RNAi-mediated gene silencing in cancer therapy

Introduction: This review presents the intriguing success of RNA interference (RNAi)-mediated gene silencing and its advantages and obstacles in cancer therapy.

Areas covered: RNAi has implications in metabolic disease, viral hepatitis, cardiovascular and cerebrovascular diseases, HIV, neurodegenerative disorders and cancer. RNAi can enhance the specificity and efficacy of therapeutic intervention for human diseases while at the same time reducing toxicity. The existing research related to gene therapy suggests encouraging prospects of a new high-efficiency and low-toxicity anti-tumor therapy. Although gene therapy is still in the experimental research phase, in the near future, this method will become an important means for the treatment of cancer therapies and it will be widely used in clinical practice.

Expert opinion: RNAi-based drug development is still in preclinical trial and several challenges limit the use of RNAi in the clinic. It is believed that further investigation of the mechanisms of RNAi-based therapies will help overcome these limitations and provide powerful weapons in the oncology clinic.     

Strategies for short hairpin RNA delivery in cancer gene therapy

RNA interference (RNAi) gene silencing can be achieved by delivering vectors that transcribe short hairpin RNA (shRNA), which stably express small interfering RNA in target cells. Therefore, shRNA is of potential therapeutic use for inhibiting cancer cells, in which aberrant expression of certain mRNA's causes problems. However, this technique has not yet been developed for cancer therapy. The major problem for clinical use is lack of effective methods of delivery. In this article, we review the current strategies for shRNA delivery for target validation and their therapeutic uses in cancer to help further the understanding of challenges confronting shRNA technology, such as different principles of RNAi technology, basic construction of shRNA-expressing vectors and delivery barriers, which exist for both local and systemic delivery stratiges. Even if there are data showing that shRNA can be used in mice, we are still a long way from its application in human cancer therapy, because serious problems remain, including biodistribution and clearance of nanoparticles following systemic delivery of shRNA-expressing vectors.     

RNA-mediated gene silencing: mechanisms and its therapeutic applications

RNA interference, part of a complicated network of interconnected pathways for cellular defence, RNA surveillance and development, has become a powerful tool for the experimental manipulation of gene expression. It is the process by which double-stranded (dsRNA) silences specific gene expression through homology-dependent degradation of cognate mRNA. The dsRNA is converted into 21nt small interfering RNAs (siRNAs), which directs a complex ribonuclease system to substrate mRNA targets. The degradation of the target mRNA is initiated with the cleavage at a position corresponding to the centre of the siRNA. Dissecting individual cellular pathways to reveal the function of numerous proteins is an approach to drug discovery. Interfering RNA (RNAi) serves as a rapid and convenient tool, which works in various organisms. RNAi technology has the potential to facilitate our understanding of biological processes and potentially lead to exciting new drugs. Here we review various experimental approaches adopted with RNAi and possible therapeutic applications.     

Oncolytic-adenovirus-expressed RNA interference for cancer therapy

Importance of the field: RNA interference (RNAi) has generated considerable excitement for its potential cancer therapeutic applications. Because of the difficulties in delivering a large amount of siRNA to cancer cells and the short half-life of siRNA, it is important to choose an efficient delivery system for transduction of siRNA into target cells. Oncolytic adenovirus offers a better platform by virtue of its high transfection efficiency and selective replication in cancer cells.

Areas covered in this review: This review focuses on the synergism between oncolytic adenovirus and siRNA antitumor responses, and discusses recent progresses in oncolytic-adenovirus-expressed siRNA.

What the reader will gain: siRNA-expressing oncolytic adenovirus can generate a significantly enhanced antitumor effect through gene knockdown and viral oncolysis.

Take home message: Due to its potency and target specificity, using siRNA delivery by oncolytic adenovirus has generated much excitement and will open new avenues for treatment of human cancer.     

Pancreatic cancer: gene therapy approaches and gene delivery systems

Importance of the field: Due to the absence of early diagnosis, the highly invasive and metastatic features and the lack of effective therapeutic modalities, the prognosis of patients with pancreatic cancer is poor. Gene therapy is currently regarded as a potential and promising therapeutic modality for pancreatic cancer.

Areas covered in this review: This article summarizes an update of gene therapy approaches and reviews the latest progress in gene delivery systems that have been tested on pancreatic cancer.

What the reader will gain: The treatment effectiveness of gene combination therapy is better than that of the regulation of single-gene or single gene therapy approaches. Naked DNA is limited because of degradation by intracellular and extracellular nucleases. Virus vectors show high transfection efficiency but are limited due to immunogenicity, inflammatory response and potential carcinogenicity. Non-viral vectors, such as cationic polymers or inorganic nanoparticles, show an important feature that they can be easily modified, and the progress of materials science will provide more and better non-viral vectors, accordingly improving the efficiency and safety of gene therapy, which will make them the most promising vectors for pancreatic cancer.     

Advances in adenovirus-mediated p53 cancer gene therapy

Introduction: The tumor suppressor p53 gene regulates diverse cellular processes, such as cell-cycle arrest, senescence, apoptosis and autophagy, and it is frequently inactivated by genetic alterations in ～ 50% of all types of human cancers. To restore wild-type p53 function in p53-inactivated tumors, adenovirus-mediated p53 gene therapy has been developed as a promising antitumor strategy in preclinical experiments and clinical studies.

Areas covered: This review focuses on the clinical relevance of replication-deficient adenovirus vectors that carry the wild-type p53 gene (Ad-p53; Advexin, Gendicine and SCH-58500) in clinical studies of patients with various cancers and the future perspectives regarding conditionally replicating adenovirus vectors expressing the wild-type p53 gene (CRAd-p53; AdDelta24-p53, SG600-p53, OBP-702) in preclinical experiments. Moreover, the recent advances in our understanding of the molecular basis for the p53-mediated tumor suppression network induced by Ad-p53 and CRAd-p53 vectors and the combination therapies for promoting the therapeutic potential of adenovirus-mediated p53 gene therapy are discussed.

Expert opinion: Exploration of the molecular mechanism underlying the p53-mediated tumor suppression network and the effective strategy for enhancing the p53-mediated cell death signaling pathway would provide novel insights into the improvement of clinical outcome in p53-based cancer gene therapy.     

An update on gene therapy for lysosomal storage disorders

Introduction: Gene therapies can be envisioned for many disorders where conventional therapies fall short. Lysosomal Storage Disorders (LSDs) are inherited, mostly monogenic, disorders resulting from deficient lysosomal enzyme or co-factor activity. Existing standard-of-care treatments for LSDs are expensive and can negatively impact quality-of-life. They also may not be sufficiently efficacious. LSDs are particularly amenable to gene therapy as modified cells can secrete functional enzyme that can also correct unmodified cells. Gene therapies may thus be able to provide sustained long-term correction for LSD patients.

Areas covered: We highlight recent advances and discuss advantages/disadvantages of gene therapies with a focus on lentiviral and adeno-associated virus vectors currently in clinical trials for LSDs. We also mention promising strategies that are close to clinical testing. We emphasize protocols using ex vivo hematopoietic stem cell-directed gene therapy, systemic/liver-directed gene therapy, and brain-directed gene therapy. We also discuss next-generation gene therapy approaches and how they may address emerging challenges in the field.

Expert opinion: Gene therapy is still in its infancy with respect to LSDs. However, efficacy and safety has been demonstrated in numerous pre-clinical studies, and promising clinical results suggest that gene therapy treatment for several LSDs is a real possibility.     

MicroRNAs as potential target gene in cancer gene therapy of gastrointestinal tumors

Introduction: MicroRNA (miRNA) is a small non-coding RNA, which negatively regulates the expression of many target genes, thereby contributing to the modulation of diverse cell fates. Recent advances in molecular biology have revealed the potential role of miRNAs in tumor initiation, progression and metastasis. Aberrant regulation of miRNAs has been frequently reported in a variety of cancers, including gastrointestinal tumors, suggesting that cancer-related miRNAs are promising as novel biomarkers for tumor diagnosis and are potential target genes for cancer gene therapy against gastrointestinal tumors.

Areas covered: The review focuses on the role of specific miRNAs (miR-192/194/215 and miR-7) in the differentiation of gastrointestinal epithelium and on the role of tumor-suppressive (miR-34, miR-143, miR-145) and oncogenic miRNAs (miR-21, miR-17-92 cluster) in gastrointestinal tumors. Furthermore, the potential role of miRNAs as novel biomarkers and target genes for cancer gene therapy against gastrointestinal tumors are discussed. We will also outline the potential clinical application of miRNAs for tumor diagnosis and cancer gene therapy against gastrointestinal tumors.

Expert opinion: Exploration of tumor-related miRNAs would provide important opportunities for the development of novel cancer gene therapies aimed at normalizing the critical miRNAs that are deregulated in gastrointestinal tumors.     

Tissue-specific promoters for cancer gene therapy

Adenoviral cancer gene therapy approaches have resulted in promising recent results. Following only a decade of intense development, some of the crucial obstacles are now being overcome. Insufficient transduction has been the main limitation of earlier approaches. A new approach for increasing transduction of tumour cells is utilisation of replication-competent oncolytic agents, such as conditionally replicating adenoviruses (CRADs). The anti-tumour effect is caused by replication of the virus per se and, thus, replication must be restricted to tumour cells to protect normal tissues from damage. Tissue-specific promoters (TSPs) represent a powerful tool for decreasing the toxicity of cancer gene therapy to normal tissues and have previously been utilised for specific mutation compensation or delivery of prodrug-converting enzymes. However, TSPs can also be used for controlling crucial viral replication regulators and consequent restriction of replication to tumour cells. Initial clinical trials have demonstrated the safety and suggested efficacy for TSP-controlled CRADs as a novel approach for cancer gene therapy.     

Human cancer gene therapy with cytokine gene-modified cells

Cytokines can impede tumour growth and activate innate and adaptive immune responses, leading to elimination of cancer cells. For many years, it was believed that systemic administration of recombinant cytokines might become a standard treatment of different cancer types. However, due to a high toxicity of therapeutic doses and a low efficacy, even in combination with chemotherapy, this strategy is generally not accepted. On the other hand, cancer gene therapy approaches utilising cells modified with cytokine genes seem to represent a novel promising approach. For the last decade, numerous Phase I and II clinical trials evaluating different therapies based on cytokine gene-modified cells have been carried out. In the early studies, several strategies have been shown to improve clinical outcomes and induce strong antitumour immune responses. Recently, a few prospective, randomised, Phase III clinical trials have been initiated in order to finally determine the efficacy of particular cancer immunogene therapy strategies. This article reviews the present status and perspectives of clinical trials of cancer immunotherapies utilising cytokine gene-modified cells.     

人骨肉瘤mta1 mRNA的siRNA载体构建及其对mta1沉默作用

目的本实验拟构建针对mta1 mRNA的siRNA重组载体,将siRNA对应的DNA发卡样双链序列克隆入载体内,位于H1启动子下游,转染入人骨肉瘤细胞内,在细胞内表达mta1靶向的siRNA分子,特异性沉默mta1的表达。方法依据GenBank中mta1基因(NM-004689)的序列,用设计分析软件进行设计候选序列,确定针对mta1的siRNA序列。合成mta1发卡样siRNA寡核苷酸,退火成双链DNA,克隆入T载体,利用α互补和T7/SP6PCR扩增筛选鉴定重组子pGEM-T-mta1;用BglⅡ和HindⅢ双酶切重组子pGEM-T-mta1和siRNA表达载体pSuperneo;将双粘mta1发卡样siRNA片段亚克隆入siRNA表达载体pSuperneo中;利用BglⅡ和HindⅢ双酶切筛选鉴定重组子pSuperneo-mta1;对插入序列进行DNA序列分析。将siRNA表达载体pSuperneo-mta1转入人骨肉瘤细胞系MG-63中,用RT-PCR方法检测细胞mta1 mRNA表达水平。结果转染pSuperneo-mta1的实验组骨肉瘤细胞中mta1 mRNA表达几乎完全被抑制,而2个对照组(无关siRNA对照组和空载体对照组)和未转染的骨肉瘤细胞系MG-63细胞中都存在较高水平的mta1 mRNA表达。结论设计出针对mta1的发卡样siRNA寡核苷酸片段,成功构建出针对mta1的siRNA表达载体pSuperneo-mta1,pSuperneo-mta1转染骨肉瘤细胞后能显著降低细胞mta1 mRNA表达。     

siRNA沉默细胞周期相关激酶基因对鼠结肠癌生长及其细胞凋亡的影响

目的探讨siRNA沉默细胞周期相关激酶(CCRK)基因对鼠结肠癌生长及其凋亡的影响。 方法将50只BALB/C(nu/nu)裸鼠皮下接种人类结肠癌SW480细胞,将其中建立移植瘤裸鼠模型成功的30只裸小鼠按照单纯随机抽样方法随机分为3组,分别接种siRNACCRK转染的人类结肠癌SW480细胞株(实验组)、正常培养的SW480细胞(空白对照组)和以转染携带无关序列片段sh RNA慢病毒的SW480细胞(阴性对照组)。第42天处死裸鼠后取下肿瘤组织,比较各组肿瘤重量、CCRKmRNA表达量、CCRK蛋白表达和细胞凋亡的变化。 结果转染siRNA组肿瘤重量、CCRK mRNA和CCRK蛋白表达[(0.54±0.15)g,1.14±0.24和0.18±0.05]表达显著低于空白对照组[(1.05 ± 0.14)g,2.41±0.42和0.49±0.07]和空siRNA载体组[(1.07 ±0 .12)g,2.39±0.47,0.47±0.09;F＝21.18,23.47,90.03;P<0.01];空白对照组与空siRNA载体组比较差异无统计学意义(q＝0.98,1.07,1.13;P>0.05)。实验组沉默CCRK基因后组织中细胞凋亡率为(21.23±1.12)%,明显高于空白对照组(6.78±0.37)%和阴性对照组(7.25±0.32)%,差异有统计学意义(F＝56.936,P<0.01);但空白对照组和空siRNA载体组之间差异无统计学意义(q＝1.78,P>0.05)。 结论CCRK靶向siRNA表达载体接种结肠癌裸鼠后能显著降低结肠癌瘤体的增长,抑制CCRK基因和蛋白表达;siRNA沉默CCRK基因能促进其凋亡,CCRK基因有望成为结直肠癌治疗的新靶点。     

In situ gene therapy for prostate cancer: immunomodulatory approaches

The development of effective treatments for prostate cancer is thwarted by the natural history of the disease. The biological and clinical potential of most individual cancers is uncertain. In many cases the disease will not progress to clinical significance but experimental and clinical studies indicate that prostate cancer can and may metastasise early in the course of the disease from relatively small foci (i.e., not necessarily the largest or index cancer). Localised prostate cancer is potentially curable with localised therapies (radical prostatectomy or irradiation therapy). However, there are no curative therapies for metastatic prostate cancer. Gene therapy, especially those approaches with an immunomodulatory component, may provide additional therapeutic options with the potential to affect both localised and systemic disease. We have pioneered the development and application of in situ gene therapy protocols using adenoviral vectors to transduce specific genes that generate cytotoxic activity and/or a systemic antitumour immune response. In addition we have completed initial studies that demonstrate the therapeutic potential of adenoviral vector-mediated gene modified cell-based vaccines. Our review discusses preclinical studies focused on the development of immunostimulatory in situ gene therapy approaches that hopefully will provide novel and effective treatments for localised and metastatic prostate cancer.     

Dissecting insect development: baculovirus-mediated gene silencing in insects

A novel concept applying baculovirus-mediated gene silencing to study insect gene function and regulation is described in this paper. A recombinant baculovirus, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), was constructed with the juvenile hormone esterase (JHE) gene from the tobacco budworm Heliothis virescens in the antisense orientation, driven by the viral p10 promoter. Infection with this recombinant greatly reduced the haemolymph JHE level and resulted in aberrant morphogenesis of final-instar H. virescens larvae. The body organization remained larval, although the cuticle became hard and brown, similar to pupal cuticle. These results demonstrated that baculovirus-mediated gene silencing can be accomplished and utilized to dissect insect development and to design a new class of baculovirus insecticides.     

Differential responses of migratory locusts to systemic RNA interference via double‐stranded RNA injection and feeding

The migratory locust, Locusta migratoria, is one of the most destructive agricultural pests and has been widely used as a model system for insect physiology, neurobiology and behavioural research. In the present study, we investigated the effects of RNA interference (RNAi) using two delivery methods for double‐stranded RNA (dsRNA) molecules, namely, injection and feeding, to develop a potential new pest control strategy. Our results showed that locusts have a sensitive and systemic response to the injection of dsRNAs in a dose‐dependent manner, but do not respond to the feeding of dsRNAs. Further experiments suggested that the ineffectiveness of dsRNA feeding was attributable to the rapid degradation of dsRNA, which was probably induced by nuclease enzymes in the locust midgut. Moreover, we identified almost all the homologous genes involved in the endocytosis‐mediated dsRNA uptake from the locust genome, which provided possible clues regarding the dsRNA uptake mechanisms from the intestine to the midgut epithelium. These findings reveal the differential response models of fourth instar locust nymphs to dsRNA delivery methods, contribute to the current understanding of insect RNAi mechanisms and provide important information for the further application of RNAi as a genetic tool and pest control strategy.     

Towards a durable RNAi gene therapy for HIV-AIDS

Background: RNA interference (RNAi) can be employed as a potent antiviral mechanism Objective: To discuss RNAi approaches to target pathogenic human viruses causing acute or chronic infections, in particular RNAi gene therapy against HIV-1. Methods: A review of relevant literature. Results/conclusions: The future of antiviral RNAi therapeutics is very promising. RNAi was discovered only a decade ago, and although we are still in the early days, the first clinical trials are already ongoing.     

MTA1基因沉默对宫颈癌细胞增殖及转移能力的影响

【】 目的 探讨MTA1基因沉默对宫颈癌细胞转移增殖的影响。方法 用慢病毒转染法稳定转染Siha细胞,转染同时进行实验分组。用RT-PCR技术和蛋白印迹法测定宫颈癌Siha、Hela细胞中MTA1 mRNA和蛋白的表达。Transwell体外侵袭实验检测转染后Siha细胞的迁移能力,MTT检测法和克隆形成实验法检测转染后Siha细胞的细胞增殖能力,FACS细胞凋亡法检测转染后Siha细胞的细胞凋亡率,PI-FACS细胞周期法检测转染后Siha细胞的各个生长周期的细胞数。结果 (1)Transwell体外侵袭实验：相比NC组,KD组Transwell转移率经T-Test分析P=2.61E-08<0.05。(2)MTT检测结果表明：相比NC组,KD组细胞增殖减缓。(3)克隆形成实验结果显示：相比NC组,KD组克隆数经T-Test分析P值=0.0006<0.05。(4)FACS细胞凋亡：相比两个对照组,KD组凋亡率经T-Test分析P<0.05。结论 MTA1基因促进宫颈癌细胞的转移和增殖,沉默MTA1基因表达能使宫颈癌细胞增殖及迁移能力下降,加速宫颈癌细胞的凋亡,为抑制肿瘤转移奠定实验基础,最后为宫颈癌的新型药物性靶向治疗提供有力的实验依据。     

Self-assembled siRNA-PLGA conjugate micelles for gene silencing

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3′ end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA-PLGA hybrid conjugates. siRNA-PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~ 20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA-PLGA micelles in aqueous solution, stable siRNA-PLGA/LPEI micelles with a size of ~ 30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA-PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.     

Vascular-targeted cancer gene therapy

As a consequence of the dramatic progress that has been made in recent years towards elucidating the diverse molecular events involved in the development and pathogenesis of malignant disease, there is now no shortage of genes that can be exploited or targeted in the context of cancer gene therapy. Many of these have been shown to be effective both in vitro and in various animal models, and a number have progressed to the clinic. The results of these later studies, although generally encouraging, are perhaps less dramatic than one might have hoped. Although a number of factors undoubtedly contribute to this finding, it is evident that a major reason relates to the difficulties implicit in achieving efficient in vivo gene transfer, particularly in a clinical context. Targeting gene therapy, not to the malignant population, but instead to the vasculature upon which the survival and growth of a tumour depends constitutes an alternative approach that overcomes some of the delivery problems associated with established tumour cell-directed strategies.