Surface-engineering of lentiviral vectors

Vectors derived from retroviridae offer particularly flexible properties in gene transfer applications given the numerous possible associations of various viral surface glycoproteins (determining cell tropism) with different types of retroviral cores (determining genome replication and integration). Lentiviral vectors should be preferred gene delivery vehicles over vectors derived from onco-retroviruses such as murine leukemia viruses (MLVs) that cannot transduce non-proliferating target cells. Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors, their interaction with the host immune system and their host range. There are however important gene transfer restrictions to some non-proliferative tissues or cell types and recent studies have shown that progenitor hematopoietic stem cells in G(0), non-activated primary blood lymphocytes or monocytes were not transducible by lentiviral vectors. Moreover, lentiviral vectors that have the capacity to deliver transgenes into specific tissues are expected to be of great value for various gene transfer applications in vivo. Several innovative approaches have been explored to overcome such problems that have given rise to novel concepts in the field and have provided promising results in preliminary evaluations in vivo. Here we review the different approaches explored to upgrade lentiviral vectors, aiming at developing vectors suitable for in vivo gene delivery.     

腺病毒载体在疫苗研究中的应用

以病毒为载体的活疫苗为疾病预防和治疗研究提供了新手段。目前用于疫苗研究的病毒载体主要包括痘苗病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体及逆转录病毒载体等。其中,重组腺病毒载体因其基因组大小适中,易于基因重组操作,繁殖滴度高,易于大量制备和保存,宿主范围广,转导效率高,安全性好,能刺激机体产生强烈的体液和细胞免疫反应等特点,而被广泛应用于重要感染性疾病及恶性肿瘤的疫苗研究。腺病毒载体在人免疫缺陷病毒(HIV)疫苗研究和临床试验中的成败更是备受关注。然而,与其他载体疫苗一样,机体对载体的免疫反应仍是阻碍腺病毒载体疫苗在临床中广泛应用的主要问题。那么,腺病毒载体解决这类问题的优势何在?我们简要综述腺病毒载体的特点及其在疫苗研究中的应用和存在的问题,为进一步优化和利用腺病毒载体在疫苗方面的研究提供参考。     

The Use of Viral Vectors in Introducing Genes into Agricultural Animal Species

The use of viral vectors is a method for introducing foreign genes into various animal species. Vectors based on retro-, adeno-, flavi-, and parvoviruses have been used for research in animal species of agricultural importance, such as chickens, quail, swine, cows, goats, sheep, fish, crustaceans, and mollusks. Viral vectors allow for efficient transgenic integration into host genome or for transient expression of the transgenic construct in somatic tissues. Because of that, viral vectors are important tools for research and potentially other biotechnology applications such as improving animal production qualities and introducing disease resistance, thus improving food quality and safety. Other uses may include generating animal models of human diseases and using animals as bioreactors for production of therapeutic proteins. Each vector type provides a unique set of advantages and limitations, which are in some cases specific to an animal species or a method of introduction. This article discusses viral vector characteristics and potential applications in agriculturally important animal species. It discusses advantages and disadvantages of using viral vectors in genetic engineering of agricultural animals.     

Improved Semliki Forest virus vectors for receptor research and gene therapy.

We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in beta-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.     

IMPROVED SEMLIKI FOREST VIRUS VECTORS FOR RECEPTOR RESEARCH AND GENE THERAPY

We have modified Semliki Forest virus (SFV) vectors to broaden their application range. Here we describe a series of site-directed mutagenesis experiments on the SFV subgenomic 26S promoter to down-regulate the heterologous gene expression. Several mutants showed a dramatic effect on transgene expression levels in BHK cells. The luciferase activity was reduced to approximately 30%, 3%, and 1% compared to the wild type promoter. Similarly, a decrease in β-galactosidase activity was observed in BHK cells and after injection into the striatum of male Wistar rats. Novel non-cytopathogenic and temperature-sensitive SFV vectors have recently been developed by introduction of point mutations in the viral nonstructural genes nsP2 and nsP4. These vectors do not show the typical shut down of host cell protein synthesis after SFV infections and therefore allow for a substantially prolonged survival of host cells. Both the mutant vectors demonstrating lower and more physiological expression levels and the non-cytopathogenic vectors should be valuable tools for various applications within receptor research. Furthermore, recent studies suggest that SFV vectors can be efficient gene delivery vehicles for gene therapy applications.     

Retroviral vector particles displaying the antigen-binding site of an antibody enable cell-type-specific gene transfer.

Retroviral vectors are the most efficient tool for stably introducing genes into vertebrate cells. However, their use is limited by the host range of the retrovirus from which they are derived. To alter the host range, we recently constructed retrovirus vector particles, derived from spleen necrosis virus, that display a single-chain antigen-binding site of an antibody (scA) on the viral surface (T.-H. T. Chu, I. Martinez, W. Sheay, and R. Dornburg, Gene Ther. 1:292-299, 1994). Using a hapten (2,4-dinitrophenol) model system, we showed that such particles are competent for infection. In this study, we repeated our experiments using an scA directed against a cell surface protein expressed on various human carcinoma cell lines. We found that such scA-displaying particles can efficiently infect human cells that express the corresponding antigen. Particles with wild-type spleen necrosis virus envelope are minimally infectious on such cells. The addition of the original monoclonal antibody to the viral vector particle solution prior to infection inhibited infection. This competition assay showed that the infection is mediated by the antibody moiety and, therefore, is antibody specific. These data indicate that retroviral vectors with antibody-envelope fusion proteins may be a valuable tool for selectively introducing genes into any target cell.     

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.     

Viral vector targeting.

The field of viral vector targeting is advancing rapidly. Recent advances include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, elucidation of the crystal structures of an adenoviral and a retroviral receptor-binding domain, and definition of strategies for inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors. Novel targeting strategies based on host range restriction and protease activation have been developed, targeted replication-competent vectors have shown promise as anti-cancer agents and the possibility of selecting targeted vectors from vector display libraries has been established.     

An EBV‐based plasmid can replicate and maintain in stem cells

Viral vectors have a wide range of applications in biology, particularly in gene therapy. Based on their integration capacity, viral vectors are classified as either integrating or non‐integrating vectors. Although integrating vectors, such as lentivectors, have the ability to direct prolonged expression of exogenous genes, manipulation of the host genome is an inappropriate feature of these gene delivery tools. Non‐integrating vectors, such as episomal replicating plasmids, can replicate and persist in host cells for long periods without any chromosomal interruption. These advantages made them good tools for gene induction purposes in gene therapy and basic studies. Due to the necessity of gene induction in stem cells for study of mammalian development and targeted differentiation, the use of integrating vectors for prolonged expression of genes of interest has been developed. Application of replicating plasmids can overcome some drawbacks associated with integrating vectors, although replication and maintenance of these plasmids can differ between cell types. Previously, it has been shown that such plasmids can be maintained in human embryonic stem cells for more than one month, but the rate of the plasmid replication during the host cell cycle has not been elucidated. In the present study, we showed that an EBV‐based plasmid can replicate simultaneously with host in pluripotent and multipotent human and mouse stem cells and can be sustained for long time periods in dividing cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1579–1585, 2015     

宿主防御肽抗病毒感染机制研究进展

宿主防御肽是一类广泛存在于脊椎动物的小分子多肽,具有广谱的抗菌活性以及抗炎、细胞趋化、促进血管生成和修复损伤等免疫调节功能。以往的研究多集中在宿主防御肽抗细菌和真菌感染的研究上。近年来大量研究发现,宿主防御肽也具有广泛的抗病毒活性,在临床各类病毒病的预防和治疗上具有潜在的应用前景。本文围绕宿主防御肽直接杀伤病毒、调节病毒感染过程和参与宿主抗病毒天然免疫调节这3个方面的作用机制进行综述,为宿主防御肽抗病毒相关研究和相关抗病毒生物药物的研发提供参考和借鉴。     

植物病毒表达载体研究进展

利用DNA或RNA植物病毒作载体表达外源蛋白是近几年发展较快的一种新的遗传转化方式,它具有以下几个优点:表达量大,表达速度快,易于进行基因操作和接种以及适用对象广泛。已发展的四种载体构建策略包括:基因取代,基因插入,融合抗原和基因互补。植物病毒表达载体可以用于基因的重组、病毒的移动和基因功能的检测等基础性研究,也可用于商业上表达多种药用蛋白或疫苗。植物病毒表达载体的稳定性主要取决于存在同源序列而引起的基因重组。本文还对病毒载体的生物安全性进行了讨论。     

体内基因转染技术在肝再生研究中的应用

肝脏是一个特殊的器官,不仅因为它独特的解剖结构和生理特征,而且它还具有无限的再生能力。在各种动物模型中,应用病毒或非病毒载体将肝细胞生长因子等基因转入体内,能增强肝再生能力,这就是肝脏基因转染技术在肝再生研究中的应用。未来的研究目标就是消除病毒载体的毒副作用和增加非病毒载体的转染率,这也是目前肝内基因转染技术中面临的主要难题;另一个研究目标就是用受体介导基因靶向肝转染,使转入基因在肝细胞中特异高表达。这些研究成果将有助于肝再生基因机制研究,以及将来临床基因治疗提供参考。     

Delivery of CRISPR/Cas9 for therapeutic genome editing

The clustered, regularly‐interspaced, short palindromic repeat (CRISPR)‐associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome‐editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome‐editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off‐target effects. Recently, non‐viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non‐viral vectors, including polymers, liposomes, cell‐penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non‐viral vectors are also discussed.     

转座子Sleeping Beauty和PiggyBac

近10年来,得益于转座子Sleeping Beauty(SB)和PiggyBac(PB)的发现和完善,转座子作为一种遗传工程工具在脊椎动物的基因遗传研究中得到广泛应用.SB和PB宿主范围极其广泛,从单细胞生物到哺乳动物都能够发挥作用.转座过程需要转座序列和转座酶的存在,类似于"剪切"、"粘贴"的方式.转座子载体系统转座时可携带一段外源DNA序列,利用这一特性可以用于实现目的基因的转移,现已广泛用于转基因动物、基因功能研究、基因治疗等领域.当转座系统与基因捕获技术相结合,不仅可研究插入突变基因的功能,还能通过所携带的报告基因获得捕获基因的表达图谱.作为非病毒载体的SB和PB转座系统,由于具有高容量、高效率和高安全性等优势,并且PB在转座后不留任何足迹,不会造成遗传物质的不可预测改变,在动物基因工程以及基因治疗方面具有诱人的前景.     

Facilitating viral vector movement enhances heterologous protein production in an established plant system

Molecular farming technology using transiently transformed Nicotiana plants offers an economical approach to the pharmaceutical industry to produce an array of protein targets including vaccine antigens and therapeutics. It can serve as a desirable alternative approach for those proteins that are challenging or too costly to produce in large quantities using other heterologous protein expression systems. However, since cost metrics are such a critical factor in selecting a production host, any system-wide modifications that can increase recombinant protein yields are key to further improving the platform and making it applicable for a wider range of target molecules. Here, we report on the development of a new approach to improve target accumulation in an established plant-based expression system that utilizes viral-based vectors to mediate transient expression in Nicotiana benthamiana. We show that by engineering the host plant to support viral vectors to spread more effectively between host cells through plasmodesmata, protein target accumulation can be increased by up to approximately 60%.     

Cross-species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live-virus vaccines

All living organisms are continuously exposed to a plethora of viruses. In general, viruses tend to be restricted to the natural host species which they infect. From time to time viruses cross the host-range barrier expanding their host range. However, in very rare cases cross-species transfer is followed by the establishment and persistence of a virus in the new host species, which may result in disease. Recent examples of viruses that have crossed the species barrier from animal reservoirs to humans are hantavirus, haemorrhagic fever viruses, arboviruses, Nipah and Hendra viruses, avian influenza virus (AI), monkeypox virus, and the SARS-associated coronavirus (SARS-CoV). The opportunities for cross-species transfer of mammalian viruses have increased in recent years due to increased contact between humans and animal reservoirs. However, it is difficult to predict when such events will take place since the viral adaptation that is needed to accomplish this is multifactorial and stochastic. Against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live-vector vaccines is a cause for concern. This review addresses a number of potential risk factors and their implications for activities with viral vectors from the perspective of cross-species transfer of viruses in nature, with emphasis on the occurrence of host-range mutants resulting from either cell culture or tropism engineering. The issues are raised with the intention to assist in risk assessments for activities with vector viruses.     

Production and purification of lentiviral vectors

Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (10(6) viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (10(9) viral particles/ml) purified preparation requires 2 more days.     

Hybrid vector designs to control the delivery, fate and expression of transgenes

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to 'gutted' and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression.     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

Gene therapy vectors: the prospects and potentials of the cut-and-paste transposons

Gene therapy applications require efficient tools for the stable delivery of genetic information into eukaryotic genomes. Most current gene delivery strategies are based on viral vectors. However, a number of drawbacks, such as the limited cargo capacity, host immune response and mutational risks, highlight the need for alternative gene delivery tools. A comprehensive gene therapy tool kit should contain a range of vectors and techniques that can be adapted to different targets and purposes. Transposons provide a potentially powerful approach. However, transposons encompass a large number of different molecular mechanisms, some of which are better suited to gene delivery applications than others. Here, we consider the range and potentials of the various mechanisms, focusing on the cut-and-paste transposons as one of the more promising avenues towards gene therapy applications. Several cut-and-paste transposition systems are currently under development. We will first consider the mechanisms of piggyBac and the hAT family elements Tol1 and Tol2, before focusing on the mariner family elements including Mos1, Himar1 and Hsmar1.