efficiency and result in an enhanced and possibly clinically worthwhile antitumor effect in patients.
GENE TRANSFER TECHNOLOGY
Several experimental approaches have been tested for suicide gene delivery into tumor cells, including viral and nonviral vectors. The gene delivery technology is developing rapidly and there have been specific developments that could be translated into genebased therapies for diseases. Gene transfer may then be facilitated by a number of methods, including viruses, liposome, calcium phosphate coprecipitation, particle bombardment, naked DNA injection, and electroporation. Currently alternative viral vectors with potential advantages over retroviruses in specific applications are under development. Adenoviruses, which can infect nondividing cells, and can be concentrated to high titers, are comparatively highly efficient vectors. Adenoassociated viruses are ubiquitous and nonpathogenic in humans and can also infect nonreplicating cells, but, like retroviruses and adenoviruses, they are limited in size of the foreign gene that can be inserted. This problem may be overcome by the use of herpes simplex group viruses and possibly even vectors based on hepatitis B virus, which has potential additional advantages of hepatotropism and an ability to integrate with host genome in vivo[7].
COMBINED THERAPY OF SUICIDE GENE AND CYTOKINE GENE FOR CANCER
Gene therapy may offer a new tool for the treatment of renal cell carcinoma (RCC). In 2001, Cao et al[13] had tested a combination of cytotoxic and antiangiogenic gene therapy for wildtype orthotopic human RCC xenografts in nude mice using intratumoral adenovirusmediated HSVtk and endostatin (ES) gene therapy. It is concluded that the inhibition of angiogenesis by using ES gene transfer together with the cytotoxic HSVtk gene therapy results in a significantly improved treatment effect in RCC compared to the single gene treatments. Guan et al[9] explored that HSVtk and GMCSF genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and demonstrated the characteristics of the transduced cells. The IRES sequence can be used to construct retroviral vectors to facilitate cotransfection of two genes. SKOV3/TKGM cells can simultaneously express the HSVtk and GMCSF genes with biological activities, which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.
In 2002, Hall et al[10] tried to enhance the NK population induced by HSVtk gene transduction and ganciclovir (GCV) and adenovirusmediated (Ad) expression of IL12 were added to Ad.HSVtk + GCV as combination gene therapy. This approach results in improved local and systemic growth suppression in a metastatic model of mouse prostate cancer (RM1). The combination of Ad.HSVtk/GCV + Ad.mIL12 results in enhanced local growth control via apoptosis due to tumor cell expression of Fas and FasL and improved antimetastatic activity secondary to a strong NK response. Suicide gene therapy in combination with prodrugs represents an attractive approach to the treatment of cancer. Unfortunately this approach is limited by the difficulty in targeting all tumor cells, especially those at the distant metastases associated with the most complex tumors. For this reason, attempts to stimulate global antitumor immune responses at the sites of effective suicide gene/prodrugmediated tumor cell destruction are appealing.
At present, based on their clinical experience with combined gene therapy of glioblastoma, Barzon et al[12] developed a retroviral vector expressing two therapeutic genes and evaluated its efficiency in vitro and in vivo. These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas. Janouskova et al[8] tested the effects of combined transduction of a suicide gene and genes coding for various immunostimulatory factors on the oncogenicity and immunogenicity of TC1 cells, and several bicistronic recombinant adenoassociated viruses were constructed. Each of these constructs carried, and in infected cells expressed, the HSVtk and the gene of one of the following immunostimulatory factors. The tumor resistance rates were related not only to the immunostimulatory gene used for the transduction, but also to the GCV treatment. The best protection was recorded in mice preinoculated with TC1 cells transduced with either B7.1 or MCP1expressing rAAV and not given GCV.
HSVtk/gancyclovir (GCV) therapy has the ability to inhibit tumor formation in animal models but the results of clinical trials have been disappointing. In 2003, to improve the performance of tk/GCV therapy, Yamaguchi et al[11] tried combination therapy designed to enhance its cytotoxic effects by introducing genes that induce apoptosis of the tumor cells through different pathways. They concentrated their efforts on the use of Bim, a BH3only member of death activators in the Bcl2 superfamily, because Bim is not involved in the pathways through which HSVtk/GCV therapy induces apoptosis in malignant glioma cells. The results indicated that BimS had the ability to improve the efficiency of HSVtk/GCV therapy in the treatment of malignant glioma and suggested that the targeting of different proapoptotic pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.
PROSPECT IN FUTURE
Because of adaptor sequenceselecting complexity, previous research had been based on enzyme restriction site ligation, which is convenient to choose vector, to obtain intended fusion gene and also convenient to finish enzyme cleavage identification of fusion gene. But the expression activity of newly established fusion gene needs further experimental research. The recent introduction of computer technology and advances of protein engineer make it easy for researchers to present deliberate design and eliminate other possible disadvantages. Two different genes[13], such as human interleukin 2 (hIL2) and HSVtk gene, were tested on this modified vector for gene transfer and expression. Further more, derivatives space structure of fusion protein is sustained in its natural state and in its best biological activity. In a word, studies show that this area of research has a bright future and it is an effective means for searching novel therapy[14].
【REFERENCES】
[1] Engelmann C, Heslan JM, Fabre M, et al. Importance, mechanisms and limitations of the distant bystander effect in cancer gene therapy of experimental liver tumors [J]. Cancer Lett, 2002; 8(179):59-69.
[2] Burrows FJ, Gore M, Smiley WR, et al. Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells [J]. Cancer Gene Ther, 2002;9:87-95.
[3] Hayashi K, Hayashi T, Sun HD, et al. Contribution of a combination of ponicidin and acyclovir/ganciclovir to the antitumor efficacy of the herpes simplex virus thymidine kinase gene therapy system [J]. Hum Gene Ther, 2002;10(13):415-423.
[4] Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy [J]. Gene Ther, 2002;9:1023-1030.
[5] Pulkkanen KJ, Laukkanen JM, Fuxe J, et al. The combination of HSVTK and endostatin gene therapy eradicates orthotopic human renal cell carcinomas in nude mice [J]. Cancer Gene Ther, 2002;9:908-916.
[6] Qiao J, Doubrovin M, Sauter BV, et al. Tumorspecific transcriptional targeting of suicide gene therapy [J]. Gene Ther, 2002;9:168-175.
[7] Xu CT, Huang LT, Pan BR. Current gene therapy for stomach carcinoma [J]. World J Gastrenterol, 2001;7:752-759.
[8] Janouskova O, Sima P, Kunke D. Combined suicide gene and immunostimulatory gene therapy using AAVmediated gene transfer to HPV16 transformed mouse cell: Decrease of oncogenicity and induction of protection [J]. Int J Oncol, 2003 ;22(3):569-577.
[9] Guan J, Ma L, Wei L. Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GMCSF and HSVTK genes [J]. Chin Med J (Engl), 2001;114:147-151.
[10] Hall SJ, Canfield SE, Yan Y, et al. A novel bystander effect involving tumor cellderived Fas and FasL interactions following Ad.HSVTK and Ad.mIL12 gene therapies in experimental prostate cancer [J]. Gene Ther, 2002;9:511-517.
[11] Yamaguchi T, Okada T, Takeuchi K, et al. Enhancement of thymidine kinasemediated killing of malignant glioma by BimS, a BH3only cell death activator [J]. Gene Ther, 2003;10(5):375-385.
[12] Barzon L, Bonaguro R, Castagliuolo I, et al. Gene therapy of thyroid cancer via retrovirallydriven combined expression of human interleukin2 and herpes simplex virus thymidine kinase [J]. Eur J Endocrinol, 2003 ;148(1):73-80.
[13] Zhang R, DeGroot LJ. An adenoviral vector expressing functional heterogeneous proteins herpes simplex viral thymidine kinase and human interleukin2 has enhanced in vivo antitumor activity against medullary thyroid carcinoma [J]. Endocr Relat Cancer, 2001;8:315-325.
[14] Yu B, Cheng SY, Zhang HZ. Construction and identification of expressing vectors containing fusion genes of Hsvtk，IL2，TNFα genes [J]. Cancer, 2001;20:1019-1023.
Correspondence to: ZHANG JianHua, Department of Life Science and Technology, Xian Jiaotong University, Xian 710049, China. Email. Peter_hua@sohu.com
Biography: WANG HongXiao
(male, born in 1968, in Weinan city, Shaanxi province, China). Tel. (029)82335086Email. Whx_ca@yahoo.com.cn
EditorPan Borong, 百拇医药(WANG HongXiao, YUAN Jia)
【Keywords】 suicide gene; gene therapy; neoplasms
INTRODUCTION
The transfer of suicide genes into tumor cells is currently being used in a variety of clinical gene therapy trials for the treatment of cancer, and suicide gene therapy is the transduction of a gene that transforms a nontoxic into a toxic substance[1]. Herpes simplex virus thymidine kinase gene (HSVtk) + ganciclovir (GCV), HSVtk phosphorylates GCV inhibit the synthesis of DNA, and this combination produces a bystander effect and has been demonstrated to be of certain effect for the treatment of tumors[2-6]. But the systemic antitumoral immune response, however, is not developed fully to eliminate tumor. The unpredictability of the bystander effect and the difficulties in transduction have kept cure rates low. The use of tissuespecific vectors to deliver the genes and the combination of the strategy with cytokines may improve the efficacy of the approach in time. Gene therapy of suicide gene combined with cytokine gene has become a new insight for the treatment of malignancies because of their complementary effect. Some data[7-12] indicated that cytokine has the ability to improve the efficiency of HSVtk/GCV therapy in the treatment of cancer and suggested that the targeting of different pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.
SUICIDE GENE THERAPY THEORY
HSVtk + ganciclovir (GCV), or HSVtk phosphorylates GCV can inhibit the synthesis of DNA[2-6]. HSVtk itself also causes a bystander effect. A bystander effect is described when nontransduced or genetically unmodified cells are killed during the death of genetically modified tumor cells transduced with a suicide gene. The bystander effect greatly enhances the efficacy of HSVtk/GCV gene therapy for cancer. Despite promising results in vitro and in vivo, the antitumor effect in clinical trials remains poor, due to very low transfection efficiency. However, high percentages of transfected cells are not mandatory for complete eradication of a tumor in vivo. Transfected tumor cells appear to be capable of inducing the death of neighboring untransfected cells. A substantial bystander effect can overcome the limitations of low transfection efficiency and result in an enhanced and possibly clinically worthwhile antitumor effect in patients.
GENE TRANSFER TECHNOLOGY
Several experimental approaches have been tested for suicide gene delivery into tumor cells, including viral and nonviral vectors. The gene delivery technology is developing rapidly and there have been specific developments that could be translated into genebased therapies for diseases. Gene transfer may then be facilitated by a number of methods, including viruses, liposome, calcium phosphate coprecipitation, particle bombardment, naked DNA injection, and electroporation. Currently alternative viral vectors with potential advantages over retroviruses in specific applications are under development. Adenoviruses, which can infect nondividing cells, and can be concentrated to high titers, are comparatively highly efficient vectors. Adenoassociated viruses are ubiquitous and nonpathogenic in humans and can also infect nonreplicating cells, but, like retroviruses and adenoviruses, they are limited in size of the foreign gene that can be inserted. This problem may be overcome by the use of herpes simplex group viruses and possibly even vectors based on hepatitis B virus, which has potential additional advantages of hepatotropism and an ability to integrate with host genome in vivo[7].
COMBINED THERAPY OF SUICIDE GENE AND CYTOKINE GENE FOR CANCER
Gene therapy may offer a new tool for the treatment of renal cell carcinoma (RCC). In 2001, Cao et al[13] had tested a combination of cytotoxic and antiangiogenic gene therapy for wildtype orthotopic human RCC xenografts in nude mice using intratumoral adenovirusmediated HSVtk and endostatin (ES) gene therapy. It is concluded that the inhibition of angiogenesis by using ES gene transfer together with the cytotoxic HSVtk gene therapy results in a significantly improved treatment effect in RCC compared to the single gene treatments. Guan et al[9] explored that HSVtk and GMCSF genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and demonstrated the characteristics of the transduced cells. The IRES sequence can be used to construct retroviral vectors to facilitate cotransfection of two genes. SKOV3/TKGM cells can simultaneously express the HSVtk and GMCSF genes with biological activities, which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.
In 2002, Hall et al[10] tried to enhance the NK population induced by HSVtk gene transduction and ganciclovir (GCV) and adenovirusmediated (Ad) expression of IL12 were added to Ad.HSVtk + GCV as combination gene therapy. This approach results in improved local and systemic growth suppression in a metastatic model of mouse prostate cancer (RM1). The combination of Ad.HSVtk/GCV + Ad.mIL12 results in enhanced local growth control via apoptosis due to tumor cell expression of Fas and FasL and improved antimetastatic activity secondary to a strong NK response. Suicide gene therapy in combination with prodrugs represents an attractive approach to the treatment of cancer. Unfortunately this approach is limited by the difficulty in targeting all tumor cells, especially those at the distant metastases associated with the most complex tumors. For this reason, attempts to stimulate global antitumor immune responses at the sites of effective suicide gene/prodrugmediated tumor cell destruction are appealing.
At present, based on their clinical experience with combined gene therapy of glioblastoma, Barzon et al[12] developed a retroviral vector expressing two therapeutic genes and evaluated its efficiency in vitro and in vivo. These findings demonstrate the feasibility and efficiency of a combined immunomodulating and suicide gene therapy approach for thyroid carcinomas. Janouskova et al[8] tested the effects of combined transduction of a suicide gene and genes coding for various immunostimulatory factors on the oncogenicity and immunogenicity of TC1 cells, and several bicistronic recombinant adenoassociated viruses were constructed. Each of these constructs carried, and in infected cells expressed, the HSVtk and the gene of one of the following immunostimulatory factors. The tumor resistance rates were related not only to the immunostimulatory gene used for the transduction, but also to the GCV treatment. The best protection was recorded in mice preinoculated with TC1 cells transduced with either B7.1 or MCP1expressing rAAV and not given GCV.
HSVtk/gancyclovir (GCV) therapy has the ability to inhibit tumor formation in animal models but the results of clinical trials have been disappointing. In 2003, to improve the performance of tk/GCV therapy, Yamaguchi et al[11] tried combination therapy designed to enhance its cytotoxic effects by introducing genes that induce apoptosis of the tumor cells through different pathways. They concentrated their efforts on the use of Bim, a BH3only member of death activators in the Bcl2 superfamily, because Bim is not involved in the pathways through which HSVtk/GCV therapy induces apoptosis in malignant glioma cells. The results indicated that BimS had the ability to improve the efficiency of HSVtk/GCV therapy in the treatment of malignant glioma and suggested that the targeting of different proapoptotic pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.
PROSPECT IN FUTURE
Because of adaptor sequenceselecting complexity, previous research had been based on enzyme restriction site ligation, which is convenient to choose vector, to obtain intended fusion gene and also convenient to finish enzyme cleavage identification of fusion gene. But the expression activity of newly established fusion gene needs further experimental research. The recent introduction of computer technology and advances of protein engineer make it easy for researchers to present deliberate design and eliminate other possible disadvantages. Two different genes[13], such as human interleukin 2 (hIL2) and HSVtk gene, were tested on this modified vector for gene transfer and expression. Further more, derivatives space structure of fusion protein is sustained in its natural state and in its best biological activity. In a word, studies show that this area of research has a bright future and it is an effective means for searching novel therapy[14].
【REFERENCES】
[1] Engelmann C, Heslan JM, Fabre M, et al. Importance, mechanisms and limitations of the distant bystander effect in cancer gene therapy of experimental liver tumors [J]. Cancer Lett, 2002; 8(179):59-69.
[2] Burrows FJ, Gore M, Smiley WR, et al. Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells [J]. Cancer Gene Ther, 2002;9:87-95.
[3] Hayashi K, Hayashi T, Sun HD, et al. Contribution of a combination of ponicidin and acyclovir/ganciclovir to the antitumor efficacy of the herpes simplex virus thymidine kinase gene therapy system [J]. Hum Gene Ther, 2002;10(13):415-423.
[4] Boucher PD, Ostruszka LJ, Murphy PJ, et al. Hydroxyurea significantly enhances tumor growth delay in vivo with herpes simplex virus thymidine kinase/ganciclovir gene therapy [J]. Gene Ther, 2002;9:1023-1030.
[5] Pulkkanen KJ, Laukkanen JM, Fuxe J, et al. The combination of HSVTK and endostatin gene therapy eradicates orthotopic human renal cell carcinomas in nude mice [J]. Cancer Gene Ther, 2002;9:908-916.
[6] Qiao J, Doubrovin M, Sauter BV, et al. Tumorspecific transcriptional targeting of suicide gene therapy [J]. Gene Ther, 2002;9:168-175.
[7] Xu CT, Huang LT, Pan BR. Current gene therapy for stomach carcinoma [J]. World J Gastrenterol, 2001;7:752-759.
[8] Janouskova O, Sima P, Kunke D. Combined suicide gene and immunostimulatory gene therapy using AAVmediated gene transfer to HPV16 transformed mouse cell: Decrease of oncogenicity and induction of protection [J]. Int J Oncol, 2003 ;22(3):569-577.
[9] Guan J, Ma L, Wei L. Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GMCSF and HSVTK genes [J]. Chin Med J (Engl), 2001;114:147-151.
[10] Hall SJ, Canfield SE, Yan Y, et al. A novel bystander effect involving tumor cellderived Fas and FasL interactions following Ad.HSVTK and Ad.mIL12 gene therapies in experimental prostate cancer [J]. Gene Ther, 2002;9:511-517.
[11] Yamaguchi T, Okada T, Takeuchi K, et al. Enhancement of thymidine kinasemediated killing of malignant glioma by BimS, a BH3only cell death activator [J]. Gene Ther, 2003;10(5):375-385.
[12] Barzon L, Bonaguro R, Castagliuolo I, et al. Gene therapy of thyroid cancer via retrovirallydriven combined expression of human interleukin2 and herpes simplex virus thymidine kinase [J]. Eur J Endocrinol, 2003 ;148(1):73-80.
[13] Zhang R, DeGroot LJ. An adenoviral vector expressing functional heterogeneous proteins herpes simplex viral thymidine kinase and human interleukin2 has enhanced in vivo antitumor activity against medullary thyroid carcinoma [J]. Endocr Relat Cancer, 2001;8:315-325.
[14] Yu B, Cheng SY, Zhang HZ. Construction and identification of expressing vectors containing fusion genes of Hsvtk，IL2，TNFα genes [J]. Cancer, 2001;20:1019-1023.
Correspondence to: ZHANG JianHua, Department of Life Science and Technology, Xian Jiaotong University, Xian 710049, China. Email. Peter_hua@sohu.com
Biography: WANG HongXiao
(male, born in 1968, in Weinan city, Shaanxi province, China). Tel. (029)82335086Email. Whx_ca@yahoo.com.cn
EditorPan Borong, 百拇医药(WANG HongXiao, YUAN Jia)