Non-viral delivery of RNA interference targeting cancer cells in cancer gene therapy

RNA interference (RNAi) is a collection of small RNA-directed mechanisms that result in sequence-specific inhibition of gene expression. RNAi delivery has demonstrated promising efficacy in the treatment of genetic disorders in cancer. Although viral vectors are currently the most efficient systems for gene therapy, potent immunogenicity, mutagenesis, and the biohazards of viral vectors remain their major risks. Various non-viral delivery vectors have been developed to provide a safer approach for gene delivery, including polymers, peptides, liposomes, and nanoparticles. However, some concerns and challenges of these non-viral gene delivery approaches remain to be overcome. In this review, we summarize the recent progress in the development of non-viral systems delivering RNAi and the currently available preclinical and clinical data, and discuss the challenges and future directions in cancer therapy.     

Progress in developing cationic vectors for non-viral systemic gene therapy against cancer

Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection and for multi-located tumor sites, i.e. metastases. Non-viral vectors based on the use of cationic lipids or polymers appear to have promising potential, given the problems of safety encountered with viral vectors. Using these non-viral vectors, the current challenge is to obtain a similarly effective transfection to viral ones. Based on the advantages and disadvantages of existing vectors and on the hurdles encountered with these carriers, the aim of this review is to describe the "perfect vector" for systemic gene therapy against cancer.     

Polysaccharide gene transfection agents

Gene delivery is a promising technique that involves in vitro or in vivo introduction of exogenous genes into cells for experimental and therapeutic purposes. Successful gene delivery depends on the development of effective and safe delivery vectors. Two main delivery systems, viral and non-viral gene carriers, are currently deployed for gene therapy. While most current gene therapy clinical trials are based on viral approaches, non-viral gene medicines have also emerged as potentially safe and effective for the treatment of a wide variety of genetic and acquired diseases. Non-viral technologies consist of plasmid-based expression systems containing a gene associated with the synthetic gene delivery vector. Polysaccharides compile a large family of heterogenic sequences of monomers with various applications and several advantages as gene delivery agents. This chapter, compiles the recent progress in polysaccharide based gene delivery, it also provides an overview and recent developments of polysaccharide employed for in vitro and in vivo delivery of therapeutically important nucleotides, e.g. plasmid DNA and small interfering RNA.     

载体与基因转染的研究进展

基因载体是指将基因或其它核酸物质运载到细胞中的工具.其化学本质可以是蛋白质或多肽、核酸、脂类、糖类、其它有机分子或它们的复合物.基因传递系统是基因治疗的重要组成部分,也是目前基因治疗的瓶颈.现有的基因载体包括两类.即病毒载体和非病毒载体.病毒载体转染效率高,但由于其转染具有免疫原性和致突变性限制了它的应用;非病毒载体系统具有低毒、低免疫原性和相对靶向性等优点,是新兴发展起来的基因转移系统.就各种载体的最新研究进展作一综述.     

Viral based gene therapy for prostate cancer

In the last few years, significant advances in gene therapy have been made as a result of advances in many areas of molecular and cell biology, including the improvement of both viral and nonviral gene delivery systems, discovery of new therapeutic genes, better understanding of mechanism of disease progression, exploration of tissue specific promoter, receptor- and antibody-mediated targeting delivery, and development of better prodrug enzyme/prodrug systems. In this article, viral based gene therapy for prostate cancer will be reviewed and discussed. The areas of emphasis in this review are: choice of viral vectors, comparison of delivery routes, development of prostate-targeted viruses, choice of therapeutic genes and strategies including corrective gene therapy (tumor suppressor gene and anti-oncogene gene approaches), suicide gene therapy, programmed cell death therapy, immunomodulation therapy, and conditional oncolytic virus approach. Among them, several examples will be discussed in detail for the scientific basis and therapeutic applications. In addition, prostate cancer gene therapy clinical trials, unresolved problems and future directions in this field will also be described.     

New therapeutic approaches based on gene transfer techniques

Conclusions The experimental results from the use of gene transfer techniques for cancer therapy are promising but, at the same time, somewhat perplexing. Progress will depend on the further understanding of the biology of tumour interaction with the immune system, as this would be important for the development of optimal strategies for harnessing an effective immune response against tumours. On the other hand, extrapolation from laboratory models to human disease is limited and, therefore, there is a requirement for well-planned clinical studies. Although numerous viral and non-viral methods of gene transfer are available currently, in order to achieve realistic therapeutic efficacy for in vivo gene therapy of cancer there is a pressing need to improve on existing vectors. Hopefully, effort being directed at this objective will result in the development of safer, more highly efficient and well-targeted vectors which in turn will lead to the reality of wide clinical application.     

Adenovirus-mediated p53 tumor suppressor gene therapy of osteosarcoma

The clinical outcome for osteosarcoma (OS) remains discouraging despite efforts to optimize treatment using conventional modalities including surgery, radiotherapy and chemotherapy. Novel therapeutic approaches based on our expanding understanding of the mechanisms of tumor cell killing have the potential to alter this situation. Tumor suppressor gene therapy aims to restore the function of a tumor suppressor gene lost or functionally inactivated in cancer cells. One such molecule, the p53 tumor suppressor gene plays a critical role in safeguarding the integrity of the genome and preventing tumorigenesis. Introduction of wild-type (wt) p53 into transformed cells has been shown to be lethal for most cancer cells in vitro, but clinical trials of p53 gene replacement have had limited success. Analysis of these clinical trials highlighted the insufficient efficacy of current vectors and low proapoptotic activity of wt p53 as a single agent in vivo. In this review, a contemporary summarization of the current status of adenovirus-mediated p53 gene therapy of OS is presented. Advancement in our understanding of p53 tumor suppressor activity, the molecular biology of chemoresistant OS, and recent advances in tumor targeting with adenoviral vectors are also addressed. Based on these parameters, prospects for future investigations are proposed.     

Adenovirus-based cancer gene therapy

Over the past decade, adenovirus (Ad)-based vectors have been used extensively in the context of cancer gene therapy. Two basic strategies have been pursued for the use of Ad vectors in cancer gene therapy: 1) approaches aimed at direct tumor cell killing through delivery of replicating oncolytic viruses or non-replicating vectors encoding tumor suppressor genes, suicide genes or anti-angiogenic genes, and 2) immunotherapeutic approaches aimed at inducing host anti-tumor immune responses that can destroy tumor cells at both primary and metastatic locations. Both strategies offer the potential of selective tumor cell destruction without damage to normal tissues. Extensive pre-clinical and clinical studies have been conducted based on these strategies. Encouraging results have been obtained but robust clinical efficacy remains elusive. Several obstacles limiting the therapeutic activity of Ad vectors have been encountered, including efficiency of tumor cell transduction and inhibition of efficacy by anti-Ad host immune responses. However, expanding knowledge in the areas of Ad biology and tumor biology continues to lead to increasingly sophisticated approaches to address these issues. A review of various Ad-based cancer gene therapy approaches and recent progress in the area are presented herein.     

Airway epithelium directed gene therapy for cystic fibrosis

Gene therapy is a promising therapeutic modality for the treatment of cystic fibrosis (CF). Despite a better understanding of the molecular organization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and mutations resulting in pathophysiological and phenotypic alterations, several forms of treatments including gene therapy have failed to yield clinical success. Major limitations for the delivery of drugs and gene therapy vectors from reaching target cells in CF patients lie in physical and immunological barriers of airway epithelium. Over the last decade, non-viral and viral gene therapy approaches have been tested in preclinical studies and human clinical trials of CF. Outcomes of these studies have helped to identify hurdles that need to be overcome before such approaches can be routinely applied to patients. In addition to the physiological and immunological barriers of airway epithelium, vector transduction is also impaired by the absence or low-abundance of cellular receptors and co-receptors for viral binding and internalization. Thus, the initial enthusiasm for gene replacement therapy for CF following cloning of the CFTR gene dampened, as more limitations were recognized. Research directed towards improving the efficiency of gene transfer technology in CF, is focused on testing of compounds to enhance vector permeability and trafficking, identification and development of vectors which can transduce through alternate pathways, identification of airway epithelium-specific targeting ligands, and the identification of stem cells for combining cell therapy and gene therapy by ex vivo methods. Details provided in this article will give a comprehensive analysis of the prospects and limitations in CF gene therapy using viral and non-viral vectors.     

Gene therapy for the prevention of ischemia/reperfusion injury in organ transplantation

Introduction of gene therapy into molecular medicine has been gaining increasing interest. Although treatment of various diseases e.g. monogenetic defects or cancer by using gene transfer technologies has been extensively probed, the clinical success has been limited. However, recent experimental data suggest that gene therapy may represent an attractive and powerful approach in preventing ischemia/reperfusion injury as well as organ rejection in transplant recipients. Easy and selective access to the donor organ facilitates the reduction of potentially harmful systemic side effects of gene therapy vectors. By introducing anti-apoptotic or cytoprotective genes, these studies focused on the protection of the transplant from the apoptotic cell death. In addition, down-regulation of adhesion molecules and/or blockade of gene expression in the graft itself also ameliorated ischemia/reperfusion injury. This review summarizes the current progress on gene therapy application in combating ischemia-reperfusion injury in organ transplantation. Although the use of viral vectors is emphasized, non-viral gene transfer techniques are also discussed. Future development of novel, low-immunogenic vectors should further contribute to the minimization of ischemia/reperfusion injury, and thus to the overall success of organ transplantation.     

Non-viral approach toward gene therapy of cystic fibrosis lung disease

Since Cystic Fibrosis (CF) is an autosomal recessive disorder due to mutations in the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, studies towards a gene therapy approach to its treatment followed immediately upon the cloning of the gene. It was demonstrated that the insertion of a single copy of the wild-type gene restored the normal phenotype in CF cells in vitro. Encouraging results were obtained in many in vivo model systems (CF transgenic mice) involving viral as well as non-viral vectors, which demonstrated the recovery of CFTR function in the airways. These results constituted the basis for human studies. Of those with a non-viral approach, a total of seven clinical trials using cationic lipids have reported data on efficiency, efficacy and safety. An effective gene transfer approach for the treatment of CF lung disease is not however imminent: low transfection efficiency and poor maintenance of gene expression are so far the main obstacles on this therapeutic path. On the other hand, no important adverse effects have been documented and repeated administration in humans is possible. The understanding of tissue and cellular barriers is a prerequisite for the development of more efficient non-viral gene therapy protocols for CF patients. While cationic lipids have been shown to be blocked by the mucous airway barrier and not be able to transfect differentiated respiratory epithelial cells, a new class of non-viral vectors, cationic polymers, are endowed with chemical and biological properties that make them more efficient in mediating gene transfer than lipids. Cationic polymers, such as polyethylenimine, are promising vectors for CF lung gene therapy.     

Progress in cationic lipid-mediated gene transfection: a series of bio-inspired lipids as an example

Over the last several years, various gene delivery systems have been developed for gene therapy applications. Although viral vector-based gene therapy has led to the greatest achievements in animal and human studies, synthetic non-viral vectors have also been developed as they offer several advantages over viral systems, including lower immunogenicity and greater nucleic acid packaging capacity. Nevertheless, the transfection efficiency of the current non-viral gene carriers still needs to be improved, especially as regards direct in vivo transfection. In particular, cationic lipid/nucleic acid complexes (termed lipoplexes) have been the subject of intensive investigation with a view to optimize their performance and to better understand their mechanisms of action, and consequently to design new approaches to overcome the critical barriers of cationic liposome-mediated gene delivery. A possible strategy may rely on considering the membrane constituents and properties of the vast variety of living organisms as a source of inspiration for the design of biocompatible, non-toxic and effective novel artificial liposomal systems. Thus, the present forward-looking review provides an overview of the progress already made during the last years in the field of cationic lipid-mediated gene transfection and also focuses on a series of novel bio-inspired lipids for both in vitro and in vivo gene transfection.     

基因治疗中的核酸药物及非病毒递送载体的研究进展

基因治疗是针对基因异常相关疾病的终极治疗技术,各种具有不同机制的核酸药物的出现为基因治疗带来了更多的可能性。但是,由于存在体内稳定性差、难以高效进入靶细胞等问题,核酸药物需要载体的帮助而进入目标细胞并到达特定的胞内位置,因此,开发安全高效的核酸递送系统是基因治疗的基石。与病毒载体相比,非病毒载体具有更高的安全性,但转染效率较低。随着纳米技术的发展,非病毒载体的效率得到了显著的提升,进入临床研究的数量逐渐增多。本文简要介绍基因治疗中的核酸药物及其递送载体,对非病毒核酸药物递送技术的瓶颈及进展做综合评述。     

Light directed gene transfer by photochemical internalisation

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.     

Advances in liver-directed gene therapy for hepatocellular carcinoma by non-viral delivery systems

Hepatocellular carcinoma (HCC) is a malignancy with a high mortality. Gene therapy provides a promising way for the treatment of HCC. Efficient gene delivery system, suitable gene target and appropriate way of administration together determine the effect of gene therapy for HCC. In recent years, employing non-viral gene delivery systems in gene therapy for HCC has attracted a lot of attention. Compared with viral vectors, non-viral gene delivery systems are nearly non-immunogenic, relatively safer, less expensive to produce and can carry a good many of genetic materials. But the transfection efficiency of these vectors still needs to be improved. And the liver targeting is another problem that needs to be solved. Attaching ligands to the non-viral vectors to enhance the targeting ability to the specific receptor and targeting to molecular targets of HCC are the effective strategies. Adopting suitable ways of administration is also a factor that plays an important role to achieve liver targeting. This review introduced the advances in liver-targeted gene therapy by non-viral vectors including the efforts to overcome the low transfection efficiency and enhance the liver targeting effect.     

Targeting tumor angiogenesis with gene therapy

A recent target of cancer gene therapy is tumor angiogenesis. An appealing feature of gene therapy targeting the tumor vasculature is that it is readily accessible, particularly when the carrier and its gene are administered systemically. Several gene-based viral and nonviral therapies that target tumor angiogenesis have demonstrated the "proof of principle" of antiangiogenic therapy in preclinical models. The utility of antiangiogenic gene therapy in a clinical setting will depend in large part on developing vectors with minimal toxicity and with increased in vivo transfection efficiency. In this review, we discuss the current status and future directions of antiangiogenic gene therapy.     

Cytokine gene transfer into dendritic cells for cancer treatment

Bone marrow-derived dendritic cells have been used to treat established experimental tumors by unleashing a cellular immune response against tumor antigens. Such antigens are artificially loaded onto dendritic cells' antigen-presenting molecules by different techniques including incubation with synthetic antigenic determinants, tumor lysates or nucleic acids encoding for those relevant antigens. Ex vivo gene transfer with viral and non-viral vectors is frequently used to obtain expression of the tumor antigens and thereby to formulate the therapeutic vaccines. Efficacy of the approaches is greatly enhanced if dendritic cells are transfected with a number of genes which encode immunostimulating factors. In some cases, such as with IL-12, IL-7 and CD40L genes, injection inside experimental malignancies of thus transfected dendritic cells induces complete tumor regression in several models. In this case tumor antigens are captured by dendritic cells by still unclear mechanisms and transported to lymphoid organs where productive antigen presentation to T-cells takes place. Many clinical trials testing dendritic cell-based vaccines against cancer are in progress and partial clinical efficacy has been already proved. Transfection of genes further strengthening the immunogenicity of such strategies will join the clinical club soon.     

Current Status and Future Directions of Gene and Cell Therapy for Cystic Fibrosis

Although the development of gene therapy for cystic fibrosis (CF) was high priority for many groups in academia and industry in the first 10-15 years after cloning the cystic fibrosis transmembrane conductance regulator (CFTR) gene, more recently active research into CF gene therapy is only being performed by a small number of committed groups. However, despite the waning enthusiasm, which is largely due to the realization that gene transfer into lungs is more difficult than originally thought, and the fact that meaningful clinical trials are expensive and difficult to perform, gene therapy continues to hold promise for the treatment of CF lung disease. Problems related to repeat administration of adenovirus and adeno-associated virus-based vectors led to a focus on non-viral vectors in clinical trials. However, the recent evidence that lentiviral vectors may be able to evade the immune system and, thereby, allow for repeat administration and long-lasting expression opens new doors for the use of viral vectors in the context of CF gene therapy. In addition, early pre-clinical studies have recently been initiated to address cell therapy-based approaches for CF. In this review, we discuss recent developments with viral and non-viral vectors and cell therapy, and provide an update on clinical gene therapy studies.     

Synthetic and natural polycations for gene therapy: state of the art and new perspectives

Currently, the major drawback of gene therapy is the gene transfection rate. The two main types of vectors that are used in gene therapy are based on viral or non-viral gene delivery systems. There are several non-viral systems that can be used to transfer foreign genetic material into the human body. In order to do so, the DNA to be transferred must escape the processes that affect the disposition of macromolecules. These processes include the interaction with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is also a potential obstacle for functional delivery to the target cell. Cationic polymers have a great potential for DNA complexation and may be useful as non-viral vectors for gene therapy applications. The objective of this review was to address the state of the art in gene therapy using synthetic and natural polycations and the latest strategies to improve the efficiency of gene transfer into the cell.     

Countering hepatitis B virus infection using RNAi: how far are we from the clinic?

Globally, persistent HBV infection is a significant cause of public health problems. Currently available HBV therapies have variable efficacy and there is a need to develop improved treatment to prevent cirrhosis and hepatocellular carcinoma. Although RNA interference (RNAi)-based approaches have shown promise, accomplishing safe and sustained silencing by RNAi activators, as well as their efficient delivery to hepatocytes have hampered clinical translation of this very promising technology. Expressed silencers may be produced in a sustained manner from stable DNA templates, which makes them suited to treatment of chronic HBV infection. DNA expression cassettes can be incorporated into both viral and non-viral vectors, but in vivo delivery of these cassettes with non-viral vectors is currently inefficient. Synthetic short interfering RNAs (siRNAs), which may be chemically modified to improve stability, specificity and efficacy, are more conveniently delivered to their cytoplasmic sites of action with synthetic non-viral vectors. However, the short duration of action of this class of RNAi activator is a drawback for treatment of chronic HBV infection. Despite the impressive progress that has been made in developing highly effective HBV gene silencers, challenges continue to face implementation of RNAi-based HBV therapy. This review will discuss the current status of the topic and consider the developments that are required to advance RNAi-based HBV therapy to clinical application.