The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/uracil phosphoribosyltransferase suicide gene

Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/uracil phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluorouracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-gamma-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.     

Intracellular prodrug gene therapy for cancer mediated by tumor cell suicide gene exosomes

Recently, we reported about exosomes possessing messenger RNA (mRNA) of suicide gene secreted from mesenchymal stem/stromal cells (MSCs) engineered to express the suicide gene—fused yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD::UPRT). The yCD::UPRT‐MSC exosomes are internalized by tumor cells and intracellularly convert prodrug 5‐fluorocytosine (5‐FC) to cytotoxic drug 5‐fluorouracil (5‐FU). Human tumor cells with the potential to metastasize release exosomes involved in the creation of a premetastatic niche at the predicted organs. We found that cancer cells stably transduced with yCD::UPRT gene by retrovirus infection released exosomes acting similarly like yCD::UPRT‐MSC exosomes. Different types of tumor cells were transduced with the yCD::UPRT gene. The homogenous cell population of yCD::UPRT‐transduced tumor cells expressed the yCD::UPRT suicide gene and secreted continuously exosomes with suicide gene mRNA in their cargo. All tumor cell suicide gene exosomes upon internalization into the recipient tumor cells induced the cell death by intracellular conversion of 5‐FC to 5‐FU and to 5‐FUMP in a dose‐dependent manner. Most of tumor cell‐derived suicide gene exosomes were tumor tropic, in 5‐FC presence they killed tumor cells but did not inhibit the growth of human skin fibroblast as well as DP‐MSCs. Tumor cell‐derived suicide gene exosomes home to their cells of origin and hold an exciting potential to become innovative specific therapy for tumors and potentially for metastases.     

Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.     

Comparative analysis of enzyme and pathway engineering strategies for 5FC-mediated suicide gene therapy applications

Bacterial- and yeast- encoded cytosine deaminases (bCD and yCD, respectively) are widely investigated suicide enzymes used in combination with the prodrug 5-fluorocytosine (5FC) to achieve localized cytotoxicity. Yet characteristics such as poor turnover rates of 5FC (bCD) and enzyme thermolability (yCD) preclude their full therapeutic potential. We previously applied regio-specific random mutagenesis and computational design to create novel bCD and yCD variants with altered substrate preference (bCD(1525)) or increased thermostability (yCD(double), yCD(triple)) to aid in overcoming these limitations. Others have utilized pathway engineering in which the microbial enzyme uracil phosphoribosyltransferase (UPRT) is fused with its respective CD, creating bCD/bUPRT or yCD/yUPRT. In this study, we evaluated whether the overlay of CD mutants onto their respective CD/UPRT fusion construct would further enhance 5FC activation, cancer cell prodrug sensitivity and bystander activity in vitro and in vivo. We show that all mutant fusion enzymes allowed for significant reductions in IC(50) values relative to their mutant CD counterparts. However, in vivo the CD mutants displayed enhanced tumor growth inhibition capacity relative to the mutant fusions, with bCD(1525) displaying the greatest tumor growth inhibition and bystander activity. In summary, mutant bCD(1525) appears to be the most effective of all bacterial or yeast CD or CD/UPRT enzymes examined and as such is likely to be the best choice to significantly improve the clinical outcome of CD/5FC suicide gene therapy applications.     

通过溶瘤病毒靶向递送BiTE引发骨肉瘤局部抗肿瘤免疫的实验研究

目的:探索共表达双特异性T细胞接合器(bispecific T cell engager,BiTE)的VSVΔ51溶瘤病毒对骨肉瘤的抗肿瘤活性.方法:将BiTE的全长序列克隆到病毒糖蛋白和聚合酶基因之间产生VSVΔ51/BiTE溶瘤病毒;使用小鼠异源移植瘤模型评估VSVΔ51/BiTE的肿瘤抑制活性;通过免疫组化检测肿...     

Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5'-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with the UPRT gene (Colon 26/UPRT cells) and tested the in vivo antitumoral effect of 5-FU in syngeneic immunocompetent mice. After 5-FU administration, tumors of Colon 26/UPRT cells regressed, whereas those of wild-type cells were unaffected. The mice that once eliminated Colon 26/UPRT tumors after 5-FU treatment rejected wild-type cells that were subsequently inoculated but not irrelevant syngeneic tumor cells. This suicide gene/prodrug system was less efficient in nude mice, suggesting that mature alphabeta T cells play a role in the antitumoral effect. The cytotoxicity mediated by the bystander effect was marginal in this system, contrary to the herpes simplex virus-thymidine kinase gene/ganciclovir system. Therefore, expression of the UPRT gene in tumor cells followed by 5-FU administration is a possible strategy for cancer gene therapy, but potentiation of the bystander effect is required for its therapeutic application.     

Replication-competent retrovirus vector-mediated prodrug activator gene therapy in experimental models of human malignant mesothelioma

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.     

肿瘤双自杀基因治疗系统pTRKH2-PsT/CD,pTRKH2-PsT/UPRT在厌氧婴儿双歧杆菌中的构建

Objective: We recombine the suicide gene CD, UPRT into plasmid pTRKH2 and clone the recombinant dual suicide gene therapy system into tumor-hypoxia-targeting vector Bifidobacterium infantis and characterize its function. Methods: CD gene, UPRT gene and lactic acid bacteria expression plasmid pTRKH2 were digested by restriction endonuclease BamH I and Sal I, and constructed recombinant plasmids pTRKH2/CD and pTRKH2/UPRT in E. coll. The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation. Identification of pTRKH2/CD and pTRKH2/UPRT was processed by dual restriction endonuclease digesting and sequencing. RT-PCR and SDS-PAGE were used to examine the expression of CD and UPRT genes at RNA and protein levels. The killing effects on Melanoma B16-F10 cells by pTRKH2/CD and pTRKH2/UPRT suicide gene therapy system with 5-FC were examined by MTT assay. Results: The CD gene and UPRT gene was successfully recombined into lactic acid bacteria expression plasmid pTRKH2. After dual endonuclease digestion of plasmid purified from the positively transfected E. co/i, two fragments of 6.9 Kb and 1.3 Kb were found for CD gene and two fragments of 6.9 Kb and 620 bp were found for UPRT gene. The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data. A fragment of 1.3 Kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobac- terium by RT-PCR. A 52 KDa protein for CD gene was identified in whole-cell protein of recombinant Bifidobacterium and a 26 KDa protein for UPRT gene was identified in supernatant fluid of recombinant Bifidobacterium. The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pTRKH2/CD and pTRKH2/UPRT dual suicide gene therapy system on Melanoma B16-F10 cells. Conclusion: CD gene and UPRT gene are suc- cessfully inserted into pTRKH2 and transfected into tumor-hypoxia-targeting vector Bifldobacterium Infantis. This dual suicide gene therapy system shows a high efficiency for tumor cells killing.     

双自杀基因对Raji淋巴瘤细胞杀伤的增强作用

目的 :观察反转录病毒介导的双自杀基因对 Raji淋巴瘤细胞的杀伤增强作用 ,探讨淋巴瘤的基因治疗方法。方法 :通过脂质体将含有双自杀基因的反转录病毒载体 p WZL neo CDglytk导入病毒包装细胞 PA317,经 G4 18筛选后大量培养产病毒的阳性克隆 PA317/ CD+ tk细胞株 ,收集病毒上清 ,浓缩后转染 Raji淋巴瘤细胞 ,再次经 G4 18筛选 ,获得稳定表达双自杀基因的 Raji/ CD+ tk细胞株。用RT- PCR检测双自杀基因的表达。给予前体药物 5 -氟胞嘧啶 (5 - flourocytosine,5 - FC)和 /或无环鸟苷(Ganciciovir,GCV)后 ,MTT法测定细胞的存活率及 CDglytk双自杀基因对 Raji细胞的杀伤作用。结果 :双自杀基因在 Raji细胞中可稳定表达 ,联合使用 5 - FC和 GCV时 Raji细胞的存活率 (13.83% )明显低于单独使用 GCV (5 0 .6 5 % )或 5 - FC(5 7.6 8% )时 ,各组比差异具有显著性 (P<0 .0 1)。结论 :反转录病毒介导双自杀基因对 Raji淋巴瘤的杀伤作用明显增强     

Combined suicide gene therapy for human colon cancer cells using adenovirus-mediated transfer of escherichia coli cytosine deaminase gene and Escherichia coli uracil phosphoribosyltransferase gene with 5-fluorocytosine

The virus-directed enzyme/prodrug system using the Escherichia coli cytosine deaminase (CD) gene and 5-fluorocytosine (5-FC) suffers from a sensitivity limitation in many tumor cells. The E. coil uracil phosphoribosyltransferase (UPRT), which is a pyrimidine salvage enzyme, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine monophosphate at the first step of its activating pathway. To improve the antitumoral effect of the CD/5-FC system, we investigated a combined suicide gene transduction therapy for human colon cancer cells using two separate adenovirus vectors expressing the E. coli CD and E. coli UPRT genes and systemic 5-FC administration (the CD, UPRT/5-FC system). The present study demonstrates that the CD, UPRT/5-FC system generates a co-operative effect of CD and UPRT, resulting in dramatic increases in both RNA- and DNA-directed active forms, including 5-fluorouridine triphosphate incorporated into RNA, 5-fluorodeoxyuridine monophosphate, and the thymidylate synthase inhibition rate, compared with the CD/5-FC system. Furthermore a significant increase in the 5-FC sensitivity of colon cancer cells was demonstrated in the CD, UPRT/5-FC system compared with the CD/5-FC system in vitro and in vivo. These results suggest that the CD, UPRT/5-FC system is a powerful approach in gene therapy for colorectal cancer.     

肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT的构建

目的 利用婴儿双歧杆菌对实体瘤低氧区的靶向效应,构建肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT /CD和pTRKH2-PsT /UPRT。方法 用PCR的方法从质粒pGEX/CD和pGEX/UPRT中扩增出CD基因和UPRT基因,双酶切CD基因、UPRT基因和质粒pTRKH2-PsT,分别连接后重组于大肠杆菌中。之后用电转化的方法将重组质粒转入婴儿双歧杆菌中。用RT-PCR检测该系统mRNA水平的表达,SDS-PAGE检测该系统在蛋白质水平的表达。在黑色素瘤B16-F10细胞上检测该系统的体外肿瘤细胞杀伤效果。结果 成功地将CD基因和UPRT基因转入了质粒pTRKH2-PsT,CD基因和UPRT基因的测序结果表明序列与Genebank公布的序列一致。RT-PCR检测到CD和UPRT mRNA水平的明显表达。在含有CD基因的婴儿双歧杆菌细胞全蛋白中发现了CD蛋白质的表达,在含有UPRT基因的婴儿双歧杆菌上清液中发现了UPRT蛋白质的表达。黑色素瘤细胞的低存活率证明了pTRKH2-PsT/CD、pTRKH2-PsT/UPRT自杀基因治疗系统对黑色素瘤的显著杀伤作用。结论 肿瘤厌氧靶向双自杀基因治疗系统pTRKH2-PsT/CD、pTRKH2-PsT/UPRT构建成功并显示出杀伤肿瘤细胞的作用。     

Chemovirotherapy for head and neck squamous cell carcinoma with EGFR-targeted and CD/UPRT-armed oncolytic measles virus

First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal antiEGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.     

Bystander effect in uracil phosphoribosyltransferase/5-fluorouracil-mediated suicide gene therapy is correlated with the level of intercellular communication

We examined whether a suicide gene/prodrug system using the uracil phosphoribosyltransferase (UPRT) of E. coli origin and 5-fluorouracil (5-FU) could achieve a bystander effect in two rodent tumor cell lines, murine colon carcinoma (Colon 26) and rat gliosarcoma (9L) cells. Cytotoxicity tests of mixed populations consisting of parent and transduced cells showed that the bystander effect was not produced in Colon 26 cells in either the UPRT/5-FU system or the herpes simplex virus-thymidine kinase/ganciclovir system but a strong bystander effect was evidenced by both suicide gene systems in 9L cells. The expression level of connexin 43, a protein that constitutes gap junctions, was high in 9L but low in Colon 26 cells. A gap junction-permeable fluorescein dye could be transferred among 9L cells but hardly at all among Colon 26 cells. Taken together, the efficacy of the bystander effect in the UPRT/5-FU system can be affected by gap junction-mediated intercellular communication.     

Cancer gene therapy and immunotherapy (review)

Gene therapy is a newly hatched field of biomedical research aimed at introducing therapeutically important genes into somatic cells of patients for the treatment of human disease. Whereas for inborn errors of metabolism transfer of a single gene can correct the disorder, cancer is a complex disease involving mutations in a number of protooncogenes and tumor suppressor genes as well as an imbalance and disarray in phosphorylation events and regulatory circuits of the cell cycle; transfer of the wild-type p53 or p21 tumor suppressor genes is a successful gene therapy approach leading to apoptotic death of cancer cells or in restrain of their chaotic growth. A different promising approach is transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene (suicide gene) and systemic treatment with the prodrug ganciclovir which is converted by HSV-tk into a toxic drug killing dividing cells. Expression of suicide genes, p53, and other therapeutic genes preferentially in cancer cells can be achieved by regulatory elements from tumor-specific genes such as carcinoembryonic antigen, BRCA1, and PSA.     

UPRT/5-FU前药转换酶系统对小鼠胃癌治疗的实验研究

目的　探讨UPRT/ 5 FU前药转换酶系统对小鼠胃癌细胞MFC的杀伤效应。方法　采用PCR技术从大肠杆菌K12基因组中扩增UPRT基因 ,并构建pLXSN逆转录病毒表达载体 ,进一步转染MFC细胞。采用MTT法检测转导UPRT基因前后 ,MFC对 5 FU的敏感性的变化。建立 6 15小鼠胃癌皮下移植瘤模型 ,腹腔内给于 5 FU ,观察治疗效果。结果　体外MTT法检测结果显示 ,转导UPRT基因后可使MFC对 5 FU的敏感性提高约 17.2 6倍。动物实验的结果表明 ,转导UPRT基因不影响MFC的致瘤性 ;经 5 FU治疗后 ,转UPRT基因的肿瘤 ,生长明显受抑 (P <0 .0 0 0 5 ) ,抑瘤率为 89.6 2 %。结论　UPRT基因修饰小鼠胃癌细胞株MFC ,能明显提高MFC对 5 FU杀伤的敏感性 ,增强 5 FU的抗瘤效应。     

hTERT启动子调控的融合自杀基因CD:UPRT载体的构建及其应用

目的 构建hTERT启动子调控的融合自杀基因CD:UPRT表达载体,研究其对人胃癌细胞SGC7901的体外靶向性杀伤作用。方法 PCR扩增hTERT核心启动子片段,克隆入荧光素酶报告基因质粒pGL3 Basic,检测hTERT启动子在人胃癌细胞SGC7901和人成纤维细胞HLF中的转录活性。构建hTERT启动子调控的CD:UPRT基因表达载体hTERT CD:UPRT,将其和CMV启动子调控的CD:UPRT基因表达载体pcDNA3.1 CD:UPRT用脂质体转染法分别转染入SGC7901和HLF细胞,筛选稳定表达细胞系,用RT PCR和Western blot方法检测CD基因的表达,用MTT法检测5 FC对转染细胞的杀伤作用。结果 成功克隆hTERT核心启动子;荧光素酶活性检测显示,hTERT启动子在SGC7901细胞中的转录活性为阳性对照的(21.50±2.15)%,而在HLF细胞中仅有背景活性。成功构建hTERT启动子调控的CD:UPRT基因表达载体,转染pcDNA3.1 CD:UPRT的SGC7901和HLF细胞以及转染hTERT CD:UPRT的SGC7901细胞在mRNA和蛋白质水平均可检测到CD基因的表达,且对5 FC敏感;而转染hTERT CD:UPRT的HLF细胞未检测到CD基因的表达,对5 FC不敏感。结论 构建的hTERT启动子调控的融合自杀基因系统CD:UPRT/5 FC能在体外靶向性杀伤SGC7901细胞。     

In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene

Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase (FCY1) and uracil phosphoribosyltransferase genes (FUR1). This suicide gene, designated FCU1, encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine-5'monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Unexpectedly, although the uracil phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1, its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1. As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1/5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用     

Enhanced antitumoral effect of adenovirus-mediated cytosine deaminase gene therapy by induction of antigen-presenting cells through stem cell factor/granulocyte-macrophage colony-stimulating factor gene transfer

Suicide gene therapy has been studied intensively for the treatment of cancer. A limited antitumoral effect was obtained by intratumoral injection of adenovirus harboring Escherichia coli cytosine deaminase gene (AdCD) in tumor-bearing mice followed by continuous administration of 5-fluorocytosine (5FC). To address the drawbacks of the limited potential for the induction of antitumoral immunity by CD suicide gene therapy, we hypothesized that antigen-presenting cells (APCs) might contribute to the efficient induction of an antitumoral immune response in tumor-bearing mice undergoing suicide gene therapy. We preinjected the mice with murine stem cell factor (SCF)-encoding adenovirus (AdSCF) and murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-encoding adenovirus (AdGM-CSF); after 7 days, the mice were inoculated with CT26 colon adenocarcinoma. AdCD was injected intratumorally into tumor-bearing mice followed by 5FC administration. The results showed that AdSCF/AdGM-CSF treatment could increase the number, surface molecule expression, and function of APCs efficiently. A more significant growth inhibition of established tumors and a prolongation of the survival period were observed in tumor-bearing mice after AdSCF/AdGM-CSF pretreatment in combination with AdCD/5FC therapy when compared with mice treated with AdSCF or AdGM-CSF in combination with AdCD/5FC, or AdCD/5FC alone (P < .01). Cytotoxic T-lymphocyte activity was induced efficiently after the combined therapy, and mRNA of tumor necrosis factor-alpha, interleukin-4, interferon-gamma, and interleukin-2 was present in the tumor mass after combined therapy, suggesting that a more potent antitumoral response was induced by enhanced APCs. Our results demonstrated that AdSCF/AdGM-CSF pretreatment could activate APCs, and that these APCs could present the tumor antigens released from AdCD/5FC-killed tumor cells and activate the antitumoral response of the host, thus increasing the therapeutic efficiency of suicide gene therapy.     

Combination of a fusogenic glycoprotein, prodrug activation, and oncolytic herpes simplex virus for enhanced local tumor control

We have previously developed an oncolytic herpes simplex virus-1 based on a clinical virus isolate, which was deleted for ICP34.5 to provide tumor selected replication and ICP47 to increase antigen presentation as well as tumor selective virus replication. A phase I/II clinical trial using a version of this virus expressing granulocyte macrophage colony-stimulating factor has shown promising results. The work reported here aimed to develop a version of this virus in which local tumor control was further increased through the combined expression of a highly potent prodrug activating gene [yeast cytosine deaminase/uracil phospho-ribosyltransferase fusion (Fcy::Fur)] and the fusogenic glycoprotein from gibbon ape leukemia virus (GALV), which it was hoped would aid the spread of the activated prodrug through the tumor. Viruses expressing the two genes individually or in combination were constructed and tested, showing (a) GALV and/or Fcy::Fur expression did not affect virus growth; (b) GALV expression causes cell fusion and increases the tumor cell killing at least 30-fold in vitro and tumor shrinkage 5- to 10-fold in vivo; (c) additional expression of Fcy::Fur combined with 5-fluorocytosine administration improves tumor shrinkage further. These results indicate, therefore, that the combined expression of the GALV protein and Fcy::Fur provides a highly potent oncolytic virus with improved capabilities for local tumor control. It is intended to enter the GALV/Fcy::Fur expressing virus into clinical development for the treatment of tumor types, such as pancreatic or lung cancer, where local control would be anticipated to be clinically advantageous.