Prospects for virus-based gene therapy for cystic fibrosis

Gene therapy for cystic fibrosis (CF) could potentially be accomplished with one of several recombinant virus vectors, including a murine retrovirus (MMuLV), adenovirus, or adeno-associated virus (AAV). All these vectors take advantage of their respective viruses' mechanisms for delivery of viral DNA to cells, evasion of lyosomal degradation, and optimization of the levels and duration of expression of viral (or vector) DNA. Each has its own unique life cycle, however. The differences among these viruses result in certain advantages and disadvantages, such as the requirement of retroviruses for active cell division, and the potential pathogenic effects from expression of certain adenovirus genes present in adenovectors. While no single vector may be optimal for CF gene therapy in humans, new techniques, such as receptor-mediated gene transfer, seek to take advantage of the desirable properties of one or more of the virus-based systems while avoiding certain potential hazards.     

应用新城疫病毒治疗肿瘤的研究进展

新城疫病毒可以特异地杀伤肿瘤细胞,而对正常细胞没有伤害,目前在临床实验中认为是安全、有效的溶瘤试剂。随着近年来反向遗传操作技术的日趋成熟,该技术开始应用到新城疫病毒溶瘤效果的优化方面,通过改造新城疫病毒的F基因,及表达重组粒细胞巨噬细胞集落刺激因子,干扰素-γ,白细胞介素-2和肿瘤坏死因子-α等肿瘤杀伤因子,使该病毒具备更加优越的肿瘤杀伤能力,成为肿瘤治疗领域一个新兴的亮点,为癌症的临床治疗提供了崭新的前景。以下将简要介绍应用反向遗传操作技术重组新城疫病毒优化肿瘤治疗效果的研究进展,以及本实验室在相关领域的研究情况。     

Herpes simplex virus vectors for gene therapy

Herpes simplex virus (HSV) has a number of advantages as a vector for delivering specific genes to the nervous system. These include its large size, wide host range, and its ability to establish long-lived asymptomatic infections in neuronal cells in which a specific region of the viral genome continues to be expressed. Unfortunately, the large size of this virus and difficulty in manipulating it has led to its use as a vector lagging behind that of other, smaller viruses such as the retroviruses. In addition, the virus's ability to replicate lytically in the brain, under some circumstances, causing encephalitis, has led to fears about its potential safety for ultimate use in humans. This review will discuss a number of new approaches that are aimed at rendering simpler the insertion of foreign genes into the virus and making it as safe as possible. Ultimately, these advances offer real hope for the use of HSV vectors in gene therapy procedures.     

腺相关病毒作为基因治疗载体在生殖系统中的分布及影响的研究进展

腺相关病毒(adeno-associated virus,AAV)作为一种基因治疗载体被广泛应用于医学相关的各个领域。大量以AAV为载体的基因治疗被应用于药物研究,但目前国内外没有相关的基因药物应用于临床,原因在于其安全性问题有待进一步研究。AAV具有器官靶向性,易于分布在靶器官,但在非靶器官也会分布。AAV在生殖系统的短暂分布降低了对其功能的影响,但不排除潜在影响。就目前的研究结果来看,AAV不会整合在生殖细胞,从而不会对子代产生影响。本文将综述目前以腺相关病毒介导的基因治疗中对生殖系统安全性的研究进展,从载体在生殖系统的分布、对生殖系统功能的影响以及对子代影响的三个方面进行阐述。     

病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

Adenovirus and adeno-associated virus vectors

Recombinant adenovirus (rAd) and recombinant adeno-associated virus (rAAV) are among the most extensively used vectors in gene therapy studies to date. These two vectors share some similar features such as a broad host range and ability to infect both proliferating and quiescent cells. However, they also possess their own unique set of properties that render them particularly attractive for gene therapy applications. rAd vectors can accommodate larger inserts, mediate transient but high levels of protein expression, and can be easily produced at high titers. Development of gutted rAd vectors has further increased the cloning capacity of these vectors. The gaining popularity of rAAV use in gene therapy can be attributed to its lack of pathogenicity and added safety due to its replication defectiveness, and its ability to mediate long-term expression in a variety of tissues. Site-specific integration, as occurs with wild-type AAV, will be a unique and valuable feature if incorporated into rAAV vectors, further improving their safety. This paper describes these properties of rAd and rAAV vectors, and discusses further development and vector improvements that continue to extend the utility of these vectors, such as cell retargeting by capsid modification, differential transduction by use of serotypes, and extension of the cloning capacity of rAAV vectors by dual vector heterodimerization.     

Gene delivery to neurons: Is herpes simplex virus the right tool for the job?

Herpes simplex virus (HSV)-derived vectors are currently being developed for the introduction of foreign DNA into neurons. HSV vectors can facilitate a range of molecular studies on postmitotic neurons and may ultimately be used for somatic cell gene therapy for certain neurologic diseases. In this article, the salient features of the pathogenesis and molecular biology of HSV relevant to its use as a vector are described, along with an overview of the methods used to derive these vectors. The accomplishments which have been made to date using the HSV vector system are discussed, with emphasis on the issues of this technology which remain to be addressed. HSV has the potential to be a most useful tool for neuronal cell transgenesis and it is likely that important neurobiological questions will be answered using this vector system.     

Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification

Large scale purification of viruses and viral vectors for gene therapy applications and viral vaccines is a major separation challenge. Here tangential flow microfiltration and ultrafiltration using flat sheet membranes has been investigated for concentration of human influenza A virus. Ultrafiltration membranes with molecular weight cutoffs of 100 and 300 kDa as well as 0.1, 0.2 and 0.45 microm microfiltration membranes have been tested. The results indicate that use of 300 kDa membranes not only concentrate the virus particles but also lead to a significant removal of host cell proteins and DNA in the permeate. Tangential flow filtration may be used to fractionate virus particles. Human influenza A virus particles are spherical with an average size of 100 nm. Use of a 0.1 microm membrane leads to passage of virus particles less than 100 nm into the permeate and an increase of larger particles in the retentate. These results suggest that control of the transmembrane pressure, membrane pore size and pore size distribution could enable isolation of intact virus particles from damaged virions. Isolation of the virus particles of interest from viral fragments and other particulate matter could result in simplification of subsequent purification steps. Larger pore size membranes such as 0.45 microm that allow the passage of all virus particles may be used to remove host cell fragments. In addition virus particles attached to these fragments will be removed. Careful selection of membrane morphology and operating conditions will be essential in order to maximize the benefit of tangential flow filtration steps in the purification of viral products from cell cultures.     

靶向肿瘤治疗的腺相关病毒载体

靶向性是肿瘤治疗取得成功的关键因素。病毒载体用于治疗肿瘤的过程中必须要求特异性作用于肿瘤细胞的同时降低对正常细胞的毒性。腺相关病毒(adeno-associated virus,AAV)较其他病毒载体具有免疫原性小、宿主范围广和介导基因可长期表达等优点,因此得到了广泛的应用。然而,AAV载体针对肿瘤的靶向性一直是近年研究的热点和难点。现就AAV载体治疗肿瘤的概况和靶向策略以及其安全性等方面作一综述。     

TMV介导的外源蛋白在植物中表达

烟草花叶病毒(Tobaccomosaicvirus,TMV)为Tobamovirus代表成员,以此病毒介导的外源蛋白在植物中表达,经过了十几年的研究和不断完善,已被证实为一种有效的表达外源蛋白的途径.这项技术已经在医用活性多肽以及疫苗的研制、功能基因的鉴定、植物体内生物合成途径的研究等方面发挥越来越重要的作用.重点阐述了TMV基因组RNA的结构和分子生物学特征,并着重对重组载体的构建及其利用加以了论述.     

Foxtail mosaic virus: A tool for gene function analysis in maize and other monocots

Many plant viruses have been engineered into vectors for use in functional genomics studies, expression of heterologous proteins, and, most recently, gene editing applications. The use of viral vectors overcomes bottlenecks associated with mutagenesis and transgenesis approaches often implemented for analysis of gene function. There are several engineered viruses that are demonstrated or suggested to be useful in maize through proof-of-concept studies. However, foxtail mosaic virus (FoMV), which has a relatively broad host range, is emerging as a particularly useful virus for gene function studies in maize and other monocot crop or weed species. A few clones of FoMV have been independently engineered, and they have different features and capabilities for virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX) of proteins. In addition, FoMV can be used to deliver functional guide RNAs in maize and other plants expressing the Cas9 protein, demonstrating its potential utility in virus-induced gene editing applications. There is a growing number of studies in which FoMV vectors are being applied for VIGS or VOX in maize and the vast majority of these are related to maize–microbe interactions. In this review, we highlight the biology and engineering of FoMV as well as its applications in maize–microbe interactions and more broadly in the context of the monocot functional genomics toolbox.     

HIV-1载体的研发沿革

自从科学家于1983年发现了人类免疫性缺陷病毒1(human immunodeficiency virus1,HIV-1)以来,随着对它的研究不断深入,其表达载体的开发也有了长足的进步。与其他逆转录病毒载体相比,如莫罗尼小鼠白血病病毒(murine leukemiavirus,MLV)载体和泡沫病毒(foamyvirus,FV)载体等,HIV-1载体具有诸多独特的优点,因而有着更广泛的应用于临床基因治疗的前景。该文对HIV-1载体的研发过程及其优缺点进行综述。     

Optimized production and concentration of lentiviral vectors containing large inserts

Generation of high titer lentiviral stocks and efficient virus concentration are central to maximize the utility of lentiviral technology. Here we evaluate published protocols for lentivirus production on a range of transfer vectors differing in size (7.5-13.2 kb). We present a modified virus production protocol robustly yielding useful titers (up to 10(7)/ml) for a range of different transfer vectors containing packaging inserts up to 7.5 kb. Moreover, we find that virus recovery after concentration by ultracentrifugation depends on the size of the packaged inserts, heavily decreasing for large packaged inserts. We describe a fast (4 h) centrifugation protocol at reduced speed allowing high virus recovery even for large and fragile lentivirus vectors. The protocols outlined in the current report should be useful for many labs interested in producing and concentrating high titer lentiviral stocks.     

重组腺相关病毒包装容量研究进展

在以病毒载体介导的基因治疗研究中,重组腺相关病毒(rAAV)因其疗效和安全性方面的优势,是最有临床应用前景的载体。但其转基因包装容量一般不能超过5.0kb,给需要转导大片段基因的应用带来了困难,限制了rAAV在基因治疗研究中的应用。随着对rAAV细胞转导生物学过程研究的不断深入,发现了一些可以突破rAAV包装容量限制的技术,如反式剪接和同源重组策略,为拓展该载体应用范围提供了可能性。另外,rAAV包装容量限制的特点还可以被用来减少生产过程中具有可复制能力的类病毒杂质的污染,为rAAV的临床安全性提供了保障。     

Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.     

昆虫内共生菌沃尔巴克氏体抗病毒研究进展

植物病毒病是危害我国蔬菜生产的第一大病害,而烟粉虱Bemisia tabaci Gennadius、蓟马和蚜虫等小型昆虫是蔬菜病毒病的主要传播媒介.虫传病毒病害的防控策略复杂且难度大,目前生产上主要依赖化学农药防治介体昆虫,预防与控制蔬菜病毒病.种植户化学杀虫药剂的不合理使用、甚至滥用,导致媒介昆虫抗药性、杀虫剂污染与残留等问题,严重威胁农产品安全与生态安全.发展高效控制媒介昆虫及其传播重大病毒病害防控技术是保障食品安全、蔬菜产业健康可持续发展的迫切需求.内共生菌沃尔巴克氏体Wolbachia是节肢动物体内广泛存在并经卵传播的革兰氏阴性胞内次生共生细菌,约65％的昆虫天然携带沃尔巴克氏体.沃尔巴克氏体对宿主昆虫具有胞质不亲和、产雌孤雌生殖、雌性化或者杀雄作用等生殖调控作用,同时抑制虫媒病毒病在昆虫体内的复制和传播.基于沃尔巴克氏体的蚊媒及蚊媒病毒病控制研究与应用取得了重大进展,现就沃尔巴克氏体抗病毒及其应用的最新进展和未来发展进行综述,为发展蔬菜害虫及其传播病毒病防控技术提供新思路.     

重组腺相关病毒生产方法研究进展

重组腺相关病毒(rAAV)作为基因治疗的载体,具有感染范围广、能在宿主细胞中长久稳定表达外源基因和非致病性等优点,因此在基因治疗领域倍受青睐。rAAV的大规模制备技术一直是限制其临床广泛应用的瓶颈。近年来,有许多改进rAAV制备工艺的尝试。我们在介绍rAAV载体制备所需各种元件的基础上,对这些方法进行了简要综述。     

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.     

肿瘤靶向治疗的新型细胞载体

肿瘤靶向性病毒作为一种特殊的肿瘤治疗药物和基因治疗载体近年来已得到长足发展,许多高效、靶向性病毒载体已被相继研究开发,但仍不能满足临床上肿瘤靶向治疗的需要,如何将这些靶向病毒准确而高效地运输到肿瘤病变部位仍然未得到充分解决.细胞因子诱导杀伤细胞(cytokine-inducedkillercells,CIK)作为肿瘤的细胞治疗方法之一已成功地在临床上得到了广泛应用.最近科学家使用CIK细胞作为病毒运载工具,成功地将病毒运载到肿瘤组织部位并显示出高效的抗肿瘤作用,该方法为病毒运输定位于肿瘤病变部位找到了突破口,实验资料显示其具有潜在的应用价值.     

利用嗜神经病毒跨突触追踪神经网络研究进展

解析大脑神经网络的连接图谱是认识大脑功能的前提。发展追踪大脑神经环路结构的技术,已成为神经科学研究中的迫切需求。基于嗜神经病毒发展而来的跨突触追踪技术,是揭示大脑神经网络结构的最有效手段,也是神经科学研究中发展十分迅速的领域。不同的嗜神经病毒类型或毒株,都有其独特的分子生物学特性、跨突触标记特性、改造方式。通过使用遗传重组改造的嗜神经病毒追踪神经环路,可以获得特定区域或特定类型神经元多级输出网络、输入网络及单级输入或输出网络。主要介绍神经科学研究中常用的神经病毒及相关的辅助工具病毒特性,及嗜神经病毒介导的各种神经回路标记技术。