双链RNA和植物对病毒的抗性

双链RNA能诱导转录后的基因沉默,是生物抵御病毒入侵、维持自身基因稳定的一种自我保护机制.把源自病毒的基因构建成反向重复结构转入植物体内,其转录出的RNA会通过分子内序列互补形成双链,将入侵病毒的同源序列降解,使转基因植株获得对病毒的高抗性.RNA干扰型抗病毒转基因植株中,转病毒基因的mRNA不存在或存在量很少,也不会翻译成有功能的病毒蛋白,因此不存在病毒RNA重组、异源包装及协生作用的潜在风险,具有较高的生物安全性.双链RNA抗病毒转基因正在成为一种高效、安全的植物抗病毒策略.     

病毒miRNA与免疫逃逸

微小RNA（microRNA,miRNA）是一种非编码的小分子RNA,长度一般在22 nt左右,通过与mRNA 3＇UTR的特异性结合介导转录后调控过程。现已鉴定出的miRNA涵盖了从植物到人类的多个物种,并参与了调节生长、免疫、凋亡等多种生命活动。最近发现,DNA病毒感染宿主时也能编码产生miRNA,并在病毒免疫逃逸中扮演着重要角色。病毒感染是一个复杂的过程,病毒需要逃脱免疫系统才能对宿主产生持续性感染,而病毒miRNA能调控宿主和自身基因表达,帮助病毒感染宿主,且因其本身没有免疫原性,而成为病毒逃避免疫应答的重要工具,但其中的分子机制尚不十分清楚。该文就病毒miRNA如何调控病毒自身与宿主基因进行免疫逃逸的近期研究作一综述。     

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.     

CRISPR-Cas9应用于病毒性传染病防控的研究进展

病毒性传染病是威胁人类健康的重要因素,迫切需要新的治疗方法来降低由急性病毒感染如鼻病毒和登革热病毒以及慢性病毒感染如人类免疫缺陷病毒1和乙型肝炎病毒引起的发病率和死亡率.随着分子生物学技术的发展,靶向序列特异性的基因编辑技术成为传染病治疗的有力工具.其中规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)-CRISPR相关蛋白9(CRISPR associated protein 9,Cas9)凭借其高效、简便、高特异性等特点被广泛应用于细胞系和动物模型中的传染病治疗,从而成为有前景的新型传染病治疗模式.目前,利用病毒和非病毒载体将Cas9以DNA、m RNA或蛋白质的形式递送到细胞中的可行性研究和评估CRISPR-Cas9体内适用性的临床试验已经在进行中.本篇综述中,我们将对CRISPR-Cas9的原理,其应用于传染病治疗的最新研究进展以及该技术面临的挑战和可预测性的解决方法等加以概述,并进一步展望其未来的发展方向.     

Natural selection and the organ-specific differentiation of HIV-1 V3 hypervariable region

The existence of organ-specific HIV-1 populations within infected hosts has been studied for many years; nonetheless results reported by different authors are somewhat discrepant. To tackle this problem, we used a population genetics approach to analyze previously published data from the V3 hypervariable region of the envelope env gene. Our results are compatible with a population subdivision by organs in 95% of individuals analyzed at autopsy. In addition, populations infecting the nervous system and testicles clearly appear as differentiated subsets of the so-called macrophage-tropic variants. Liver and kidney may harbor differentiated populations as well. Although it is widely accepted that organ compartmentalization arises as a consequence of different selective pressures imposed by different organs, a definitive demonstration has not yet been provided. Our analysis of the pattern of synonymous and nonsynonymous nucleotide substitutions provides evidence supporting this hypothesis, without discarding the role of other evolutionary processes. In contrast, positive selection does not seem to be the mechanism responsible for the evolution of patient-specific sequences.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.     

Using viral genomics to develop viral gene products as a novel class of drugs to treat human ailments

A novel strategy for drug discovery and advancement has been developed by exploiting viral genomics. By manipulating cell function through the therapeutic administration of specific gene products from viruses, an important new class of therapeutics could be developed for the treatments of major human diseases. As it may turn out in the human genome arena, the groups which can establish a stronghold in the field would be in the best position to exploit this new drug development strategy.     

Production,Processing, and Characterization of Synthetic AAV Gene Therapy Vectors

Over the last two decades, gene therapy vectors based on wild-type Adeno-associated viruses (AAV) are safe and efficacious in numerous clinical trials and are translated into three approved gene therapy products. Concomitantly, a large body of preclinical work has illustrated the power and potential of engineered synthetic AAV capsids that often excel in terms of an organ or cell specificity, the efficiency of in vitro or in vivo gene transfer, and/or reactivity with anti-AAV immune responses. In turn, this has created a demand for new, scalable, easy-to-implement, and plug-and-play platform processes that are compatible with the rapidly increasing range of AAV capsid variants. Here, the focus is on recent advances in methodologies for downstream processing and characterization of natural or synthetic AAV vectors, comprising different chromatography techniques and thermostability measurements. To illustrate the breadth of this portfolio, two chimeric capsids are used as representative examples that are derived through forward- or backwards-directed molecular evolution, namely, AAV-DJ and Anc80. Collectively, this ever-expanding arsenal of technologies promises to facilitate the development of the next AAV vector generation derived from synthetic capsids and to accelerate their manufacturing, and to thus boost the field of human gene therapy.     

Development of novel poly(ethylene glycol)-based vehicles for gene delivery

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.     

植物病毒诱导的基因沉默在基因功能研究中的应用

传统的植物遗传转化方法周期长、工作量大、过程繁琐,不利于基因功能的快速高通量鉴定．近年来随着基因沉默机制研究的深入和不断发展,利用病毒诱导的基因沉默(Virus induced gene silencing,VIGS)进行植物功能基因组研究作为一种快速、高通量的反向遗传学工具已被广泛应用在烟草、马铃薯、番茄等植物中, 在大规模的植物基因组功能鉴定中展示了广阔的应用前景．综述了 VIGS 的作用机制、植物病毒栽体、转化方法以及在植物基因功能研究等方面的应用及前景．     

RNA interference for antiviral therapy

Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double-stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene-specific therapeutics.     

基因治疗的病毒载体系统

病毒载体是基因治疗中应用最为广泛的载体系统。该文就RNA病毒载体、DNA病毒载体和杂合病毒载体的生物学特性、基因组特点以及其在基因治疗中的优缺点进行综述,并对病毒载体系统的发展前景进行了展望。     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.