Clinical advances of siRNA therapeutics

Small interfering RNA (siRNA) enables efficient target gene silencing by employing a RNA interference (RNAi) mechanism, which can compromise gene expression and regulate gene activity by cleaving mRNA or repressing its translation. Twenty years after the discovery of RNAi in 1998, ONPATTRO? (patisiran) (Alnylam Pharmaceuticals, Inc.), a lipid formulated siRNA modality, was approved for the first time by United States Food and Drug Administration and the European Commission in 2018. With this milestone achievement, siRNA therapeutics will soar in the coming years. Here, we review the discovery and the mechanisms of RNAi, briefly describe the delivery technologies of siRNA, and summarize recent clinical advances of siRNA therapeutics.     

How to make siRNA lipoplexes efficient? Add a DNA cargo

Background

We recently reported an efficient formulation of siRNA targeting TNF-α, that was able to restore immunological balance in a mouse arthritis model following intravenous injection.

Method

Since this efficient formulation included the pre association of siRNA with a DNA cargo, we decided to extensively characterise siRNA lipoplexes with or without DNA cargo, in order to better understand the DNA cargo enhancing effect.

Results

We showed that addition of DNA cargo to siRNA lipoplexes led to specific gene extinction in vitro, using reduced siRNA concentration. This procedure is also applicable to other lipid vectors, like Lipofectamine or DMRIE-C. No structural modification could be observed in siRNA lipoplexes upon addition of DNA cargo using dynamic light scattering or transmission electronic microscopy. Nevertheless, we observed some slight differences, in the amount of lipid required to obtain neutrality of the complex and in stability of the complex towards incubation with heparan sulfate.

Conclusions

These results suggest that the addition of DNA cargo to siRNA complexes is an easy procedure that leads to more efficient complexes to transfer siRNA at low concentration and in the presence of serum.     

Transfection of siRNA into <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (Rotifera)

Lake Taihu is one of the most contaminated lakes in China. Surface sediment data show that the northern area of the Lake has the worst heavy metals pollution, and high heavy metal concentrations were attributed to discharge of untreated and partially treated industrial waste water from cities to the north of the lake. To study geochemical features and pollution history of heavy metals, total content and chemical fractionations of Cu, Fe, Mn, Ni, Pb, and Zn were analyzed for core sediments from western Lake Taihu using the speciation extraction procedure, proposed by the Commission of the European Communities Bureau of Reference (BCR), together with grain size and organic carbon measurements. Results show that sediments are composed of organic-poor clayey-fine silts for Cores MS and DLS, and have similar geochemical features shown by heavy metals. Cu, Fe, Ni, and Zn mainly are associated with the residue fraction, Mn is concentrated in the exchangeable-carbonate and residue fractions, and Pb is concentrated in the Fe–Mn oxide fraction and organic-sulfide fraction. The fractions of Ni, Pb, and Zn bound to Fe–Mn oxide show significant correlations with Mn from the Fe–Mn oxide fraction, and the organic-sulfide fractions of Cu, Mn, Ni, Pb, and Zn are correlated with TOC. The increase of Cu, Mn, Ni, Pb and Zn content and percentage of extractable fractions in the upper layers of the sediments are correlated with anthropogenic input of heavy metals due to rapid industrial development. This coincides with rapid economic development in the Taihu basin since late 1970s. Heavy metals in the surface sediments have certain potential biological toxicity as shown by the higher SEM/AVS ratio.     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

Rigid nanoparticle-based delivery of anti-cancer siRNA: Challenges and opportunities

Gene therapy is a promising strategy to treat various genetic and acquired diseases. Small interfering RNA (siRNA) is a revolutionary tool for gene therapy and the analysis of gene function. However, the development of a safe, efficient, and targetable non-viral siRNA delivery system remains a major challenge in gene therapy. An ideal delivery system should be able to encapsulate and protect the siRNA cargo from serum proteins, exhibit target tissue and cell specificity, penetrate the cell membrane, and release its cargo in the desired intracellular compartment. Nanomedicine has the potential to deal with these challenges faced by siRNA delivery. The unique characteristics of rigid nanoparticles mostly inorganic nanoparticles and allotropes of carbon nanomaterials, including high surface area, facile surface modification, controllable size, and excellent magnetic/optical/electrical properties, make them promising candidates for targeted siRNA delivery. In this review, recent progresses on rigid nanoparticle-based siRNA delivery systems will be summarized.     

哺乳动物中RNA干扰沉默基因表达的研究进展

RNA干扰及相关基因沉默导致的代谢通路变化已经彻底改变了人们对基因调控的理解。基因沉默技术已经被用来作为一种研究工具来控制某些细胞基因的表达,特异的si RNA导入不同的细胞需要不同的转染载体才能达到最大的转染效率,并且不会产生较大的细胞毒性。近年来尤其是碳纳米管等新型纳米材料载体的应用拓展了人们对传统脂质体和病毒载体的认识。首先介绍RNA干扰技术及其发展历程,随后对利用几种不同的载体来设计和进行RNA干扰试验进行了比较,最后对最初的脂质体到生物病毒再到高分子纳米材料介导的RNA干扰在疾病的治疗和临床诊断进行了展望。     

用聚乙烯亚胺反向转染siRNA表达盒进行RNA干扰的研究

目的:建立适用于大规模RNA干扰(RNAi)筛选的siRNA生成与导入技术。方法:以绿色荧光蛋白(GFP)为模型,用PCR方法生成了包含U6启动子、互补的反义链和正义链以及终止序列的特异性siRNA表达盒(SEC),对聚乙烯亚胺(PEI)介导的转染SEC的方法及其效率进行研究。结果:PEI对细胞的毒性呈剂量依赖关系,当PEI与DNA的N/P=7.53,即PEI与DNA等量时,转染效率达到最高。PEI与脂质体LipofectAMINETM2000的转染效率无显著差异(P>0.05),对SEC的转染效率显著高于质粒载体(P<0.01)。反向转染的转染效率显著高于常规转染(P<0.01)。利用PEI将表达GFP特异性siRNA的SEC反向转染可稳定表达GFP的HeLa细胞株(HeLa-EGFP),荧光染色和Western印迹检测均表明可显著抑制GFP的表达。结论:PEI介导的SEC反向转染具有简便、快捷、经济的优点,可满足大规模RNAi筛选的需要。     

Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.     

基于核酸自组装纳米结构的siRNA药物递送研究进展

RNA干扰(RNA interference,RNAi)作为转录后调节机制,可靶向mRNA进行剪切降解从而发挥基因沉默效应.siRNA (small interference RNA)因其高效性和特异性而被广泛应用于药物研究中.目前,研究者们已开发了多种阳离子载体用于siRNA递送.但由于siRNA双链结构具有相对较强的刚性结构,且阴离子电荷密度较低,无法与阳离子载体形成稳定、致密的复合物,使得siRNA的应用仍面临诸多挑战,如细胞摄取率低、靶向特异性差、递送过程不稳定、潜在的细胞毒性以及易诱发免疫反应等.近年来,核酸自组装纳米结构由于其结构灵活且负电荷密度较高而受到广泛关注,有望实现siRNA药物的高效递送和基因沉默.本文综述了近年来基于核酸自组装纳米结构的siRNA递送的研究进展及其应用.     

The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region

The gene-silencing effect of short interfering RNA (siRNA) is known to vary strongly with the targeted position of the mRNA. A number of hypotheses have been suggested to explain this phenomenon. We would like to test if this positional effect is mainly due to the secondary structure of the mRNA at the target site. We proposed that this structural factor can be characterized by a single parameter called "the hydrogen bond (H-b) index," which represents the average number of hydrogen bonds formed between nucleotides in the target region and the rest of the mRNA. This index can be determined using a computational approach. We tested the correlation between the H-b index and the gene-silencing effects on three genes (Bcl-2, hTF, and cyclin B1) using a variety of siRNAs. We found that the gene-silencing effect is inversely dependent on the H-b index, indicating that the local mRNA structure at the targeted site is the main cause of the positional effect. Based on this finding, we suggest that the H-b index can be a useful guideline for future siRNA design.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

Synthesis and transfection activity of novel galactosylated polycationic lipid

In this study, we synthesized a new galactosylated cationic lipid and investigated its biological activity. The structure of lipid combines both spermine residue for DNA compaction and galactose moiety for the improvement of aggregation behavior of lipoplexes. Lipid was low toxic for different mammalian cells, and was able both to compact plasmid DNA and to mediate cellular accumulation of various nucleic acids (ODN, pDNA and siRNA) exhibiting biological activity (transgene expression, gene silencing).     

The potential therapeutic of siRNA eye drops in ocular diseases

In recent years, researchers have expressed an ongoing interest in developing RNA interference (RNAi) technology for therapeutic gene suppression in various diseases. Preclinical studies in animal models and cultured cell studies indicated that RNAi technology was an effective experimental tool against a variety of ocular diseases, and some small interference RNA (siRNA) drugs have been entered into clinical trials in Stage I and Stage II. However, in these studies siRNAs were delivered into ocular tissues via either systemic or subconjunctival/intravitreous injection, which is invasive and harmful if repeated. Based on this evidence, we hypothesize that topical application of siRNA eye drops may be a safe and effective therapeutic option in ocular surface diseases with temporary changes of gene expression. Furthermore, siRNA eye drops targeting different genes may simultaneously treat several ocular surface diseases.     

肠上皮细胞RNA干扰Hsp70基因转染条件的研究

旨在研究肠上皮细胞(IEC-6)RNA干扰效率的特染条件,并对4对Hsp70干抚序列进行筛选,为进一步以RNA干扰(RNAi)技术研究Hsp70对胃肠道的保护功能以及介导信号通路的机理打下基础.以24孔培养板培养IEC-6细胞,利用阳高子脂质体特染FAM-siRNA片段进入IEC-6细胞,采用荧光倒置显微镜和流式细胞仪检测FAM-siRNA和特染试剂Lipofectamine 2000不同剂量比例对特染效率的影响,并进一步采用RT-PCR和Western blotting方法对Hsp70干扰序列进行筛选.试验结果表明,采用脂质体转染法可获得较高的转染效率,其中以80 nmol/L FAM-siRNA+1μL Lipofectamine 2000组的转染效率最高(85.77％).针对4个不同靶位点,进行Hsp70基因干涉,结果表明,4个组中Hsp70 mRNA的表达显著下降,其中oligo 4组的基因表达极显著降低.Western blotting的结果表明,oligo 2、oligo3和oligo4组中的Hap70蛋白表达极显著降低.最终确定80 nmol/L FAM-siRNA+1μL Lipofectamine 2000为最佳转染条件,以oligo 4(Hap70的基因位点为3672 -3692)为最佳RNA干扰靶基因.     

siRNA, miRNA and HIV: promises and challenges

Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing.The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNAtargeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNAspecified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells‘ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).     

哺乳动物细胞小干扰RNA库的建立和应用

双链小干扰RNA（siRNA）在多种类型细胞中介导特异性的基因沉默,这一现象的发现为深入研究单个基因的功能提供了重要的方法学基础,从而得到了广泛的应用.最近的文献报道了全基因组siRNA库的建立,为高通量基因功能分析和研究提供了新的方法,成为新的研究热点.小干扰RNA库可以用来筛选和研究介导细胞复杂表型和生物学过程的关键基因,通过建立一系列具有目的表型的细胞系,有可能对特定细胞信号调节通路进行更为全面的解析.本文综述了目前在siRNA建库方法方面的进展,并探讨了建立小干扰RNA库中的关键问题.     

Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1

Plant viruses encode RNA silencing suppressors (VSRs) to counteract the antiviral RNA silencing response. Based on in-vitro studies, several VSRs were proposed to suppress silencing through direct binding of short-interfering RNAs (siRNAs). Because their expression also frequently hinders endogenous miRNA-mediated regulation and stabilizes labile miRNA* strands, VSRs have been assumed to prevent both siRNA and miRNA loading into their common effector protein, AGO1, through sequestration of small RNA (sRNA) duplexes in vivo. These assumptions, however, have not been formally tested experimentally. Here, we present a systematic in planta analysis comparing the effects of four distinct VSRs in Arabidopsis. While all of the VSRs tested compromised loading of siRNAs into AGO1, only P19 was found to concurrently prevent miRNA loading, consistent with a VSR strategy primarily based on sRNA sequestration. By contrast, we provide multiple lines of evidence that the action of the other VSRs tested is unlikely to entail siRNA sequestration, indicating that in-vitro binding assays and in-vivo miRNA* stabilization are not reliable indicator of VSR action. The contrasted effects of VSRs on siRNA versus miRNA loading into AGO1 also imply the existence of two distinct pools of cellular AGO1 that are specifically loaded by each class of sRNAs. These findings have important implications for our current understanding of RNA silencing and of its suppression in plants.