siRNA体内递送研究进展

RNA干扰(RNA interference,RNAi),是一种在动植物中存在的通过双链RNA诱导同源特异性序列转录后基因沉默的过程。虽然小干扰RNA (siRNA) 较单链反义寡核苷酸显示出更好的稳定性与基因沉默效果,但是作为新型的基因治疗药物,靶向递送siRNA是药物进入临床应用最主要的环节,siRNA体内有效作用发挥的关键在于它在体内能否高效递送至靶细胞并与靶基因结合。目前研究主要集中在siRNA的修饰方式与递送载体研究,以提高其体内的稳定性与靶向性。文中主要综述了siRNA的体内靶向递送障碍以及近几年siRNA非病毒递送载体脂质体、阳离子多聚物、纳米粒、胶束等方面的研究进展。     

适配子介导siRNA靶向递送系统的研究进展

RNA干扰技术(RNAi)作为一种新型的基因治疗技术已应用到许多疾病的体外研究中,但靶向递送技术仍是目前制约小干扰RNA(siRNA)药物成功进入临床的关键技术问题。寡核苷酸适配子具有高亲和性、高靶向性,成为递送siRNA的重要手段。本文综述了siRNA体内递送的主要障碍,以及近年来适配子介导的siRNA靶向递送系统的最新研究进展。     

siRNA非病毒递送载体的研究现状

RNA干扰(RNA interference,RNAi)是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mRNA产生特异性降解,进而引起不同水平的基因沉默。RNAi已经用于肿瘤、病毒感染、乙型肝炎等多种疾病的治疗。小干扰RNA(siRNA)是RNAi的效应分子,可在体内诱导RNAi效应。但是裸siRNA在体内容易被核酶(RNase)降解,且半衰期短,转染效率低。因此,siRNA需要借助递送载体进入细胞发挥治疗作用。病毒载体在基因治疗中有潜在的免疫原性、致突变等副作用。所以,非病毒载体成为当前的研究热点。本文对siRNA非病毒递送载体的研究现状进行了综述。     

siRNA作为基因治疗药物的研究难题

RNA干扰（RNAi）现象的发现与研究，为基因治疗带来新的契机，虽然小干扰RNA（siRNA）较单链反义寡核苷酸显示了更好的稳定性与基因沉默效果，但却同样面临基因治疗药物存在的共同问题，如体内的靶向性与有效性、完善的定量分析方法等等。因此，siRNA作为治疗药物还有诸多困难需要克服。目前的研究主要集中在：修饰方式与递送系统研究，以提高siRNA体内的稳定性与靶向性；siRNA定量分析方法学研究，以考察其体内药动学行为并进一步阐明其体内作用机制。     

siRNA新型非病毒载体递送策略新进展

小干扰RNA(siRNA)是一个靶向治疗和精确医学的代表性治疗工具，可通过序列特异性的RNA干扰(RNAi)沉默任何疾病相关基因的表达。然而，它的治疗前景历来受到体内半衰期短、递送困难和安全问题的限制。非病毒载体介导的药物递送已经成为克服这些局限性的一个成功策略，可实现siRNA在体内的有效递送，高效沉默靶基因。目前，已有多种药物处于临床试验中，4种基于siRNA的新型疗法已获得美国FDA的批准，标志着靶向疗法新时代的开始。该文概述了近年来基于siRNA的非病毒载体递送策略的新进展及其应用，并展望了siRNA药物研究的未来发展趋势。     

siRNA药物研究进展

小干扰RNA(small interfering RNA,siRNA)药物作为小核酸药物研发的热点,凭借基因沉默效率高、不良反应可控、合成方便等优点,得到了广泛应用.siRNA裸序列不稳定,在体内递送困难,不易到达靶点发挥作用,成为早期siRNA药物研发的阻力.近年来,siRNA的稳定性修饰以及高效递送系统的开发,大大...     

siRNA抗HBV感染领域中的化学修饰和体内传递研究进展

世界人口中约有3.5亿感染乙肝病毒,乙肝病毒感染是急慢性肝炎的主要原因,其与肝纤维化和肝细胞癌的发生密切相关。目前的治疗药物不良反应多或易产生耐药性,因此迫切需要找到新的治疗手段。本文依据近5年来国内外文献进行分析、归纳、总结,综述了治疗HBV感染的小干扰RNA（small RNA interference,siRNA）药物在化学修饰和体内传递方面新进展。RNAi途径可实现病毒基因转录后沉默,特异性和有效性地抑制HBV基因的表达和复制,是治疗HBV感染的一种新颖而有效的途径。而小干扰RNA（siRNA）的化学修饰和体内传递研究,可提高其在体内的稳定性和靶向性。     

小干扰RNA的非病毒载体:从实验室走向临床

小干扰RNA（siRNA）作为RNA干扰技术的重要效应分子,具有治疗多种疾病的巨大潜力。目前已有3款siRNA药物获得美国食品药品监督管理局批准上市,数十款siRNA药物处于临床试验阶段。脂质纳米颗粒、N-乙酰半乳糖胺（GalNAc）-siRNA共轭物、高分子聚合物以及仿生载体等非病毒载体为解决siRNA体内易降解、易清除、免疫原性以及细胞膜渗透能力弱等问题提供了良好的解决策略。随着对疾病基因谱和非病毒载体的不断深入研究,越来越多的siRNA药物进入临床前和临床试验研究。文中对siRNA药物发展历程、体内应用过程中的生物学屏障、递送载体结构与功能进行系统梳理,以期对siRNA药物的未来发展提供借鉴。     

高分子聚合物用于siRNA递送系统的研究进展

小干扰RNA（siRNA）具有沉默互补的目标信使RNA（mRNA）从而抑制疾病相关基因表达的作用。因其高效性及特异性,siRNA具有作为基因药物的巨大潜力,但其在体内易受到血管屏障、内涵体及RNA酶等因素的影响,从而难以发挥药效。因此,设计高效的能够负载siRNA的纳米载体以使其富集至靶标是目前siRNA药物研发的重要任务,高分子聚合物纳米载体是目前研究热点之一。本文就高分子聚合物纳米载体实现siRNA药物递送的研究进展进行综述。     

不对称RNA干扰研究进展

RNA干扰是生物医学研究,特别是基因功能研究中的重要工具.目前在哺乳细胞和临床研究中的RNA干扰技术主要由相同长度对称的双链小干扰RNA(siRNA)介导.正义链和反义链长度不一致的不对称双链小干扰RNA(aiRNA)能更有效地使相应的靶基因沉默,并且降低脱靶效应,提高体内外稳定性,成为疾病治疗的新希望.本文就aiRNA的研究进展作一综述.     

Nonviral vector-mediated RNA interference: Its gene silencing characteristics and important factors to achieve RNAi-based gene therapy

RNA interference (RNAi) is a potent and specific gene silencing event in which small interfering RNA (siRNA) degrades target mRNA. Therefore, RNAi is of potential use as a therapeutic approach for the treatment of a variety of diseases in which aberrant expression of mRNA causes a problem. RNAi can be achieved by delivering siRNA or vectors that transcribe siRNA or short-hairpin RNA (shRNA). The aim of this review is to examine the potential of nonviral vector-mediated RNAi technology in treating diseases. The characteristics of plasmid DNA expressing shRNA were compared with those of siRNA, focusing on the duration of gene silencing, delivery to target cells and target specificity. Recent progresses in prolonging the RNAi effect, improving the delivery to target cells and increasing the specificity of RNAi in vivo are also reviewed.     

Efficient in vitro siRNA delivery and intramuscular gene silencing using PEG-modified PAMAM dendrimers

Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the results from the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and GFP transgenic mice.     

Inhalable siRNA: potential as a therapeutic agent in the lungs

RNA interference (RNAi) is gaining increasing popularity both as a molecular biology tool and as a potential therapeutic agent. RNAi is a naturally occurring gene regulatory mechanism, which has a number of advantages over other gene/antisense therapies including specificity of inhibition, potency, the small size of the molecules and the diminished risk of toxic effects, e.g., immune responses. Targeted, local delivery of RNAi to the lungs via inhalation offers a unique opportunity to treat a range of previously untreatable or poorly controlled respiratory conditions. In this timely review we look at the potential applications of RNAi in the lungs for the treatment of a range of diseases including inflammatory and immune conditions, cystic fibrosis, infectious disease and cancer. In 2006 Alnylam initiated the first phase 1 clinical study of an inhaled siRNA for the treatment of respiratory syncytial virus. If its potential as a therapeutic is to be realized, then safe and efficient means of targeted delivery of small interfering RNA (siRNA) to the lungs must be developed. Therefore in this review we also present the latest developments in siRNA delivery to airway cells in vitro and the work to date on in vivo delivery of siRNA to the lungs for the treatment of a range of diseases.     

The cutting-edge technologies of siRNA delivery and their application in clinical trials

siRNA therapeutics allows precise regulation of disease specific gene expression to treat various diseases. Although gene silencing approaches using siRNA therapeutics shows some promising results in the treatment of gene-related diseases, the practical applications has been limited by problems such as inefficient in vivo delivery to target cells and nonspecific immune responses after systemic or local administration. To overcome these issues, various in vivo delivery platforms have been introduced. Here we provide an overview for three different platform technologies for the in vivo delivery of therapeutic siRNAs (siRNA–GalNAc conjugate, SAMiRNA technology, and LNP-based delivery method) and their applications in the treatment of various diseases. In addition, a brief introduction to some rare diseases and mechanisms of siRNA therapeutics-mediated treatment is described.     

Recent progress in copolymer-mediated siRNA delivery

RNAi-mediated gene silencing has great potential for treating various diseases, including cancer, by delivering a specific short interfering RNA (siRNA) to knock down pathogenic mRNAs and suppress protein translation. Although many researchers are dedicated to devising polymer-based vehicles for exogenous in vitro siRNA transfection, few synthetic vehicles are feasible in vivo. Recent studies have presented copolymer-based vectors that are minimally immunogenic and facilitate highly efficient internalizing of exogenous siRNA, compared with homopolymer-based vectors. Cationic segments, organelle-escape units, and degradable fragments are essential to a copolymer-based vehicle for siRNA delivery. The majority of these cationic segments are derived from polyamines, including polylysine, polyarginine, chitosan, polyethylenimines and polyamidoamine dendrimers. Not only do these cationic polyamines protect siRNA, they can also promote disruption of endosomal membranes. Degradable fragments of copolymers must be derived from various polyelectrolytes to release the siRNA once the complexes enter the cytoplasm. This review describes recent progress in copolymer-mediated siRNA delivery, including various building blocks for biocompatible copolymers for efficient in vitro siRNA delivery, and a useful basis for addressing the challenges of in vivo siRNA delivery.     

Pulmonary delivery of therapeutic siRNA

Small interfering RNA (siRNA) has a huge potential for the treatment or prevention of various lung diseases. Once the RNA molecules have successfully entered the target cells, they could inhibit the expression of specific gene sequence through RNA interference (RNAi) mechanism and generate therapeutic effects. The biggest obstacle to translating siRNA therapy from the laboratories into the clinics is delivery. An ideal delivery agent should protect the siRNA from enzymatic degradation, facilitate cellular uptake and promote endosomal escape inside the cells, with negligible toxicity. Lung targeting could be achieved by systemic delivery or pulmonary delivery. The latter route of administration could potentially enhance siRNA retention in the lungs and reduce systemic toxic effects. However the presence of mucus, the mucociliary clearance actions and the high degree branching of the airways present major barriers to targeted pulmonary delivery. The delivery systems need to be designed carefully in order to maximize the siRNA deposition to the diseased area of the airways. In most of the pulmonary siRNA therapy studies in vivo, siRNA was delivered either intratracheally or intranasally. Very limited work was done on the formulation of siRNA for inhalation which is believed to be the direction for future development. This review focuses on the latest development of pulmonary delivery of siRNA for the treatment of various lung diseases.     

Polyethylenimine as a promising vector for targeted siRNA delivery

Recent discovery of RNA interference (RNAi) technology for gene therapy has triggered explosive research efforts towards development of small interfering RNA (siRNA) as therapeutic modality for gene silencing. Owing to its large molecular weight (~13 kDa), polyanionic nature (~40 negative phosphate groups) and rapid enzymatic degradation, delivery of siRNA remains an unresolved issue. Hence, there arises a need of an appropriate delivery vector to overcome the intrinsic, poor intracellular uptake and limited in vitro and in vivo stability. Amongst the various non-viral delivery vectors, the application of polymeric vectors such as polyethylenimine (PEI) or its derivatives has attracted much attention due to its high transfection efficiency and ease of manipulation. PEI has been extensively investigated for DNA delivery, only recently this polymer has been employed for siRNA delivery. This review will focus on studies done on PEI to deliver siRNA, with emphasis on the targeted, self-assembled polymeric nanoparticles with promising potential to evolve as therapeutic tool in gene therapy.     

整合素配体RGD修饰的阳离子脂质体作为siRNA输送载体的体外研究

本研究构建了能够靶向肿瘤新生血管的RGD修饰阳离子脂质体（RGD-Lipo），作为靶向耐药相关基因MDR1的siRNA输送载体并评价其相关的药剂学性质。该脂质体与siRNA形成的复合物粒径控制在200nm以内，并且对其中所包载的siRNA具有一定的保护作用。体外实验结果表明，经RGD修饰的脂质体（RGD-Lipo）细胞粘附能力显著增强，并可增加细胞内siRNA的转染效果。与利用前插法进行靶向修饰相比，利用后插法进行RGD修饰可有效地改善siRNA的溶酶体释放效率。细胞毒试验结果表明，后插法制备的pRGD-Lipo-siRNA能够有效逆转人卵巢癌SKV03／A细胞的耐药性，并增加阿霉素药物在细胞内的蓄积。体外研究结果证实，使用pRGD-Lipo-siRNA有利于提高耐药细胞对化疗药阿霉素的敏感度，并将有可能应用于临床耐药肿瘤的治疗。