Single-step, multiple retroviral transduction of human T cells

Background

Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells.

Methods

Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T‐cell line (MT‐2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double‐ and triple‐transduced cells were isolated by fluorescence cell sorting.

Results

Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), β‐galactosidase, and truncated versions of human nerve growth factor receptor (ΔNGFR) and human growth hormone receptor (ΔGHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti‐CD3. The transient production of viral particles encoding EGFP, ΔNGFR, and ΔGHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells.

Conclusions

This study describes new experimental conditions for efficient single‐step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches. Copyright © 2002 John Wiley & Sons, Ltd.

     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzedin vitro andin vivo; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy     

1例耐多药复杂重组人类免疫缺陷病毒1型亚型感染病例的抗病毒治疗

随着抗病毒治疗时间的延长,如何处理人类免疫缺陷病毒（HIV）耐药患者是临床医师面对的一个挑战。本文报道对1例耐多药复杂重组HIV-1亚型感染病例成功进行抗病毒治疗的过程,总结了抗病毒依从性的重要性,即只有规律服药才能达到好的治疗效果。临床上可应用病毒基因耐药检测来指导抗病毒药物的选择,但应注意体外实验与体内实际情况可能存在的差异。此外,需进一步研究一些少见亚型毒株对药物的反应及对药物压力的逃避机制。     

Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDR1 ribozyme gene mediated by retrovirus

According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effe     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

猪源肠外致病性大肠杆菌耐药性分析及耐药基因和I类整合子检测

【背景】由于抗生素的长期大量且不合理使用,猪源肠外致病性大肠杆菌(extraintestinal pathogenic Escherichia coli,ExPEC)多重耐药性日趋严重。【目的】探究猪源ExPEC的耐药性及其与耐药基因和I类整合子的相关性。【方法】采用微量肉汤稀释法测定54株猪源ExPEC对22种抗生素的最低抑菌浓度(minimal inhibitory concentration,MIC)和最低杀菌浓度(minimum bactericidal concentration,MBC);依据药敏试验结果确定相关耐药基因,采用PCR方法检测染色体DNA和质粒DNA上的耐药基因及I类整合子分布情况。【结果】54株猪源ExPEC对青霉素、氟苯尼考、氨苄西林、阿莫西林高度耐药,其中52株对甲氧苄氨嘧啶、复方新诺明高度耐药,它们的MIC值均大于256μg/mL,无MBC值;对头孢唑林、四环素、头孢氨苄、大观霉素、链霉素的MIC值在1-256μg/mL之间,MBC值分别为8、16、32、64、128、256μg/mL,均可耐受11种以上抗生素,其中以耐受17种为主,占比为18.52%...     

Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein

Neuronal progenitor cells (NPC) are particularly suited as the target population for genetic and cellular therapy of neurological disorders such as Parkinson's disease or stroke. However, genetic modification of these cells using retroviral vectors remains a great challenge because of the low transduction rate and the need for fetal calf serum (FCS) during the transduction process that induces the cell differentiation to mature neurons. To overcome these problems, we developed a new retrovirus production system in which the simplified retroviral vector GCDNsap engineered to be resistant to denovo methylation was packaged in the vesicular stomatitis virus G protein (VSV-G), concentrated by centrifugation, and resuspended in serum-free medium (StemPro-34 SFM). In transduction experiments using enhanced green fluorescent protein (EGFP) as a marker, the concentrated FCS-free virus supernatant infected NPC at a high rate, while maintaining the ability of these cells to self-renew and differentiate in vitro. When such cells were grafted into mouse brains, EGFP-expressing NPC were detected in the region around the injection site at 8 weeks post transplantation. These findings suggest that the gene transfer system described here may provide a useful tool to genetically modify NPC for treatments of neurological disorders.     

Selective gene therapy for human lung adenocarcinoma by tumor-specific expression of herpes simplex virus thymidine kinase gene

According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious "bystander effect" in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice     

Retroviral-mediated gene transfer in primary murine and human T-lymphocytes

Recombinant retroviruses are efficient vectors for introducing genes into many mammalian cell types. They are useful in the context of clinical as well as experimental applications, owing to the ability to generate high-titer and helper-free viral stocks. Retroviral vectors are especially appropriate for the transduction of primary lymphocytes, because gene transfer is stable and mediated by nonimmunogenic vectors. Stable integration in chromosomes of cells undergoing clonal expansion ensures that the foreign genetic material will be faithfully transmitted to the cells’ progeny. However, oncoretroviral vectors derived from murine leukemia viruses (MLV) require target cell division to integrate. Here we review factors that determine retroviral modiated gene transfer efficiency in primary T-lymphocytes, in particular T cell activation status, viral receptor expression, and culture conditions.     

Combined therapy of p53-wt and drug in an orthotopic multidrug-resistant human lung cancer model

Balb/c nu/nu mice were inoculated intratracheally with multidrug-resistant human lung cancer cells GLK containing p53 mutation at codon 245 and treated with intratracheal instillation of p53-wt retroviral vector (pDOR53W) to increase cell chemosensitivity, and then with intraperitoneal injection of doxorubicin. 30 d after tumor cell inoculation, 75% of the control mice showed macroscopic tumors in the lung. Sole pDOR53W suppressed GLK tumor formation in 68 % of mice; sole doxorubicin 33. 3 % , but the combination of pDOR53W and doxorubicin 88.9%. The exogenous p53 sequence was detected and confirmed in the tumor that grew after treatment with pDOR53W retroviral vector by PCR and Southern blot hybridization with p53 cDNA. These results suggested that di-rect administration of a retroviral vector expressing p53-wt combined with treatment of anticancer agent was an effec-tive therapeutic method for multidrug-resistant human lung cancer.     

Roles of noncoding RNAs in drug resistance in multiple myeloma

Despite the administration of new effective drugs in recent years, relapse and drug resistance are still the main obstacles in multiple myeloma (MM) treatment, making MM an incurable disease. To overcome drug resistance in MM, it is critical to understand the underlying mechanisms of malfunctioning gene expression and develop novel targeted therapies. During the past few decades, with the discovery and characterization of noncoding RNAs (ncRNAs), the landscape of dysregulated ncRNAs of cancers as well as their biological and pathobiological functions in tumorigenesis and drug resistance have been recognized. Studies about ncRNAs improved the understanding of variations of drug response among individuals at a level distinguished from genetic polymorphism, and provided with new orientations for targeted therapies. In this review, we will summarize the emerging impact and underlying molecular mechanisms of the most relevant classes of ncRNAs in drug resistance of MM, and discuss the potential as well as strategies of treating ncRNAs as therapeutic targets.     

Increasing drug resistance in human lung cancer cells by mutant-type p53 gene mediated by retrovirus

Human mutant-type (mt) p53 cDNA was synthesized and cloned from human lung cancer cell line GL containing mt-p53 gene by using polymerase chain reaction (PCR). It was confirmed that the mt-p53 cDNA con-tained the complete coding sequence of p53 gene but mutated at codon 245 (G→T) and resulted in glycine to cysteine by sequencing analysis. The retroviral vector pD53M of the mt-p53 was constructed and introduced into the drug-sen-sitive human lung cancer cells GAO in which p53 gene did not mutate. The transfected GAO cells strongly expressed mutant-type p53 protein by immunohistochemistry, showing that pD53M vector could steadily express in GAO cells. The drug resistance to several anticancer agents of GAO cells infected by pD53M increased in varying degrees, with the highest increase of 4-fold, in vitro and in vivo. By quantitative PCR and flow cytometry (FCM) analyses, the expression of MDR1 gene and the activity of P-glycoprotein (Pgp) did not increase, the expression of MRP gene and the activity of m     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

ncRNAs associated with drug resistance and the therapy of digestive system neoplasms

Digestive system cancer remains a common cancer and the main cause of cancer-related death worldwide. Drug resistance is a major challenge in the therapy of digestive system cancer, and represents a primary obstacle in the treatment of cancer by restricting the efficiency of both traditional chemotherapy and biological therapies. Existing studies indicate that noncoding RNAs play an important role in the evolution and progression of drug resistance in digestive system cancer, mainly by modulating drug transporter-related proteins, DNA damage repair, cell-cycle-related proteins, cell apoptosis-related proteins, drug target-related proteins, and the tumor microenvironment. In this review, we address the potential mechanisms of ncRNAs underlying drug resistance in digestive system tumors and discuss the possible application of ncRNAs against drug resistance in digestive system tumors.     

Targeted vectors for gene therapy of cancer and retroviral infections

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.