Development of retroviral vectors as safe, targeted gene delivery systems

The transfer of genes of potential therapeutic benefit is presently being attempted in the clinic to treat a number of genetic and virally induced diseases. Many of these protocols use retroviral vectors derived from murine leukemia retroviruses as gene delivery systems. Although these viral delivery systems are well suited for this purpose, a number of their characteristics, some of which are discussed here, are still troublesome. Future retroviral vectors will incorporate nonretroviral features and will be tailored to desired needs for specific uses. These vectors will be safer, more efficient, and targeted in their delivery. Further, expression of the therapeutic genes carried will be limited to the specific target cell type. Some of the recent advances that have been made towards this goal are reviewed here.     

Viral vectors in cancer immunotherapy: which vector for which strategy?

Gene therapy involves the transfer of genetic information to a target cell to facilitate the production of therapeutic proteins and is now a realistic prospect as a cancer treatment. Gene transfer may be achieved through the use of both viral and non-viral delivery methods and the role of this method in the gene therapy of cancer has been demonstrated. Viruses represent an attractive vehicle for cancer gene therapy due to their high efficiency of gene delivery. Many viruses can mediate long term gene expression, while some are also capable of infecting both dividing and non-dividing cells. Given the broadly differing capabilities of various viral vectors, it is imperative that the functionality of the virus meets the requirements of the specific treatment. A number of immunogene therapy strategies have been undertaken, utilising a range of viral vectors, and studies carried out in animal models and patients have demonstrated the therapeutic potential of viral vectors to carry genes to cancer cells and induce anti-tumour immune responses. This review critically discusses the advances in the viral vector mediated delivery of immunostimulatory molecules directly to tumour cells, the use of viral vectors to modify tumour cells, the creation of whole cell vaccines and the direct delivery of tumour antigens in animal models and clinical trials, specifically in the context of the suitability of vector types for specific strategies.     

Gene therapy in soft tissue reconstruction

Gene therapy is defined as the introduction of a therapeutic gene into a cell, whose expression can lead to a cure of a disease or offer a transient advantage for tissue growth and regeneration. The delivery of genes can be undertaken for a number of purposes, usually it is attempted to enhance or add a function to a cell or a tissue or to delete or reduce another function. In this brief overview we describe various vehicles and techniques that have been developed to deliver therapeutic genes into cells, such as viral vectors and physical/chemical gene delivery methods including naked DNA and particle-mediated gene transfer, the microseeding technique and the application of lipids. Furthermore we review the potential utility of gene therapy from the perspective of a reconstructive surgeon. Several tissues will be discussed, particularly muscle, tendon, nerve, bone, skin and wounds.     

Plasmid-mediated muscle-targeted gene therapy for circulating therapeutic protein replacement: a tale of the tortoise and the hare?

There is now conclusive evidence that gene therapy can lead to real clinical benefit. Initial enthusiasm has been muted by set-backs related to viral vectors including retroviral oncogenesis and adenoviral inflammatory response. Plasmid-mediated muscle-targeted gene transfer offers the potential of a cost-effective pharmaceutical grade therapy delivered by simple intramuscular injection without the need for anaesthetic, cell culture, transplantation or immunosuppression. This approach is particularly appropriate for long-term circulating therapeutic protein replacement currently requiring repeated injection therapy. Wide-ranging clinical applications include haemophilia, chronic anaemia, growth hormone deficiency and diabetes. Inadequate transgene expression, unregulated protein delivery and immune response have been major limiting factors. Recent innovations including in situ electroporation enabling sustained systemic protein delivery within the therapeutic range are reviewed. Pharmacological and physiological approaches to regulation are discussed in addition to the role of innate and humoral immunity. Translation of advances in all of these areas to clinical success will enable muscle-targeted gene therapy to capitalise on its inherent strengths and realise its long-standing promise.     

Viral vectors for gene-directed enzyme prodrug therapy

Conventional cancer treatments are often hampered by a lack of tumour selectivity, resulting in toxicity to healthy tissue. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy approach that aims to improve the selectivity of chemotherapy by enabling cancer cells to convert non-cytotoxic prodrugs to cytotoxic drugs. Many enzyme/prodrug systems have been described, some of which have already been tested in clinical trials. A key component of GDEPT is a foreign enzyme that is expressed selectively at the tumour site where it converts the prodrug into the cytotoxic agent. The gene encoding the prodrug-activating enzyme needs to be expressed selectively and efficiently in tumour cells in order to spare normal tissue from damage. Substantial efforts have been made to develop gene therapy vectors that are capable of targeting cancer cells. A large number of gene delivery systems have been described for GDEPT: Viral vectors are the most advanced. They include replication-deficient and replication-selective (oncolytic) viruses. Recent advances in engineering viruses for GDEPT are reviewed in this article and data from both preclinical studies and clinical trials are discussed.     

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.     

Polycation-based gene therapy: current knowledge and new perspectives

At present, gene transfection insufficient efficiency is a major drawback of non-viral gene therapy. The 2 main types of delivery systems deployed in gene therapy are based on viral or non-viral gene carriers. Several non-viral modalities can transfer foreign genetic material into the human body. To do so, polycation-based gene delivery methods must achieve sufficient efficiency in the transportation of therapeutic genes across various extracellular and intracellular barriers. These barriers include interactions with blood components, vascular endothelial cells and uptake by the reticuloendothelial system. Furthermore, the degradation of therapeutic DNA by serum nucleases is a potential obstacle for functional delivery to target cells. Cationic polymers constitute one of the most promising approaches to the use of viral vectors for gene therapy. A better understanding of the mechanisms by which DNA can escape from endosomes and traffic to enter the nucleus has triggered new strategies of synthesis and has revitalized research into new polycation-based systems. The objective of this review is to address the state of the art in gene therapy with synthetic and natural polycations and the latest advances to improve gene transfer efficiency in cells.     

Cellular Vehicles for Cancer Gene Therapy

Cancer is a difficult target for any therapeutic strategy; therefore, there is a continuous search for new therapeutic modalities, for application either alone or in combination. In this regard, gene-based therapy is a new approach that offers hope of improved control of tumors. Intensive research to apply gene therapy for cancer treatment has led to identification of the most important technical and theoretical barriers that need to be overcome for clinical success. One of the central unresolved challenges remains the issue of specific and efficient delivery of genes to target cells or tissues, emphasizing the importance of the gene carrier. Along with different viral and non-viral vector systems, mammalian cells have also been considered as vehicles for delivery of anti-cancer therapeutics. The cell-based delivery approach was introduced as the first attempt to apply gene therapy to cancer treatment, and in general, has followed most of the ups and downs of gene therapy applications, progressing alongside new knowledge gained in this field. As a result, significant progress has been made in some aspects of the cell-based approach, while the development of other essential issues is only just gaining speed. It appears that the initial phase of development of cell-based protocols - the achievement of efficient ex vivo cell loading with therapeutics - has largely been fulfilled. However, the desired efficacy of cell-based strategies in general has not yet been reached, and specificity of tumor homing needs to be improved considerably. There is hope that advances in related scientific fields will promote the utilization of cells as powerful and versatile vehicles for cancer gene therapy.     

Transductional targeting with recombinant adenovirus vectors

Replication-deficient adenoviruses are considered as gene delivery vectors for the genetic treatment of a variety of diseases. The ability of such vectors to mediate efficient expression of therapeutic genes in a broad spectrum of dividing and non-dividing cell types constitutes an advantage over alternative gene transfer vectors. However, this broad tissue tropism may also turn disadvantageous when genes encoding potentially harmful proteins (e.g. cytokines, toxic proteins) are expressed in surrounding normal tissues. Therefore, specific restrictions of the viral tropism would represent a significant technological advance towards safer and more efficient gene delivery vectors, in particular for cancer gene therapy applications. In this review, we summarize various strategies used to selectively modify the natural tropism of recombinant adenoviruses. The advantages, limitations and potential impact on gene therapy operations of such modified vectors are discussed.     

Clinical applications of gene therapy in head and neck cancer

Despite advances in surgery, radiotherapy, and the incorporation of novel systemic agents into treatment, long-term outcomes of patients with head and neck cancer remain unsatisfactory. The growing understanding of head and neck cancer biology suggests that targeting molecular events governing carcinogenesis or tumor progression may provide novel therapeutic approaches for head and neck cancer. Squamous cell carcinoma of the head and neck (SCCHN) is characterized by locoregional spread and is clinically accessible, making it an attractive target for intratumoral gene therapy, a potentially efficacious experimental treatment. Systemic delivery of gene therapy may be also possible, albeit with several limitations. In this review we will discuss the rationale, delivery methods, and accumulated clinical data with cancer gene therapy in SCCHN.     

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn’s disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.     

基因强化骨组织工程中的病毒载体

背景：不同的基因输送策略也被应用到骨组织工程中以修复破坏的骨组织,作为最有效率的基因转运载体,病毒载体在骨组织工程中的应用方兴未艾。 目的：系统回顾和讨论目前基因强化骨组织工程中常用的病毒载体相关应用。 方法：利用PubMed数据库对2002年1月至2015年1月的相关文献进行了检索,检索的文章主要聚焦在病毒载体基因转导方法和其在骨组织工程中的应用。对腺病毒、反转录病毒、腺相关病毒和嵌合病毒在骨组织工程的相关应用及不足进行了讨论。总共24篇相关文献被纳入此篇综述。 结果与结论：总结了近年来病毒载体联合基因治疗促进骨组织再生的研究工作。讨论了包括装载目的基因的病毒载体联合种子细胞例如间充质干细胞植入支架材料修复骨缺损。研究表明,基因强化的骨组织工程比传统组织工程具有更多的优点;病毒载体介导的基因转染效率比普通载体更高;病毒载体介导的基因强化骨组织工程用于人体的安全性仍需要漫长的临床观察研究。病毒载体系统仍然是最有效的将外源基因转入种子细胞的手段之一。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Immune response to helper dependent adenoviral mediated liver gene therapy: challenges and prospects

Adenovirus-mediated gene therapy holds significant potential especially for applications requiring high levels of target tissue transduction. While significant advances in clinical adenoviral gene therapy applications have been made in cancer, the clinical translation of adenoviral gene replacement therapy for genetic disease has lagged. Encouragingly, advances in vector production have led to the development of Helper-Dependent ("gutted" or "high capacity") adenoviral vectors (HDV) deleted of all viral coding genes. HDV significantly reduces the chronic toxicity associated with early generation adenoviral vectors that has been most significant after systemic administration in both small and large animal models. However, the field remains confounded by innate immune responses inherent to adenovirus, and more generally, to the adaptive immune response to transgene. Together they decrease the effective therapeutic index for any particular treatment. This review summarizes the current advances toward understanding the decisive cell and molecular mechanisms underlying the acute toxicity to systemic HDV administration. We focus on the complex immune response and consequences of systemic vector delivery in the context of liver-directed monogenic disease therapy. Future development of interventions to avoid the innate immune response, including vector and pharmacologic manipulations, should further contribute to minimizing vector toxicity while maximizing the efficacy of systemic HDV gene transfer.     

Laboratory formulated magnetic nanoparticles for enhancement of viral gene expression in suspension cell line

One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology including viral and non-viral vectors have been made, an ideal vector system has not yet been constructed. Due to the growing concerns over the toxicity and immunogenicity of viral DNA delivery systems, DNA delivery via improve viral routes has become more desirable and advantageous. The ideal improve viral DNA delivery system should be a synthetic materials plus viral vectors. The materials should also be biocompatible, efficient, and modular so that it is tunable to various applications in both research and clinical settings. The successful steps towards this improve viral DNA delivery system is demonstrated: a magnetofection system mediated by modified cationic chitosan-coated iron oxide nanoparticles. Dense colloidal cationic iron oxide nanoparticles serve as an uptake-enhancing component by physical concentration at the cell surface in presence of external magnetic fields; enhanced viral gene expression (3-100-fold) due to the particles is seen as compared to virus vector alone with little virus dose.     

病毒介导的免疫治疗癌症进展

病毒主要作为基因转染载体在免疫治疗中发挥重要作用.本文详细地介绍了病毒载体类型,病毒介导的对肿瘤细胞、宿主细胞的调节,病毒疫苗等免疫治疗癌症的研究进展,可为癌症免疫治疗的基础和临床研究提供参考.     

Gene silencing in the development of personalized cancer treatment: the targets, the agents and the delivery systems

The advent of sophisticated experimental tools that can probe the molecular pathology of cancer has revealed a number of genes and gene families that could prove attractive targets for cancer therapy. Thus, gene silencing strategies have been envisioned to treat cancer by targeting the cancer cell's capacity to: (I) resist conventional treatment methods (chemotherapy and radiotherapy), (II) promote angiogenesis, and (III) metastasize and/or to survive microenvironments that normally would promote cell apoptosis/necrosis. The realization of such strategies is limited by the lack of pharmaceutically-viable technologies that enable the safe and effective delivery of gene-targeting agents to neoplastic cells following systemic administration. There are many reasons for this, including an incomplete understanding of how cancer cells respond when genes are silenced. Further the pharmacokinetic and pharmacodynamic attributes of gene therapy products are not well understood. This review will discuss gene therapy strategies that have been developed based on gene inhibition by the use of antisense oligonucleotides, ribozymes and RNA interference (RNAi). In this context, several particularly promising targets will be described, with a focus on strategies that have progressed to the stage where clinical trials have been initiated. The review highlights product development strategies that emphasize non-viral systemic formulations and the potential for delivery systems to become an enabling technology for development of effective gene therapy products.     

Advances in gene therapy for bladder cancer

The efficacy of various currcntly available therapeutic strategies for bladder cancer is not always suffcient, especially for the advanced disease, recurrent superficial cancer, and treatment-resistant carcinoma in situ. Advances in genetic and molecular biology have led to novel approaches for cancer treatment. Gene therapy is currently one of the most promising strategies against various malignancies, and several clinical trials have been approved worldwide. Various strategies for modulating the genetic state have been applied in bladder cancer treatment, and encouraging results have been demonstrated both in vitro and in vivo. Although the therapeutic genes work dramatically when the transgenes are effectively expressed in the targeted cells, however, a sufficient rate of transduction cannot always be achieved. The most significant obstacle for clinical application of cancer gene therapy might be the method for sufficient delivery and expression of the therapeutic genes. Bladder is an easily accessible organ because of its anatomy; however, a glycosaminoglycan (GAG) layer on the bladder mucosa may protect integration of exo-delivered genetic vectors. Various strategies are applied for improving the transduction efficacy of the therapeutic genes into the bladder cancer cells. These strategies include the modification of adenoviral fibers, cotransduction of the materials for enhancing the viral infectivity, and disruption of the GAG layer. Recent advances in the field of gene therapy for bladder cancer are briefly summarized in this review.     

Gene therapy for colorectal cancer: therapeutic potential

Colorectal cancer is a leading cause of cancer mortality in Western countries. Gene therapy has been proposed as a potential novel treatment modality for colorectal cancer, but it is still in an early stage of development. The preclinical data have been promising and numerous clinical trials are underway. This brief review aims to summarise the current status of clinical trials of different gene therapy strategies, including immune stimulation, mutant gene correction, prodrug activation and oncolytic virus therapy, for patients with colorectal cancer. Data from phase I trials have proven the safety of the reagents but have not yet demonstrated significant therapeutic benefit. In order to achieve this and extend the scope of the treatment, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery and accuracy of gene targeting.     

Gene Therapy for Brain Tumors

Gene therapy has opened new doors for treatment of neoplastic diseases. This new approach seems very attractive, especially for glioblastomas, since treatment of these brain tumors has failed using conventional therapy regimens. Many different modes of gene therapy for brain tumors have been tested in culture and in vivo. Many of these approaches are based on previously established anti-neoplastic principles, like prodrug activating enzymes, inhibition of tumor neovascularization, and enhancement of the normally weak anti-tumor immune response. Delivery of genes to tumor cells has been mediated by a number of viral and synthetic vectors. The most widely used paradigm is based on the activation of ganciclovir to a cytotoxic compound by a viral enzyme, thymidine kinase, which is expressed by tumor cells, after the gene has been introduced by a retroviral vector. This paradigm has proven to be a potent therapy with minimal side effects in several rodent brain tumor models, and has proceeded to phase 1 clinical trials. In this review, current gene therapy strategies and vector systems for treatment of brain tumors will be described and discussed in light of further developments needed to make this new treatment modality clinically efficacious.