Minimum requirements for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors

The development of gene delivery vectors based on feline immunodeficiency virus (FIV) is an attractive alternative to vectors based on primate sources for the delivery of genes into humans. To investigate the requirements for efficient transduction of dividing and nondividing cells by vector particles based on FIV, a series of packaging and vector constructs was generated for which viral gene expression was minimized and from which unnecessary cis-acting sequences were deleted. Pseudotyped vector particles produced in 293T cells were used to transduce various target cells, including contact-inhibited human skin fibroblasts and growth-arrested HT1080 cells. FIV vectors in which the U3 promoter was replaced with the cytomegalovirus promoter gave rise to over 50-fold-higher titers than FIV vectors containing the complete FIV 5' long terminal repeat (LTR). Comparison of the transduction efficiencies of vectors containing different portions of the FIV Gag coding region indicates that at least a functional part of the FIV packaging signal (Psi) is located within an area which includes the 5' LTR and the first 350 bp of gag. Transduction efficiencies of vectors prepared without FIV vif and orf2 accessory gene expression did not differ substantially from those of vectors prepared with accessory gene expression in either dividing or nondividing cells. The requirement for FIV rev-RRE was, however, demonstrated by the inefficient production of vector particles in the absence of rev expression. Together, these results demonstrate the efficient transduction of nondividing cells in vitro by a multiply attenuated FIV vector and contribute to an understanding of the minimum requirements for efficient vector production and infectivity. In addition, we describe the ability of an FIV vector to deliver genes in vivo into hamster muscle tissue.     

Virus-mediated gene transfer to induce therapeutic angiogenesis: Where do we stand?

The potential to induce therapeutic angiogenesis through gene transfer has engendered much excitement as a possible treatment for tissue ischemia. After 10 years of clinical experimentation, however, it now appears clear that several crucial issues are still to be resolved prior to achieving clinical success. These include the understanding of whether functional blood vessels might arise as a result of the delivery of a single angiogenic factor or require more complex cytokine combinations, the identification of the proper timing of therapeutic gene expression and, most notably, the development of more efficacious gene delivery tools. Viral vectors based on the adeno-associated virus (AAV) appear particularly suitable to address the last requirement, since they display a specific tropism for skeletal muscle cells and cardiomyocytes, and drive expression of the therapeutic genes in these cells for indefinite periods of time. In this review, I discuss the current applications of gene therapy for cardiovascular disorders, with particular attention to the possible improvements in the technologies involved in virus-mediated gene transfer.     

Sequence-specific modification of mouse genomic DNA mediated by gene targeting techniques

The major impact of the human genome sequence is the understanding of disease etiology with deduced therapy. The completion of this project has shifted the interest from the sequencing and identification of genes to the exploration of gene function, signalling the beginning of the post-genomic era. Contrasting with the spectacular progress in the identification of many morbid genes, today therapeutic progress is still lagging behind. The goal of all gene therapy protocols is to repair the precise genetic defect without additional modification of the genome. The main strategy has traditionally been focused on the introduction of an expression system designed to express a specific protein, defective in the transfected cell. But the numerous deficiencies associated with gene augmentation have resulted in the development of alternative approaches to treat inherited and acquired genetic disorders. Among these one is represented by gene repair based on homologous recombination (HR). Simply stated, the process involves targeting the mutation in situ for gene correction and for restoration of a normal gene function. Homologous recombination is an efficient means for genomic manipulation of prokaryotes, yeast and some lower eukaryotes. By contrast, in higher eukaryotes it is less efficient than in the prokaryotic system, with non-homologous recombination being 10-50 fold higher. However, recent advances in gene targeting and novel strategies have led to the suggestion that gene correction based on HR might be used as clinical therapy for genetic disease. This site-specific gene repair approach could represent an alternative gene therapy strategy in respect to those involving the use of retroviral or lentiviral vectors to introduce therapeutic genes and linked regulatory sequences into random sites within the target cell genome. In fact, gene therapy approaches involving addition of a gene by viral or nonviral vectors often give a short duration of gene expression and are difficult to target to specific populations of cells. The purpose of this paper is to review oligonucleotide-based gene targeting technologies and their applications on modifying the mouse genome.     

冠状病毒载体研究进展

随着定向重组技术和反向遗传学系统的发展,利用冠状病毒独特的转录机制表达外源基因成为可能。目前已经开发出了两类基于冠状病毒的表达载体,即辅助病毒依赖的表达载体系统和单基因组表达载体系统。通过对冠状病毒感染性cDNA进行改造可以获得外源基因的高效(50μg/106细胞)、稳定(30代)表达。此外,冠状病毒载体以下几个特征使其成为非常具有吸引力的载体:①通过删除非结构基因、组特异性基因可以将冠状病毒转化为无毒力的病毒;②通过对S蛋白的改造可以改变冠状病毒的组织和物种嗜性,从而将外源基因定向表达到不同的组织器官或物种。因此,冠状病毒对于疫苗开发以及基因治疗是前景非常好的载体。     

Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors

Gene therapy has provided great potential to revolutionize the treatment of many diseases. This therapy is strongly relied on whether a delivery vector efficiently and safely directs the therapeutic genes into the target tissue/cells. Nonviral gene delivery vectors have been emerging as a realistic alternative to the use of viral analogs with the potential of a clinically relevant output. Dendritic polymers were employed as nonviral vectors due to their branched and layered architectures, globular shape and multivalent groups on their surface, showing promise in gene delivery. In the present review, we try to bring out the recent trend of studies on functional and biodegradable dendritic polymers as nontoxic and efficient gene delivery vectors. By regulating dendritic polymer design and preparation, together with recent progress in the design of biodegradable polymers, it is possible to precisely manipulate their architectures, molecular weight and chemical composition, resulting in predictable tuning of their biocompatibility as well as gene transfection activities. The multifunctional and biodegradable dendritic polymers possessing the desirable characteristics are expected to overcome extra- and intracellular obstacles, and as efficient and nontoxic gene delivery vectors to move into the clinical arena.     

Gene delivery and gene therapy with herpes simplex virus-based vectors

The development of efficient means of delivery genes in vivo is essential both for testing gene function in the intact animal and for human gene therapy procedures. A number of viral and non-viral gene delivery methods have been developed for this purpose. Of those herpes simplex virus (HSV)-based vectors have particular advantages for gene delivery to the nervous system including their ability to infect non-dividing neurones and establish asymptomatic latent infections. Moreover, considerable progress has been made, firstly, in disabling HSV vectors so as to prevent the damaging effects of wild type virus and secondly, to ensure long-term expression of the inserted transgene(s). These vectors thus offer a valuable tool for testing gene function in neuronal cells in vivo and may ultimately be safe enough for use in human gene therapy procedures.     

Retroviral vectors for persistent expression in vivo.

Retroviral vectors provide a safe and efficient method of introducing genes of therapeutic interest into dividing cells. The principle limitation of these vectors in the past has been poor gene expression in vivo. This problem has been overcome recently through the use of tissue-specific enhancers in commonly used retroviral vectors. In this review we discuss both the relevant biology and some of the practical applications of retroviral vectors in gene therapy.     

Helper-dependent adenoviral vectors in experimental gene therapy

In the majority of potential applications gene therapy will require an effective transfer of a transgene in vivo resulting in high-level and long-term transgene expression, all in the absence of significant toxicity or inflammatory responses. The most efficient vehicles for delivery of foreign genes to the target tissues are modified adenoviruses. Adenoviral vectors of the first generation, despite the high infection efficacy, have an essential drawback: they induce strong immune response, which leads to short term expression of the transgene, and limits their usefulness in clinical trials. In contrast, helper-dependent adenoviral vectors (HdAd) lacking all viral coding sequences display only minimal immunogenicity and negligible side-effects, allowing for long-term transgene expression. Thus, HdAd vehicles have become the carrier of choice for adenoviral vector-mediated experimental gene therapy, effectively used in animal models for delivery of transgenes into the liver, skeletal muscle, myocardium or brain. Strong and long-lasting expression of therapeutic genes has allowed for successful treatment of dyslipidemias, muscular dystrophy, obesity, hemophilia, and diabetes. Additionally, the large cloning capacity of HdAd, up to 37 kb, facilitates the use of physiologically regulated, endogenous promoters, instead of artificial viral promoter sequences. This enables also generation of the single vectors expressing multiple genes, which can be potentially useful for treatment of polygenic diseases. In this review we characterize the basic features of HdAd vectors and describe some of their experimental and potential clinical applications.     

Designing gene delivery vectors for cardiovascular gene therapy

Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy.     

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.     

Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors

Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

Baculovirus host-range

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified. Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range.     

Nucleocytoplasmic transport of DNA: enhancing non-viral gene transfer

Gene therapy, the correction of dysfunctional or deleted genes by supplying the lacking component, has long been awaited as a means to permanently treat or reverse many genetic disorders. To achieve this, therapeutic DNA must be delivered to the nucleus of cells using a safe and efficient delivery vector. Although viral-based vectors have been utilized extensively due to their innate ability to deliver DNA to intact cells, safety considerations, such as pathogenicity, oncogenicity and the stimulation of an immunological response in the host, remain problematical. There has, however, been much progress in the development of safe non-viral gene-delivery vectors, although they remain less efficient than the viral counterparts. The major limitations of non-viral gene transfer reside in the fact that it must be tailored to overcome the intracellular barriers to DNA delivery that viruses already master, including the cellular and nuclear membranes. In particular, nuclear transport of the therapeutic DNA is known to be the rate-limiting step in the gene-delivery process. Despite this, much progress had been made in recent years in developing novel means to overcome these barriers and efficiently deliver DNA to the nuclei of intact cells. This review focuses on the nucleocytoplasmic delivery of DNA and mechanisms to enhance to non-viral-mediated gene transfer.     

肿瘤基因治疗的靶向策略

对肿瘤组织的靶向性可以提高基因治疗的效果 ,避免对正常组织的损伤 ,并且能降低作为载体的微生物对机体的危害。对于瘤内注射的给药方法 ,靶向性似乎显得不是特别重要 ,但是如果要系统给药 ,靶向性是很关键的一个问题。靶向基因治疗肿瘤可以通过靶向基因导入和靶向基因表达来实现。近年来 ,在靶向基因导入方面的研究有很多进展 ,例如 ,用双亲性的桥连分子协助腺病毒和逆转录病毒靶向转导 ;在各种病毒载体的衣壳蛋白中插入靶向性的小肽或较大的多肽靶向结构域 ;增殖病毒作为一种很有前途的抗肿瘤制剂可有效地靶向杀伤肿瘤细胞。受体介导的DNA或DNA 脂质体复合物的靶向系统和其他一些靶向性的有疗效的载体 ,如细菌 ,也处于研究中。其中的一些载体已经进入临床实验。为了实现基因的靶向可调控表达 ,组织或肿瘤特异性的启动子和人工合成的可调控表达系统被用来调控治疗基因的表达。反义核酸、核酶以及脱氧核酶 (DNAzyme)被用来靶向抑制与肿瘤发生密切相关基因的表达。     

Improved gene delivery to human saphenous vein cells and tissue using a peptide-modified adenoviral vector

The establishment of efficient gene delivery to target human tissue is a major obstacle for transition of gene therapy from the pre-clinical phases to the clinic. The poor long-term patency rates for coronary artery bypass grafting (CABG) is a major clinical problem that lacks an effective and proven pharmacological intervention. Late vein graft failure occurs due to neointima formation and accelerated atherosclerosis. Since CABG allows a clinical window of opportunity to genetically modify vein ex vivo prior to grafting it represents an ideal opportunity to develop gene-based therapies. Adenoviral vectors have been frequently used for gene delivery to vein ex vivo and pre-clinical studies have shown effective blockade in neointima development by overexpression of candidate therapeutic genes. However, high titers of adenovirus are required to achieve sufficient gene delivery to provide therapeutic benefit. Improvement in the uptake of adenovirus into the vessel wall would therefore be of benefit. Here we determined the ability of an adenovirus serotype 5 vector genetically-engineered with the RGD-4C integrin targeting peptide inserted into the HI loop (Ad-RGD) to improve the transduction of human saphenous vein smooth muscle cells (HSVSMC), endothelial cells (HSVEC) and intact saphenous vein compared to a non-modified virus (Ad-CTL). We exposed each cell type to virus for 10, 30 or 60 mins and measured transgene at 24 h post infection. For both HSVSMC and HSVEC Ad-RGD mediated increased transduction, with the largest increases observed in HSVSMC. When the experiments were repeated with intact human saphenous vein (the ultimate clinical target for gene therapy), again Ad-RGD mediated higher levels of transduction, at all clinically relevant exposures times (10, 30 and 60 mins tissue:virus exposure). Our study demonstrates the ability of peptide-modified Ad vectors to improve transduction to human vein graft cells and tissue and has important implications for gene therapy for CABG.     

Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host''s chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.