肿瘤基因治疗的研究进展

肿瘤是严重影响人类身体健康的重大疾病之一,肿瘤的发生发展是一个复杂的涉及到众多基因的过程,肿瘤的基因治疗也已经成为肿瘤治疗的研究热点之一。目前,肿瘤基因治疗的策略主要包括以下几个方面:基因沉默治疗、抑癌基因治疗、免疫基因治疗、自杀基因疗法、抑制肿瘤血管生成基因治疗、肿瘤多药耐药基因治疗、抗端粒酶疗法和多基因联合疗法等。我们简要地对上述策略及相关研究进展进行综述。     

高血压基因治疗研究进展

高血压是一种多基因病症,传统的化学药物治疗有很多的缺点,拟议中的基因治疗方案是针对高血压易感基因设计出反义寡核苷酸（AS ODN）,将其导入体内后可以阻断特定基因表达,从而发挥治疗作用,这种治疗方法的特点是持续时间长,效果好,无副作用,但是由于有一些技术上的难题,此法目前正处于实验阶段。     

小分子RNA与肿瘤治疗

肿瘤是各种原因导致多基因突变累积和相互作用形成的基因网络调控的结果,通过干预致瘤因子可能达到治疗肿瘤或改变肿瘤特性的目的。小分子RNA因可调控基因表达而被作为基因治疗的有效武器。本文结合我们课题组在小分子RNA方面的研究成果,对外源性的siRNA和内源性的miRNA在肿瘤治疗、治疗前景,以及siRNA和miRNA的联系与区别等方面作一综述。     

Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands

Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF‐A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF‐A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF‐A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand‐binding domain (LBD), a p65 activation domain, and an artificial zinc‐finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4′‐dyhydroxybenzil (DHB), a small molecule, non‐steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF‐A induction ratio of >100‐fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands. Biotechnol. Bioeng. 2013; 110: 1419–1429. © 2012 Wiley Periodicals, Inc.     

昆虫杆状病毒应用于哺乳动物基因治疗的研究进展

杆状病毒是一类宿主特异性的昆虫病毒。昆虫杆状病毒表达系统是一个高效的真核表达系统,被广泛用于在昆虫细胞或昆虫幼虫中生产外源蛋白质。杆状病毒不能感染哺乳动物,却可以进入不同物种和组织来源的多种哺乳动物细胞,并在合适的哺乳动物启动子控制下表达外源基因。杆状病毒在哺乳动物细胞中不能复制,对细胞没有毒性,加上杆状病毒本身具有基因组大、可操作性好等优点,作为哺乳动物基因治疗的载体,将治疗基因传递给哺乳动物细胞已受到了广泛关注。在此就杆状病毒作为基因治疗载体的最新研究进展进行了阐述并探讨其发展趋势。     

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro‐chemo‐gene therapy

Electroporation improves the anti‐tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation‐mediated chemotherapeutics with interleukin 12 (IL‐12) plasmid DNA produces a strong yet safe anti‐tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL‐12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL‐12 gene therapy for long‐term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro‐chemo‐gene therapy (ECGT) with IL‐12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response‐based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti‐tumour response for successfully affecting not only the treated tumours, but also non‐treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.     

Gene therapy in rheumatoid arthritis: Strategies to select therapeutic genes

Significant advances have been achieved in recent years to ameliorate rheumatoid arthritis (RA) in animal models using gene therapy approaches rather than biological treatments. Although biological agents serve as antirheumatic drugs with suppressing proinflammatory cytokine activities, they are usually accompanied by systemic immune suppression resulting from continuous or high systemic dose injections of biological agents. Therefore, gene transfer approaches have opened an interesting perspective to deliver one or multiple genes in a target-specific or inducible manner for the sustained intra-articular expression of therapeutic products. Accordingly, many studies have focused on gene transferring methods in animal models by using one of the available approaches. In this study, the important strategies used to select effective genes for RA gene therapy have been outlined. Given the work done in this field, the future looks bright for gene therapy as a new method in the clinical treatment of autoimmune diseases such as RA, and by ongoing efforts in this field, we hope to achieve feasible, safe, and effective treatment methods.     

Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side

Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB’s genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.     

Over-expression of PTEN on proliferation and apoptosis in canine mammary tumors cells

Phosphatase and tensin homolog (PTEN) is an important tumor-suppressor gene which constitutes an important PI3K/Akt pathway by regulating the signaling of multiple biological processes, including apoptosis, metabolism, cell proliferation, and cell growth has been gaining increasing attention. However, the role of PTEN in regulating apoptosis of canine mammary tumors cells still needs further investigation. In this experiment, the effect of PTEN on proliferation and apoptosis in canine mammary tumors (CMT) cells was analyzed. As a result, gene and protein expression levels of apoptosis-related genes were detected. Eukaryotic expression vector pcDNA3.1+-PTEN were successfully constructed and stably transferred into canine CMT cells after geneticin (G418) selection. After pcDNA3.1+-PTEN transfection, compared with control group, the cells proliferation was inhibited and the cell apoptosis was increased in CMT cells. The expression of p-Akt was decreased and the apoptosis-related genes, such as caspase-3, caspase-9, and Bax, were increased. These data serve to demonstrate the function of PTEN on apoptosis and gene regulatory in PI3K/Akt pathway in CMT cells. Collectively, our data link the tumor-suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signaling molecules whose signal target is the functional inactivation of the apoptosis gene product.     

当前肿瘤基因治疗中存在的主要问题及其解决途径

本文概述了当前肿瘤基因治疗研究中存在的一些主要问题,如绝大多数治疗方案中目的基因只有一个,肿瘤基因治疗缺乏靶向性,基因转移载体的效率、安全性及容量等问题。讨论了解决这些问题的主要途径,即肿瘤多基因联合治疗、直接体内途径基因治疗与靶向基因治疗、基因转移载体的改造。     

Small interfering RNA–mediated gene suppression as a therapeutic intervention in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the lethal and difficult-to-cure cancers worldwide. Owing to the late diagnosis and drug resistance of malignant hepatocytes, treatment of this cancer by conventional chemotherapy agents is challenging, and researchers are seeking new alternative treatment options to overcome therapy resistance in this neoplasm. RNA interference (RNAi) is a potent and specific approach in targeting gene expression and has emerged as a novel therapeutic tool for many diseases, including cancers. Small interfering RNA (siRNA) is a type of RNAi that is produced intracellularly from exogenous synthetic oligonucleotides and can selectively knock down target gene expression in a sequence-specific manner. Various factors play roles in the initiation and progression of HCC and provide multiple candidate targets for siRNA intervention. In addition, due to the liver's unique architecture and availability of some hepatic siRNA delivery methods, this organ has received much more attention as a target tissue for such oligonucleotide action. Recent advances in designing nanoparticle systems for the in vivo delivery of siRNAs have markedly enhanced the potency of siRNA-mediated gene silencing under clinical development for HCC therapy. The utility of siRNAs as anti-HCC agents is the subject of the current review. siRNA-based gene therapies could be one of the main feasible approaches for HCC therapy in the future.     

降钙素基因相关肽转基因预防小鼠自身免疫性糖尿病发病及其抗氧化应激机制

有证据表明,活性氧(reactive oxygens pecies,ROS)参与了胰岛β细胞自身免疫损伤,是自身免疫性糖尿病发病的重要原因之一。一氧化氮(NO)和过氧化氢(H2O2)介导了致炎性细胞因子对胰岛B细胞的损害,引起脂质过氧化反应,进而损伤胰岛细胞。降钙素基因相关肽(calcitonin gene-related peptide,CGRP)在心肌细胞中可抑制ROS生成而具有细胞保护作用。通过CGRP裸质粒肌肉注射体细胞电针强化转基因方法,使骨骼肌持续表达CGRP,观察其对小剂量多次注射链脲佐菌素(streptozotocin,STZ)造成的小鼠自身免疫性糖尿病发病的影响,进一步测定胰腺局部活性氧含量以及抗氧化酶的改变,探讨其抗氧化应激机制。结果发现,CGRP裸质粒直接注射入小鼠双后肢胫前肌。继以程控电针刺激导入(体内电穿孔法),可使血浆和骨骼肌组织CGRP表达水平显著增高,且持续4周以上;注射STZ同时给予CGRP转基因治疗可减轻胰岛β细胞损伤,显著降低自身免疫性糖尿病的发病率及血糖水平;CGRP转基因可显著抑制自身免疫性糖尿病小鼠胰腺局部活性氧和丙二醛的生成,增加过氧化氢酶(CAT)及超氧化物歧化酶(superoxide dismutase,SOD)的活性。结果提示,CGRP裸质粒直接注射、电针辅助导人转基因可获得CGRF持续高水平的表达,能够预防小鼠自身免疫性糖尿病的发病,其机制之一可能为CGRP抑制了活性氧对胰岛β细胞的损伤。     

Gene therapy in cancer

Gene therapy, recently frequently investigated, is an alternative treatment method that introduces therapeutic genes into a cancer cell or tissue to cause cell death or slow down the growth of the cancer. This treatment has various strategies such as therapeutic gene activation or silencing of unwanted or defective genes; therefore a wide variety of genes and viral or nonviral vectors are being used in studies. Gene therapy strategies in cancer can be classified as inhibition of oncogene activation, activation of tumor suppressor gene, immunotherapy, suicide gene therapy and antiangiogenic gene therapy. In this review, we explain gene therapy, gene therapy strategies in cancer, approved gene medicines for cancer treatment and future of gene therapy in cancer. Today gene therapy has not yet reached the level of replacing conventional therapies. However, with a better understanding of the mechanism of cancer to determine the right treatment and target, in the future gene therapy, used as monotherapy or in combination with another existing treatment options, is likely to be used as a new medical procedure that will make cancer a controllable disease.     

Rational design of microRNA–siRNA chimeras for multifunctional target suppression

MicroRNAs (miRNAs) are involved in a variety of human diseases by simultaneously suppressing many gene targets. Thus, the therapeutic value of miRNAs has been intensely studied. However, there are potential limitations with miRNA-based therapeutics such as a relatively moderate impact on gene target regulation and cellular phenotypic control. To address these issues, we proposed to design new chimeric small RNAs (aiRNAs) by incorporating sequences from both miRNAs and siRNAs. These aiRNAs not only inherited functions from natural miRNAs, but also gained new functions of gene knockdown in an siRNA-like fashion. The improved efficacy of multifunctional aiRNAs was demonstrated in our study by design and testing of an aiRNA that inherited the functions of both miR-200a and an AKT1-targeting siRNA for simultaneous suppression of cancer cell motility and proliferation. The general principles of aiRNA design were further validated by engineering new aiRNAs mimicking another miRNA, miR-9. By regulating multiple cellular functions, aiRNAs could be used as an improved tool over miRNAs to target disease-related genes, thus alleviating our dependency on a limited number of miRNAs for the development of RNAi-based therapeutics.     

A novel approach for gene therapy: engraftment of fibroblasts containing the artificial chromosome expression system at the site of inflammation

BACKGROUND: Rheumatoid arthritis is characterized by inflammation of the synovial tissue. High systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints. Gene transfer might provide a more efficient delivery system for genes encoding therapeutic proteins. METHODS: The artificial chromosome expression system (ACE System) is a new non-integrating, non-viral gene expression system which functions like a natural chromosome. This technology offers advantages over current expression systems because it allows stable and predictable expression of proteins encoded by single or multiple genes over long periods of time. We are developing ex vivo gene therapy using murine artificial chromosomes containing a reporter gene (LacZ and red fluorescent protein (RFP)) for local delivery of genes in rats with adjuvant arthritis (AA). RESULTS: The delivery of the intact ACE System into rat fibroblast-like synoviocytes (FLS) and rat skin fibroblasts (RSF) was detected within 24 to 48 h post-transfection. After growing cells under selection, clones expressing LacZ and RFP were identified. Furthermore, we investigated the feasibility of local delivery of a reporter gene to the joints of rats with AA by ex vivo gene therapy. This resulted in engraftment of the injected cells in the synovial tissue microarchitecture and expression of the reporter gene. CONCLUSIONS: This work demonstrates the potential feasibility of treating arthritis and other inflammatory diseases using fibroblasts containing the ACE System as a non-viral vector for gene therapy.     

A novel approach for the construction of multiple shRNA expression vectors

The application of RNA interference (RNAi) as a research and therapeutic tool depends on its ability to silence genes in a sequence-specific manner. Recent studies have reported that the effective knockdown of genes can be achieved by multiple short hairpin RNAs (shRNAs) in a single vector. Moreover, this approach can depress several genes simultaneously. However, current methods for the construction of multiple shRNA vectors often suffer from vector instability and are time-consuming. Here, we describe a simple, quick and low-cost approach to construct a single vector expressing four shRNA sequences driven by four different promoters. Using this vector, we were able to improve the gene silencing efficiency and make it possible to silence four different genes simultaneously, further expanding the application spectrum of RNAi, both in functional studies and therapeutic strategies.