Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery

Nanotechnology, although not a new concept, has gained significant momentum in recent years. This stems partly from the realization that nanosystems have significantly different biological properties from large-sized systems (e.g. implants or microparticles) that could be used effectively to overcome problems in drug and gene therapy. In drug therapy, we face the problems of inefficacy or nonspecific effects; hence, nanosystems are being developed for targeted drug therapy. In gene therapy using non-viral systems, the main issues are relatively transient gene expression and lower efficiency than viral vectors. Research efforts have focused on understanding the barriers in gene delivery so that non-viral systems can be developed that are as effective as viral systems in gene transfection. Understanding the molecular mechanisms that underlie the interactions of nanosystems with the cell, their uptake properties and retention will be crucial for the successful development of these systems.     

Chimeric viral vectors--the best of both worlds?

Gene therapy to correct defective genes requires efficient gene delivery and long-term gene expression. The vector systems currently available have not allowed the simultaneous provision of both of these goals. Several groups are now developing chimeric viral vector systems that incorporate the favorable attributes of two different viral vectors. These chimeric vectors might allow the goals for specific gene therapy applications to be realized.     

米非司酮基因诱导调控系统

在基因表达和基因治疗研究中,需要目的基因在特定的时间以适当的水平实现其表达,目的基因过度表达或不适当的表达将影响实验结果,在疾病治疗中甚至会产生致命的副作用,实现对目的基因的表达时间和表达水平的精确调控是一个非常关键的问题。目前生物学家们已构建了多种新型的基因表达调控系统,其中米非司酮诱导调控系统具有诱导效率高,背景表达低,安全性高等诸多优点,是基因调控研究中的重要进展,也是目前最有应用前景的调控系统之一。本文就其结构设计和应用研究方面的进展作一简要综述。     

Gene therapy: a battle against biological barriers

One factor critical to successful human gene therapy is development of efficient gene delivery systems. Although numerous vector systems for gene transfer have been developed, a perfect vector system has not yet been constructed. Difficulties of in vivo gene transfer appear to result from resistance of living cells to invasion by foreign materials and from interference of cellular functions. We should analyze what barriers in tissues affect in vivo gene transfection and how to solve these problems for gene therapy. In this review article, the biological barriers to in vivo gene transfection are discussed and possible solutions to each barrier are discussed with respect to construction of a perfect gene therapy vector system.     

Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems

Positively and tightly regulated gene expression is essential for gene function and gene therapy research. The currently-used inducible gene expression systems include tetracycline (Tet-on and T-REx), ecdysone, antiprogestin and dimerizer-based systems. Adenovirus (Ad) vectors play an important role in gene function and gene therapy research for their various advantages over other vector systems. Previously, we reported the inferiority of the Tet-on system as an inducible gene expression system in the context of Ad vectors in comparison with the Tet-off system. In this study, to identify an optimal system for regulated gene expression from Ad vectors, we made a rigorous direct comparison of these five inducible gene expression systems in three cell lines using the luciferase reporter gene. The highest sensitivity to the respective inducer was that of the dimerizer system, followed by the antiprogestin system. The lowest basal expression and the highest induction factor were both characteristic of the dimerizer system. Furthermore, the dimerizer and T-REx systems exhibited much higher induced expression levels than the other three systems. The elucidation of the characteristic features of each system should provide important information for widespread and feasible application of these systems. Overall, these results suggest the most appropriate inducible gene expression system in the context of Ad vectors to be the dimerizer system.     

基因治疗的输送屏障及输送载体的研究

基因治疗是将可具有治疗性的基因导入病变细胞以达到治疗遗传性疾病或获得性功能缺损疾病的治疗手段,是一种极具潜力的新型治疗方法。然而基因治疗面临着一系列一陆床应用障碍,其中缺乏理想的基因输送载体是首要问题。绝大多数基因治疗方案受困缺乏安全有效的基因输送手段,载体要达到目的地发挥作用,需要克服一系列复杂的体内生物屏障,包括细胞外屏障和细胞内屏障。目前基因输送载体主要分为病毒载体和非病毒载体,其中病毒载体天然进化至可进入宿主细胞,具有输送效率高,靶向性好的特点,但存在长期安全性的缺点。非病毒载体主要包括阳离子脂质体和阳离子聚合物,由于易于制备和无免疫原性、安全性好,被认为是更有潜力的输送载体,是目前研究的重点。本文结合基因治疗输送屏障的理论基础及临床研究,对基因输送载体系统的现状进行了综述。     

Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker.

Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.     

Current developments in the design of onco-retrovirus and lentivirus vector systems for hematopoietic cell gene therapy

Over the past dozen years, the majority of clinical gene therapy trials for inherited genetic diseases and cancer therapy have been performed using murine onco-retrovirus as the gene delivery vector. The earliest systems used were relatively inefficient in both the rates of transduction and expression of the transgene. Formidable obstacles inherent in the cell biology and/or the immunology of the target cell systems limited the efficacy of gene therapy for many target diseases. Development of novel retrovirus gene transfer systems that are in progress have begun to overcome these obstacles. Evidence of this progress is the recent successful functional correction of the immune T and B lymphocyte deficiency in patients with X-linked severe combined immunodeficiency (X-SCID) and adenosine deaminase (ADA)-deficient SCID following onco-retrovirus vector ex vivo transduction of autologous marrow stem cells [Science 296 (2002) 2410; Science 288 (2000) 669; N. Engl. J. Med. 346 (2002) 1185]. These achievements of prolonged clinical benefit from gene therapy were tempered by the finding of insertional mutageneses in two of the treated X-SCID patients [N. Engl. J. Med. 348 (2003) 255].     

A concise peer into the background, initial thoughts and practices of human gene therapy

The concept of human gene therapy came on the heels of fundamental discoveries on the nature and working of the gene. However, realistic prospects to correct the underlying cause of recessive genetic disorders through the transfer of wild-type alleles of defective genes had to wait for the arrival of recombinant DNA technology. These techniques permitted the isolation and insertion of genes into the first recombinant delivery systems. The realization that viruses are natural gene carriers provided inspiration for gene therapy and, as engineered vectors, viruses became prominent gene delivery vehicles. Nonetheless, when put in the context of human and non-human primate studies, all vectors fell short of success regardless of their viral or non-viral origin. Recognition of issues such as inefficient gene transfer and short-lived or scant expression in the relevant cell type(s) prompted researchers to refine and develop several gene delivery systems, in particular those based on retroviruses, adeno-associated viruses and adenoviruses. Concomitantly, available technology was deployed to tackle disorders that require few genetically corrected cells to attain therapy.     

基因治疗的应用及研究进展

基因治疗是一种新的治疗手段,可用于癌症、遗传性疾病、感染性疾病、心血管疾病和自身免疫性疾病等的治疗。癌症基因治疗是基因治疗的主要应用领域。过去几年里,全球基因治疗临床试验取得了很大的进步,也遇到了很多困难。未来基因治疗的主要目标是发展安全和高效的基因导入系统,它们能将外源遗传物质靶向性地导入特异的细胞。简要综述了基因治疗研究和应用的进展、困难及其发展前景。     

Regulated expression systems for gene therapy

Gene therapy is a promising and rapidly developing field of modern medicine and is expected to improve or even cure the diseases that are incurable with classical therapies. The logics of the development of gene therapy in the nearest future will require the systems wherein a regulation is possible for expression of therapeutic genes. The review considers the currently available regulated gene therapeutic systems, which can be divided into two main classes. One includes the systems wherein external inducers are used to trigger therapeutic gene expression. Systems of the other class are autoregulated and function without an external inducer. The most important first-class expression systems are based on the regulation by tetracycline, rapamycin derivative-induced dimerization, steroid hormones, regulatory RNAs, and physical factors. The most important systems of the second class are regulated by oxygen or glucose levels.     

Bio and nanotechnological strategies for tumor-targeted gene therapy

Gene therapy is a new medical approach for the treatment of tumors. For safe and efficient gene therapy, therapeutic genes need to be delivered efficiently into the target tumor cells. Development of gene delivery systems to specifically recognize and target tumor cells and to distinguish them from normal cells, especially in the same tissue or organ, is one of the most important issues regarding the present gene delivery methodologies. The enhanced permeability and retention (EPR) effect using the characteristics of angiogenic tumor blood vessels, as well as gene delivery systems recognizing hyperactivated receptors or intracellular signals, is broadly applied to tumor-targeted gene therapy. In addition, bacterial vectors can be a useful means for targeting hypoxic or anoxic regions of a tumor.     

基因治疗的发展现状、问题和展望

基因治疗是一种新的治疗手段,可以治疗多种疾病,包括癌症、遗传性疾病、感染性疾病、心血管疾病和自身免疫性疾病。癌症基因治疗是基因治疗的主要应用领域。过去几年里,全球基因治疗临床试验取得了很大的进步。实际上,基因治疗也遇到了很多困难。未来,基因治疗的主要目标是发展安全和高效的基因导入系统,它们能将外源遗传物质靶向性地导入到特异的细胞。本文主要综述基因治疗所取得的突出进展、所遇到的困难和发展前景。     

Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.     

Gene therapy using herpes simplex virus-based vectors

Gene therapy involves the use of specific genes to treat human diseases and is thus critically dependent on efficient gene delivery systems. Although a variety of systems for such gene delivery are under development, HSV has unique advantages in terms of its large genome size and for gene delivery in the nervous system because of its ability to enter a latent state in neuronal cells. Considerable progress has been made in the effective disablement of this virus whilst retaining its ability to deliver genes and in producing long term expression of the foreign gene. Although much remains to be achieved in the further disablement of the virus and its testing in rodent and primate models of human diseases, it is likely that these viruses may ultimately be of use in human gene therapy procedures particularly for otherwise intractable neurological diseases.     

The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies

Regulation of heterologous gene expression is of prime importance for a wide variety of basic and applied biological research areas including functional genomics, tissue engineering, gene therapy, and biopharmaceutical manufacturing. Initial gene regulation strategies employed endogenous responsive elements, which resulted in pleiotropic interference of transgene expression with host regulatory networks. Current regulation systems are binary and consist of chimeric transactivators and responsive target promoters of heterologous bacterial or insect origin, or they contain artificially designed components. Regulation of generic systems is based on binding of a transactivator to its cognate promoter, which is modulated by specific molecules such as antibiotics or hormones and brings the transactivation domain into contact with a minimal promoter, thereby inducing target gene expression. Binary gene regulation concepts have been significantly refined in recent years with a focus to improve their regulation performance and their compatibility with human-therapeutic use. In this review we present a detailed analysis of currently available mammalian gene regulation systems and document progress that has pioneered the use of such systems in various aspects of human therapy.     

Multifunctional nanocomplexes for gene transfer and gene therapy

DNA formulated into aggregates with polycationic reagents are referred to by a variety of terms including non-viral vectors, synthetic vectors, lipoplexes, polyplexes and more recently nanoparticles. The capacity for delivery of multiple genes, genomic-sized constructs and siRNA delivery, with a diversity of possible formulations, as well as the possibilities of improved efficiency of in vivo gene deliveries, means that nanoparticles, or nanocomplexes to reflect self-assembling systems, will be investigated with increasing vigour in the coming years. This review briefly outlines the applications and challenges for nanoparticle technologies in the field of gene therapy then focuses on the development of a specific kind of formulation, receptor-targeted nanocomplex (RTN), that we have found to be particularly useful in our gene therapy research. An overriding guiding concept that has emerged in the development of synthetic nanodelivery systems is the idea to develop formulations and structures that mimic viruses, whilst retaining the safety elements of synthetic, non-viral systems. RTNs have been optimised and developed for airway epithelial transfection, leading towards gene therapy for cystic fibrosis and for vascular transfection in vein grafts used in bypass surgery. The modular design of the RTN platform further allows for the testing of specific hypotheses relating to the structure and functional role of components in the formation of stable particles and in the transfection pathway, leading to their ultimate disassembly in the nucleus.     

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.     

Gene therapy of cardiovascular disorders

More than 300 protocols have been developed for human gene therapy, but, it has not yet been proved to be a successful therapeutic strategy. One of the most important barriers to success is the development of efficient gene delivery systems. We have developed HVJ-liposomes by combining fusion proteins of HVJ (Hemagglutinating virus of Japan; Sendai virus) with liposomes containing DNA. This vector system has been very effective for in vivo gene delivery, especially in cardiovascular systems. Using HVJ-liposomes, we have reported successful gene therapy experiments such as prevention of restenosis after balloon injury, suppression of dysfunction of vein graft, and experimental ischemic disorders. Indeed, the success in the treatment of arteriosclerosis obliterance by VEGF (vascular endothelial growth factor) gene transfer was reported recently. These cardiovascular gene therapy strategies appear to be very promising therapeutics in future.