BACKGROUND & AIMS: :Incorporation of a central polypurine tract (cPPT) and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression. We compared the effects of these elements individually and together on transduction efficiency and gene expression, using lentivirus vectors pseudotyped with vesicular stomatitis virus G protein (VSV-G) and encoding enhanced green fluorescent protein (GFP) and rat erythropoietin (EPO). The transduction efficiency was greater than 2-fold higher in the vector containing the PRE element, 3-fold higher in vector encoding the cPPT element, and 5-fold increased in the GFP virus containing both cPPT and PRE elements relative to the parent virus. In comparison with parent vector the mean fluorescence intensity (MFI) of GFP expression was 7-fold higher in cells transduced with virus containing PRE, 6-fold increased in cells transduced with virus containing cPPT, and 42-fold increased in GFP-virus containing both cPPT and PRE elements. EPO-virus containing a PRE element showed a nearly 5-fold increase in EPO secretion over the parent vector, and the vector encoding both PRE and cPPT showed a 65-fold increase. Thus, lentivirus vectors incorporating both PRE and cPPT showed expression levels significantly increased over the sum of the components alone, suggesting a synergistic effect.

背景与目标： 将慢病毒载体整合到中央多嘌呤区（cPPT）和转录后调控元件（PRE）中，可提高转导效率和转基因表达。我们使用伪装了水泡性口炎病毒G蛋白（VSV-G）并编码增强型绿色荧光蛋白（GFP）和大鼠促红细胞生成素（EPO）的慢病毒载体，比较了这些元素单独以及一起对转导效率和基因表达的影响。相对于亲本病毒，在含有PRE元素的载体中，转导效率高出2倍，在编码cPPT元素的载体中，转导效率高出3倍，在同时含有cPPT和PRE元素的GFP病毒中，转导效率提高了5倍。与亲代载体相比，用含PRE的病毒转导的细胞中GFP表达的平均荧光强度（MFI）高7倍，用含cPPT的病毒转导的细胞中GFP表达的平均荧光强度高6倍，而在含cPPT的病毒转导的细胞中GFP表达的平均荧光强度增加42倍。 cPPT和PRE元素。含有PRE元件的EPO病毒的EPO分泌量比亲本载体增加了近5倍，而编码PRE和cPPT的载体均表现出了65倍的增加。因此，掺有PRE和cPPT的慢病毒载体显示表达水平明显高于单独组分的总和，表明具有协同作用。

BACKGROUND & AIMS: :Pathologic lesions caused by catheter-based revascularization procedures for occlusive artery disease include disruption of the endothelium, exposure of extracellular matrix (ECM) proteins, and proliferation of vascular smooth muscle cells, which lead to neointima formation and restenosis. We have developed matrix-collagen-targeted retroviral vectors that are able to accumulate at sites of vascular injury (Hall et al., Hum. Gene Ther. 1997;8:2183-2192; Hall et al., Hum. Gene Ther. 2000;11:983-993). The primary tissue-targeting motif, adapted from the physiological surveillance sequence found in von Willebrand factor, served to localize and concentrate the vector within vascular lesions. In the present study, we evaluated the efficiency of this vector-targeting system in rats with nonligated balloon-injured carotid arteries. Both intraarterial (by retrograde femoral artery catheterization) and intravenous (via femoral vein) injection of a matrix-targeted vector enhanced transduction of neointimal cells ( approximately 20%) at severely denuded areas when compared with the nontargeted vector (<1%). Further, intraarterial instillation of a matrix-targeted, but not a nontargeted, vector bearing an antisense cyclin G1 construct inhibited neointima lesion formation in the injured carotid arteries. Taken together, these data indicate that strategic targeting of retroviral vectors to vascular lesions would have therapeutic potential in the management of vascular restenosis and many other disorders of uncontrolled proliferation where endothelial disruption, ECM remodeling, and collagen deposition form the nexus for preferential vector localization and concentration in vivo.

背景与目标： 闭塞性动脉疾病的基于导管的血运重建术引起的病理损害包括内皮破坏，细胞外基质（ECM）蛋白暴露以及血管平滑肌细胞增殖，从而导致新内膜形成和再狭窄。我们已经开发了靶向胶原蛋白的逆转录病毒载体，它们能够在血管损伤部位积聚（Hall等，Hum。Gene Ther。1997; 8：2183-2192； Hall等，Hum.Gene Ther。2000 ; 11：983-993）。适应于von Willebrand因子中发现的生理学监视序列的主要组织靶向基序可将载体定位并集中在血管病变内。在本研究中，我们评估了这种载体靶向系统在未结扎球囊损伤颈动脉的大鼠中的效率。与非靶向载体（<1％）相比，动脉内注射（通过逆行股动脉导管插入术）和静脉内（通过股静脉）注射在严重裸露区域的新内膜细胞（约20％）的转导都增强了内膜细胞的转导。此外，动脉内滴注带有反义细胞周期蛋白G1构建体的靶向基质的载体，而非非靶向载体，抑制了受损颈动脉中新内膜病变的形成。综上所述，这些数据表明，将逆转录病毒载体战略性地靶向血管病变将具有治疗血管再狭窄和许多其他不受控制的增生性疾病的治疗潜力，其中内皮细胞破坏，ECM重塑和胶原蛋白沉积形成了优先载体定位和相关性的纽带。体内浓度。

BACKGROUND & AIMS: :In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both alphav integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

背景与目标： ：在这项研究中，我们分析了将Ad载体重新靶向人胰腺癌的两种方法，目的是提高基因转移效率。首先，我们通过流式细胞术分析了表皮生长因子受体（EGFR）在原发性和已建立的胰腺癌细胞上的表达，揭示了这些细胞表面上EGFR的高表达水平。我们显示，使用由人源EGF（sCAR-EGF）基因融合的截短的柯萨奇和腺病毒受体（sCAR）的重组可溶性形式形成的双特异性融合蛋白，通过双特异性融合蛋白形成的EGFR靶向进入途径将其重定向至EGFR，从而提高了基因转移效率胰腺癌细胞。由于流式细胞仪显示不存在CAR表达，但在胰腺癌细胞上同时存在两种alphav整合素中的至少一种，因此使用具有RGD（Arg-Gly-Asp）的基因修饰的Ad载体研究了第二种靶向方法插入到纤维结的HI-环中的含-肽。该RGD靶向广告（AdlucRGD）通过与细胞整联蛋白结合，从而显着增强了基因转移，从而揭示了与CAR无关的有效感染。这些发现与基于Ad载体的胰腺癌基因治疗策略直接相关。

BACKGROUND & AIMS: BACKGROUND:Knockdown resistance (kdr) is a well-characterized target-site insecticide resistance mechanism that is associated with DDT and pyrethroid resistance. Even though insecticide resistance to pyrethroids and DDT have been reported in Anopheles albimanus, Anopheles benarrochi sensu lato (s.l.), Anopheles darlingi, Anopheles nuneztovari s.l., and Anopheles pseudopunctipennis s.l. malaria vectors in Latin America, there is a knowledge gap on the role that kdr resistance mechanisms play in this resistance. The aim of this study was to establish the role that kdr mechanisms play in pyrethroid and DDT resistance in the main malaria vectors in Colombia, in addition to previously reported metabolic resistance mechanisms, such as mixed function oxidases (MFO) and nonspecific esterases (NSE) enzyme families. METHODS:Surviving (n = 62) and dead (n = 67) An. nuneztovari s.l., An. darlingi and An. albimanus mosquitoes exposed to diagnostic concentrations of DDT and pyrethroid insecticides were used to amplify and sequence a ~ 225 bp fragment of the voltage-gated sodium channels (VGSC) gene. This fragment spanning codons 1010, 1013 and 1014 at the S6 segment of domain II to identify point mutations, which have been associated with insecticide resistance in different species of Anopheles malaria vectors. RESULTS:No kdr mutations were detected in the coding sequence of this fragment in 129 samples, 62 surviving mosquitoes and 67 dead mosquitoes, of An. darlingi, An. nuneztovari s.l. and An. albimanus. CONCLUSION:Mutations in the VGSC gene, most frequently reported in other species of the genus Anopheles resistant to pyrethroid and DDT, are not associated with the low-intensity resistance detected to these insecticides in some populations of the main malaria vectors in Colombia. These results suggest that metabolic resistance mechanisms previously reported in these populations might be responsible for the resistance observed.

背景与目标： 背景：击倒抗性（kdrdown）是一种特征明确的靶点杀虫剂抗性机制，与DDT和拟除虫菊酯抗性相关。尽管在白bi按蚊，按蚊Bennarrochi sensu lato（s.l.），达令按蚊Annueles narlingtovari s.l.和拟按蚊Anopheles pseudopunctipennis s.l.中已经报告了对拟除虫菊酯和DDT的杀虫剂抗性。在拉丁美洲的疟疾媒介中，关于kdr抗性机制在这种抗性中所起作用的知识差距很大。这项研究的目的是确定哥伦比亚除主要报道的代谢抗性机制（如混合功能氧化酶（MFO）和非特异性酯酶（NSE））外，kdr机制在哥伦比亚主要疟疾媒介中对拟除虫菊酯和滴滴涕抗性中的作用。酶家族。
方法：存活（n = 62）和死亡（n = 67）。 nuneztovari s.l.，An。达令吉和安。暴露于诊断浓度的滴滴涕和拟除虫菊酯杀虫剂的白bi蚊被用来扩增和测序电压门控钠通道（VGSC）基因的〜225 bp片段。该片段跨越域II的S6区段的密码子1010、1013和1014，以识别点突变，该突变已与不同种类的按蚊疟疾载体中的杀虫剂抗性相关。
结果：在An的129个样品，62个存活的蚊子和67个死亡的蚊子中，在该片段的编码序列中未检测到kdr突变。达令吉努涅斯托瓦里湖和。 albimanus。
结论：VGSC基因的突变（在拟除虫菊酯和滴滴涕耐药的其他按蚊属的其他物种中最常报道）与在哥伦比亚一些主要疟疾媒介人群中对这些杀虫剂的低强度耐药性无关。这些结果表明，先前在这些人群中报道的代谢抗性机制可能是观察到的抗性的原因。

BACKGROUND & AIMS: :In the past decade, adenovirus vectors have generated tremendous interest, especially in gene therapy applications. In the so-called 'first generation' adenovirus vectors, the transgenes are inserted in place of the E1 region, or less often the E3 region. Although second-generation and helper-dependent adenovirus vectors will probably prevail in the future in applications that require long-term gene expression, first generation adenovirus vectors will remain very useful in other settings, such as cancer and vaccination, or simply to transfect cell lines that are refractory to other transfection methods. Until a few years ago, the construction of first generation adenovirus vectors was a labor-intensive and time-consuming process. More than 20 methods have appeared that facilitate their construction and are reviewed below.

背景与目标： 在过去的十年中，腺病毒载体引起了极大的兴趣，特别是在基因治疗应用中。在所谓的“第一代”腺病毒载体中，转基因被插入到E1区域，或更不常见的是E3区域。尽管第二代和依赖于助手的腺病毒载体可能会在将来需要长期表达基因的应用中盛行，但第一代腺病毒载体在其他环境（例如癌症和疫苗接种）或仅用于转染细胞系中仍将非常有用其他转染方法无法耐受的直到几年前，第一代腺病毒载体的构建还是一项劳动密集型且耗时的过程。已经出现了20多种有助于其构建的方法，下面将进行综述。

BACKGROUND & AIMS: :Currently, mosquito vector control is facing a number of key challenges, including the rapid development of resistance to synthetic pesticides and the recent spread of aggressive arbovirus outbreaks. The biosynthesis of silver nanoparticles (AgNPs) is currently considered an environmental friendly alternative to the employ of pyrethroids, carbamates and microbial agents (e.g. Bacillus thuringiensis var. israelensis), since AgNPs are easy to produce, effective and stable in the aquatic environment. However, their biophysical features showed wide variations according to the botanical agent using for the green synthesis, outlining the importance of screening local floral resources used as reducing and stabilizing agents. In this study, we focused on the biophysical properties and the mosquitocidal action of Quisqualis indica-fabricated AgNPs. AgNPs were characterized using spectroscopic (UV, FTIR, XRD) and microscopic (AFM, SEM, TEM and EDX) techniques. AFM, SEM and TEM confirmed the synthesis of poly-dispersed AgNPs with spherical shape and size ranging from 1 to 30nm. XRD shed light on the crystalline structure of these AgNPs. The acute toxicity of Quisqualis indica extract and AgNPs was evaluated against malaria, arbovirus, and filariasis vectors, Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus, as well as on three important non-target aquatic organisms. The Q. indica leaf extract showed moderate larvicidal effectiveness on Cx. quinquefasciatus (LC50=220.42), Ae. aegypti (LC50=203.63) and An. stephensi (LC50=185.98). Q. indica-fabricated AgNPs showed high toxicity against Cx. quinquefasciatus (LC50=14.63), Ae. aegypti (LC50=13.55) and An. stephensi (LC50=12.52), respectively. Notably, Q. indica-synthesized AgNPs were moderately toxic to non-target aquatic mosquito predators Anisops bouvieri (LC50=653.05μg/mL), Diplonychus indicus (LC50=860.94μg/mL) and Gambusia affinis (LC50=2183.16μg/mL), if compared to the targeted mosquitoes. Overall, the proposed one-pot biogenic fabrication of AgNPs using Q. indica is a low-cost and eco-friendly tool in the fight against Zika virus, malaria and filariasis vectors, with little impact against non-target aquatic mosquito predators.

背景与目标： 目前，蚊媒的控制面临许多关键挑战，包括对合成农药的抗性迅速发展以及近期侵略性虫媒病毒爆发的蔓延。银纳米颗粒（AgNPs）的生物合成目前被认为是替代拟除虫菊酯，氨基甲酸酯和微生物制剂（例如苏云金芽孢杆菌var.israelensis）的一种环境友好替代方法，因为AgNPs在水生环境中易于生产，有效且稳定。然而，根据用于绿色合成的植物剂，它们的生物物理特征显示出很大的差异，突出了筛选用作还原剂和稳定剂的当地花卉资源的重要性。在这项研究中，我们集中于Quisqualis d制造的AgNPs的生物物理特性和灭蚊作用。 AgNPs使用光谱技术（UV，FTIR，XRD）和微观技术（AFM，SEM，TEM和EDX）进行表征。 AFM，SEM和TEM证实了球形和尺寸范围为1至30nm的多分散AgNP的合成。 XRD揭示了这些AgNP的晶体结构。评价了Quisqualis indica提取物和AgNPs对疟疾，虫媒病毒和丝虫病载体，Stephensi按蚊，埃及伊蚊和库克西库克斯犬以及三种重要的非目标水生生物的急性毒性。 in叶提取物对Cx表现出中等杀幼虫效果。 quinquefasciatus（LC50 = 220.42），Ae。埃及（LC50 = 203.63）和An。斯蒂芬斯（LC50 = 185.98）。 Q. Ag制造的AgNP对Cx表现出高毒性。 quinquefasciatus（LC50 = 14.63），Ae。埃及（LC50 = 13.55）和An。斯蒂芬斯（LC50 = 12.52）。值得注意的是，Q合成的AgNPs对非目标水生捕食性蚊食美洲大按蚊（LC50 =653.05μg/ mL），印度洋双翅目（LC50 =860.94μg/ mL）和革兰（Gambusia affinis）（LC50 =2183.16μg/ mL）具有中等毒性。 ，如果与目标蚊子相比。总体而言，拟议的使用印度Q草的一锅式AgNP的生物制备是对抗寨卡病毒，疟疾和丝虫病载体的一种低成本且生态友好的工具，对非目标水生蚊虫几乎没有影响。

BACKGROUND & AIMS: :RNA editing is a source of molecular diversity that regulates the functional repertoire of animal transcriptomes. Multiple studies in Drosophila have revealed that conserved editing events can be a source of evolutionary adaptations, and there is a solid body of evidence linking editing and the fine-tuning of neural genes, which are often targeted by insecticides used in vector control. Yet, despite these suggestive connections, genome-wide analyses of editing in insect vectors are conspicuously lacking. Future advances will require complementing the growing wealth of vector genomes with targeted transcriptome analyses. Here, we review recent investigations of the genetic footprints of adaptive RNA editing in insects and provide an overview of new methodologies applicable to studies of RNA editing in insect vectors.

背景与目标： RNA编辑是调节动物转录组功能的分子多样性的一个来源。在果蝇中的多项研究表明，保守的编辑事件可能是进化适应的来源，并且存在将编辑和神经基因的微调联系起来的可靠证据，而神经基因通常是载体控制中使用的杀虫剂所针对的。然而，尽管有这些暗示性的联系，但是仍然明显缺乏对昆虫载体编辑的全基因组分析。未来的发展将需要通过靶向转录组分析来补充日益丰富的载体基因组。在这里，我们回顾了昆虫中自适应RNA编辑的遗传足迹的最新研究，并提供了适用于昆虫载体RNA编辑研究的新方法的概述。

BACKGROUND & AIMS: :Up until recently, Australia was considered free of Leishmania due to the absence of phlebotomine sandfly species (Diptera: Phlebotominae) known to transmit Leishmania parasites in other parts of the world. The discovery of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) in Northern Australia sparked questions as to the existence of alternative vectors of Leishmania. This has added to the complexity of fully understanding the parasite's interaction with its vector, which is known to be very specific. Previous findings demonstrated L. macropodum infection beyond the blood meal stage in the day-biting midges Forcipomyia (Lasiohelea) Kieffer (Diptera: Ceratopogonidae) implicating them in the parasite's life cycle. Currently, there is no conclusive evidence demonstrating this suspected vector to transmit L. macropodum to a naïve host. Therefore, this research aimed to investigate the vector competency of day-biting midge F. (Lasiohelea) to transmit L. macropodum utilising a novel technology that preserves nucleic acids. Honey-soaked Flinders Technology Associates (FTA®) filter-paper cards were used to obtain saliva expectorated from biting midges while sugar-feeding. F. (Lasiohelea) were aspirated directly off macropods from a known Leishmania-transmission site and were kept in a waxed-paper container holding a honey-coated FTA® card for feeding. Insect identification and Taqman quantitative real-time PCR (qPCR) screening assays revealed L. macropodum DNA in F. (Lasiohelea) up to 7 days post field-collection, and in an unidentified biting midge, previously known as F. (Lasiohelea) sp.1. Moreover, 7/145 (4.83%) of FTA® cards were confirmed positive with L. macropodum DNA after exposure to field-collected F. (Lasiohelea). Additionally, FTA® cards were found to be a valuable surveillance tool, given the ease of use in the field and laboratory. Overall, our findings support previous reports on L. macropodum transmission by an alternative vector to phlebotomine sandflies. Further studies identifying and isolating infective L. macropodum promastigotes is necessary to resolve questions on the L. macropodum vector.

背景与目标： ：直到最近，由于在世界其他地方没有已知传播利什曼原虫寄生虫的毒理mine沙蝇物种（双翅目：Phlebotominae），澳大利亚被认为没有利什曼原虫。在北澳大利亚发现利什曼原虫（Mundinia）macropodum（Kinetoplastida：Trypanosomatidae）引发了人们对利什曼原虫替代载体的存在的疑问。这增加了充分了解寄生虫与其媒介的相互作用的复杂性，这是众所周知的。先前的发现表明，在咬咬mid虫（Lasiohelea）Kieffer（双翅目：Ceratopogonidae）的食血期之后，L。macropodum感染超过了寄生虫的生命周期。目前，尚无确凿证据表明该可疑载体将大脚乳杆菌传播至幼稚宿主。因此，本研究旨在利用一种保存核酸的新技术来研究咬日mid（Lasiohelea）传播大花胶球菌的载体能力。蜂蜜浸透的Flinders Technology Associates（FTA®）滤纸卡用于在喂糖时从咬咬mid虫中排出唾液。将F.（Lasiohelea）从已知的利什曼原虫传播地点直接从巨足类动物中吸出，并保存在装有蜂蜜涂层FTA®卡的蜡纸容器中进行喂养。昆虫鉴定和Taqman定量实时PCR（qPCR）筛选分析揭示了田间采集后长达7天的F.（Lasiohelea）中的L. macropodum DNA，以及在以前被称为F.（Lasiohelea）sp的不明咬人蚊中。 .1。此外，在暴露于田间采集的F.（Lasiohelea）后，证实了L. macropodum DNA的FTA®卡有7/145（4.83％）阳性。此外，由于FTA®卡在现场和实验室易于使用，因此被认为是一种有价值的监视工具。总的来说，我们的研究结果支持了以前关于巨脚乳杆菌通过除草胺sand替代载体传播的报道。进一步的研究，以鉴定和分离感染性大脚紫苏前鞭毛体对于解决关于大脚紫苏载体的问题是必要的。

BACKGROUND & AIMS: :A new Doppler echocardiographically based method has been developed to quantify volume flow rate by surface integration of velocity vectors (SIVV). Electrocardiographic-gated color Doppler images acquired in two orthogonal planes were used to estimate volume flow rate through a bowl-shaped surface at a given time and distance from the probe. To provide in vitro validation, the method was tested in a hydraulic model representing a pulsatile flow system with a restrictive orifice. Accurate estimates of stroke volume (+/- 10%) were obtained in a window between 1.2 and 1.6 cm proximal to the orifice, just before the region of prestenotic acceleration. By use of the Bernoulli's equation, the estimated flows were used to generate pressure gradient waveforms across the orifice, which agreed well with the measured flows. To demonstrate in vivo applicability, the SIVV method was applied retrospectively to the determination of stroke volume and subaortic flow from the apical three-chamber and five-chamber views in two patients. Stroke volume estimates along the left ventricular outflow tract showed a characteristic similar to that in the in vitro study and agreed well with those obtained by the Fick oxygen method. The region where accurate measurements can be obtained is affected by instrumental factors including Nyquist velocity limit, wall motion filter cutoff, and color flow sector angle. The SIVV principle should be useful for quantitative assessment of the severity of valvular abnormalities and noninvasive measurement of pulsatile volume flows in general.

背景与目标： ：已经开发了一种基于多普勒超声心动图的新方法，可以通过对速度矢量（SIVV）进行表面积分来量化体积流量。在两个正交平面中采集的心电门控彩色多普勒图像用于估计在给定时间和距探头的距离上通过碗状表面的体积流量。为了提供体外验证，该方法在液压模型中进行了测试，该液压模型表示带有节流孔的脉动流系统。在靠近小孔的区域内，在靠近孔口1.2到1.6厘米之间的窗口中获得了对搏动量的准确估计（±10％）。通过使用伯努利方程，估计流量用于生成穿过孔口的压力梯度波形，该波形与测得的流量非常吻合。为了证明在体内的适用性，将SI​​VV方法追溯应用于两名患者从心尖三腔和五腔视图确定中风量和主动脉流量。沿左心室流出道的中风量估计值显示了与体外研究相似的特征，并且与通过Fick氧气法获得的结果相吻合。可以得到准确测量结果的区域受仪器因素的影响，这些因素包括奈奎斯特速度极限，壁运动滤波器截止和色流扇形角。一般而言，SIVV原理可用于定量评估瓣膜异常的严重程度和无创测量搏动量流量。

BACKGROUND & AIMS: :Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.

背景与目标： ：开发能够以细胞特异性方式转导靶细胞的γ-逆转录病毒载体的工程方法，可能会影响基因治疗临床应用的未来以及对转移基因载体生物学的理解。两个分子事件对于控制γ-逆转录病毒载体进入靶细胞至关重要：与细胞表面受体的结合以及随后病毒载体膜和细胞膜的融合。在本报告中，我们评估了将结合膜的抗体和融合分子分别提供结合和融合功能的方法掺入用于目标基因递送的γ-逆转录病毒载体中的方法。抗CD20抗体和Sindbis病毒糖蛋白衍生的融合蛋白可以有效地在病毒载体表面共展示。带有抗CD20抗体的载体赋予它们与表达CD20的细胞的结合特异性。对于展示抗体和融合蛋白的γ-逆转录病毒载体，观察到了向表达CD20的细胞增强的体外转导。我们发现，融合蛋白的生物学活性在这种靶向策略的效率中起着重要作用，并且能够工程化融合蛋白的几种突变形式，这些突变体表现出升高的融合功能，从而提高了靶向转导的整体效率。我们设计了一种动物模型来表明，将这种工程载体皮下注射到异种移植有靶细胞的区域，可以在体内实现靶向基因的递送。两者合计，我们证明了针对工程学针对γ-逆转录病毒载体的灵活模块化的两分子策略作为原理证明。

BACKGROUND & AIMS: :Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in pre-clinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations. Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance. Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile.

背景与目标： ：重组腺相关病毒（rAAV）作为人类疾病临床前模型中基因转移的载体显示出巨大希望，目前正在人类临床试验中进行评估。结果，越来越多的注意力转向了优化rAAV滴度，纯度和稳定性的重要任务。我们已经观察到二价阳离子依赖性对于不同rAAV载体的热稳定性的巨大变化。为了进一步研究该观察结果，在存在和不存在二价阳离子的情况下，确定了8种不同载体构建体的热稳定性，其大小范围为野生型基因组大小的73％至107％（4.68千个碱基）。含有较小基因组（即<85％的野生型）的病毒粒子是相对二价阳离子，具有热稳定性。相反，含有接近或超过野生型大小（即＞ 95％的野生型）的重组基因组的病毒体依赖于二价阳离子的热稳定性。基因组序列似乎也是较大rAAV载体热稳定性的一个因素。这些观察具有实际和理论意义。在rAAV纯化和储存过程中使用的所有缓冲溶液中均应包含二价阳离子，并避免不必要的热暴露。这些数据还表明，不应假定特定病毒的不同重组体具有相同的热稳定性。

BACKGROUND & AIMS: :A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.

背景与目标： ：无复制能力的腺病毒（Ad）载体引起了人们对基因治疗和免疫治疗的兴趣。使用Ad载体的主要限制是先天免疫应答，它会引起炎症性细胞因子的产生和组织损伤。然而，先天免疫应答的确切机制尚待阐明。在这里，我们显示血清5型人类Ad载体根据细胞类型通过髓系分化因子88（MyD88）/ Toll样受体（TLR）-9依赖性和/或非依赖性方式引发先天性免疫反应。用Ad载体刺激后，与野生型DC相比，MyD88或TLR9缺陷型树突状细胞（DC）中白介素（IL）-6和IL-12的产量显着降低。此外，在缺乏MyD88或TLR9的粒细胞巨噬细胞集落刺激因子（GM-CSF）-DC中，成熟标记蛋白（例如CD40，CD80，CD86和MHC II类）的表面表达与野生型DC。另一方面，用Ad载体感染后，缺乏MyD88或TLR9的腹膜巨噬细胞产生与野生型巨噬细胞相同水平的IL-6。我们在DC和巨噬细胞之间未发现与先天免疫有关的分子（例如MyD88，TLR3，TLR7和TLR9）的mRNA表达水平有任何差异。向缺乏MyD88或TLR9的小鼠中静脉注射表达荧光素酶的Ad载体可导致与野生型小鼠几乎可比的IL-6和IL-12产生水平以及荧光素酶表达。这些结果表明，Ad载体可以通过MyD88 / TLR9依赖性和非依赖性机制激活先天免疫。

BACKGROUND & AIMS: :Both AIDS and cancer are linked to immune dysfunctions of the body which are characterized by the persistence of disease-afflicted cells. To effect a cure with novel gene therapy approaches, these diseased cells must be eliminated either directly or indirectly using cytotoxic or suicide genes, or via activation of specific immune functional cells. Retroviral vectors are useful tools for long-term genome modification owing to their ability to integrate into host chromosomes. However, most oncoretroviruses, including murine leukemia virus (MLV), require cell division to facilitate nuclear entry; this has restricted the application of murine oncoretroviral vectors to cell targets that are actively dividing. Accordingly, gene transfer into hematopoietic stem cells (HSCs) and terminally differentiated cells such as muscles, neurons and dendritic cells (DCs) has been limited with the conventional oncoretroviral vectors. The lentiviral family of retroviruses, including human immunodeficiency virus type 1 (HIV-1), has been developed into useful gene transfer tools. Lentiviral vectors carry several nuclear entry viral proteins, and therefore can target slowly-dividing and non-dividing cells. To activate immune response against cancer or HIV infection, long-term marking of the target cells is not necessary. However, to establish intracellular defense to prevent HIV infection, prolonged genetic modification of target cells such as HSCs will be required. Due to the poor transduction efficiency and the problem of transgene silencing over time with oncoretroviral vectors, most gene therapy studies for AIDS and cancer using oncoretroviral vectors remain proof-of-concept studies. Here we will discuss recent developments in the use of retroviral vectors, including HIV-1-derived lentiviral vectors, for the treatment of AIDS and cancer, and their future therapeutic potential.

背景与目标： 艾滋病和癌症都与人体免疫功能低下有关，其特征是疾病细胞持续存在。为了用新颖的基因疗法来治愈，必须使用细胞毒性或自杀基因或通过激活特异性免疫功能细胞直接或间接消除这些患病细胞。逆转录病毒载体由于其整合入宿主染色体的能力而成为长期基因组修饰的有用工具。然而，包括鼠白血病病毒（MLV）在内的大多数癌变病毒都需要细胞分裂以促进核进入。这限制了鼠类核仁病毒载体在积极分裂的细胞靶标中的应用。因此，基因转移到造血干细胞（HSC）和终末分化的细胞如肌肉，神经元和树突细胞（DC）中已经受到常规的癌基因病毒载体的限制。逆转录病毒的慢病毒家族，包括1型人类免疫缺陷病毒（HIV-1），已被开发成有用的基因转移工具。慢病毒载体携带几种核进入病毒蛋白，因此可以靶向缓慢分裂和非分裂的细胞。要激活针对癌症或HIV感染的免疫反应，不需要对靶细胞进行长期标记。然而，为了建立防止HIV感染的细胞内防御，将需要对靶细胞如HSC进行长时间的基因修饰。由于不良反应的转导效率以及使用核转录病毒载体随时间推移转基因沉默的问题，大多数使用核转录病毒载体进行的艾滋病和癌症基因治疗研究仍是概念验证研究。在这里，我们将讨论使用逆转录病毒载体（包括HIV-1衍生的慢病毒载体）治疗AIDS和癌症的最新进展及其未来的治疗潜力。

BACKGROUND & AIMS: :Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-gamma-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.

背景与目标： 杆状病毒载体能够转导多种哺乳动物细胞类型并表达在异源启动子控制下的转基因。在这项研究中，我们评估了杆状病毒载体在疟疾疫苗接种中的潜力。为了诱导有效的CD4（）和CD8（）T细胞应答，我们产生了一系列载体，这些载体在病毒体被膜中显示恶性疟原虫环子孢子（CS）蛋白和/或在哺乳动物细胞转导时允许CS表达。我们发现杆状病毒载体可以转导专业的抗原呈递细胞并触发其成熟，这是有效抗原呈递的前提。在向小鼠肌肉内注射后，既展示又表达CS的载体比CS诱导的抗CS抗体效价更高（免疫球蛋白（IgG）1和IgG2a类型），并且产生的CS特异性干扰素-γT细胞频率更高。仅显示或仅表示CS的向量。杆状病毒CS展示/表达载体在使用来自天真的供体的人外周血单核细胞体外诱导CS特异性CD4（）和CD8（）T细胞应答方面也表现优异。这加上在人类中不存在针对杆状病毒的预先存在的免疫力，在哺乳动物细胞中不存在病毒基因表达以及杆状病毒毒粒的相对较低的免疫原性，使得这些载体成为用于疫苗接种的有前途的工具。此外，以低成本在无血清培养基中大量生产的能力为该载体系统增加了进一步的优势。

BACKGROUND & AIMS: :An imposing obstacle to gene therapy is the inability to transduce all of the necessary cells in a target organ. This certainly applies to gene transfer to the brain, especially when one considers the challenges involved in scaling up transduction from animal models to use in the clinic. Non-neurotropic viral gene transfer vectors (e.g., adenovirus, adeno-associated virus, and lentivirus) do not spread very far in the nervous system, and consequently these vectors transduce brain regions mostly near the injection site in adult animals. This indicates that numerous, well-spaced injections would be required to achieve widespread transduction in a large brain with these vectors. In contrast, herpes simplex virus type 1 (HSV-1) is a promising vector for widespread gene transfer to the brain owing to the innate ability of the virus to spread through the nervous system and form latent infections in neurons that last for the lifetime of the infected individual. In this review, we summarize the published literature of the transduction patterns produced by attenuated HSV-1 vectors in small animals as a function of the injection site, and discuss the implications of the distribution for widespread gene transfer to the large animal brain.

背景与目标： ：基因治疗的一个潜在障碍是无法在靶器官中转导所有必需的细胞。这当然适用于基因向大脑的转移，尤其是当考虑到将动物模型的转导规模扩大到临床使用时所面临的挑战时。非神经营养性病毒基因转移载体（例如，腺病毒，腺伴随病毒和慢病毒）在神经系统中不会传播很远，因此，这些载体主要在成年动物的注射部位附近转导脑区域。这表明，使用这些载体在大脑中实现广泛的转导将需要大量间隔良好的注射。相比之下，单纯疱疹病毒1型（HSV-1）是一种有前途的载体，可以广泛地将基因转移到大脑，原因是该病毒具有先天的能力，可以在神经系统中传播并在神经元中形成潜伏的感染，这种感染可以持续一生。受感染的个人。在这篇综述中，我们总结了已减毒的HSV-1载体在小动物中产生的转导模式作为注射部位的函数的已发表文献，并讨论了该分布对于将基因广泛转移到大动物脑中的意义。