Multimerized siRNA cross-linked by gold nanoparticles

In this study, siRNAs terminated with thiol groups were multimerized and cross-linked using ～5 nm gold nanoparticles (AuNPs) via Au-S chemisorption that can be intracellularly reduced. AuNPs immobilized with single-stranded antisense siRNA were assembled with those with single-stranded sense siRNA via complementary hybridization or assembled with those with single-stranded dimeric sense siRNA. The multimerized siRNA cross-linked by AuNPs showed increased charge density and enhanced enzymatic stability, and exhibited good complexation behaviors with a polycationic carrier, linear polyethylenimine (L-PEI). The resultant multi-siRNA/AuNPs/L-PEI polyelectrolyte complexes exhibited far greater gene silencing efficiencies of green fluorescent protein (GFP) and vascular endothelial growth factor (VEGF) compared to naked siRNA complexes. They could also be visualized by micro-CT imaging. The results suggest that AuNP-mediated multimerization of siRNAs could be a rational approach to achieve both gene silencing and imaging at a target tissue simultaneously.     

Multifunctional siRNA delivery system: Polyelectrolyte complex micelles of six‐arm PEG conjugate of siRNA and cell penetrating peptide with crosslinked fusogenic peptide

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six‐arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG‐siRNA). The 6PEG‐siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG‐siRNA‐Hph1). The 6PEG‐siRNA‐Hph1 conjugate was electrostatically complexed with cationic self‐crosslinked fusogenic KALA peptide (cl‐KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG‐siRNA‐Hph1/cl‐KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA‐MB‐435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Enhanced gene silencing by the application of multiple specific small interfering RNAs

Small interfering RNA duplexes (siRNA) induce gene silencing in various eukaryotic cells, although usually in an incomplete manner. Using chemically synthesized siRNAs targeting the HIV-1 co-receptor CXCR4 or the apoptosis-inducing Fas-ligand (FasL), co-transfection of cells with two or more siRNA duplexes targeting different sites on the same mRNA resulted in an enhanced gene silencing compared with each single siRNA. This was shown in the down-regulation of protein and mRNA expression, and functionally in the inhibition of CXCR4-mediated HIV infection and of FasL-mediated cell apoptosis. Transfection efficiency determined for the FasL-specific siRNAs was dose-dependent and varied among the siRNAs tested, but was not the main reason for the enhanced gene silencing.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Intracellular small interfering RNA delivery using genetically engineered double‐stranded RNA binding protein domain

Background

A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double‐stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.

Methods

A double‐stranded RNA binding domain (dsRBD) of human double‐stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines.

Results

The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down‐regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation.

Conclusions

The results suggest that dsRBD‐based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

用绿色荧光蛋白（GFP）作为报告分子筛选有效的siRNA

 建立一种利用绿色荧光蛋白（GFP）作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子（MIF）基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.     

A novel siRNA validation system for functional screening and identification of effective RNAi probes in mammalian cells

Small interfering RNAs (siRNAs) have become the most powerful and widely used gene silencing reagents for reverse functional genomics and molecular therapeutics. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily dependent on the effectiveness and specificity of the RNAi targeting sequence. However, only a limited number of siRNAs is capable of inducing highly effective and sequence-specific gene silencing by RNA interference (RNAi) mechanism. In addition, the efficacy of siRNA-induced gene silencing can only be experimentally measured based on inhibition of the target gene expression. Therefore, it is important to establish a fully robust and comparative validating system for determining the efficacy of designed siRNAs. In this study, we have developed a reliable and quantitative reporter-based siRNA validation system that consists of a short synthetic DNA fragment containing an RNAi targeting sequence of interest and two expression vectors for targeting reporter and triggering siRNA expression. The efficacy of the siRNAs is measured by their abilities to inhibit expression of the targeting reporter gene with easily quantified readouts including enhanced green fluorescence protein (EGFP) and firefly luciferase. Using fully analyzed siRNAs against human hepatitis B virus (HBV) surface antigen (HBsAg) and tumor suppressor protein p53, we have demonstrated that this system could effectively and faithfully report the efficacy of the corresponding siRNAs. In addition, we have further applied this system for screening and identification of the highly effective siRNAs that could specifically inhibit expression of mouse matrix metalloproteinase-7 (MMP-7), Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and human serine/threonine kinase AKT1. Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals.     

Specific gene silencing using small interfering RNAs in fish embryos

Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. Here we demonstrate a siRNA-mediated gene silencing technique in rainbow trout embryos. We found that siRNAs effectively suppressed the transient expression of episomally located foreign GFP genes at an early developmental stage and inhibited the expression of GFP genes in stable transgenic trout embryos. Similar gene silencing was observed with an siRNA against the endogenous tyrosinase A gene. siRNAs interfered with the expression of maternally inherited mRNA. siRNAs did not affect non-relevant gene expression and siRNAs with a 4 base mismatch did not affect target gene expression. siRNA gene silencing is therefore highly sequence-specific. Our findings are the first evidence that siRNA-mediated gene silencing is effective in fish. This technique could be a powerful tool for studying gene function during embryonic development in aquacultural fish species, zebrafish, and medaka.     

Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

RNAi in primary human chondrocytes: Efficiencies, kinetics, and non-specific effects of siRNA-mediated gene suppression

RNAi-mediated gene silencing is a recent, powerful tool to investigate gene function. Controlling for experimental factors such as transfection efficiencies, siRNA concentration, gene suppression levels, gene suppression kinetics, or non-specific effects is key to robust results. In this methods paper, we compare the efficiencies of different transfection reagents in primary human chondrocytes (PHCs). We investigated TAK1 gene suppression efficiencies and kinetics on the mRNA and protein level depending on the siRNA concentration used. Furthermore, we evaluated PKR, IL-6, and TNF-alpha induction, as well as IkappaB degradation and NFkappaB activation as control parameters of non-specific siRNA effects. PKR and IL-6 proved to be appropriate markers of cellular inflammatory responses resulting from siRNA transfection. In addition, we compared different siRNAs (silencing, non-silencing, classic 21-mer, and 25-mer stealth siRNA) with respect to their capacity to induce cellular inflammatory responses. We found the occurrence of cellular responses in PHCs to be a function of the specific siRNA sequence in use. Hence, it is essential to analyze and to compare gene silencing siRNAs and control siRNAs with respect to their off-target effects prior to any functional gene validation.     

Consumption of hydrogen-rich water alleviates renal injury in spontaneous hypertensive rats

The aim of this study was to investigate the inhibitory effect of a siRNA cocktail targeting Vascular endothelial growth factor (VEGF) and Human epidermal growth factor receptor 2 (HER2) on cell proliferation, induced apoptosis and the expression of VEGF and HER2 in human gastric carcinoma cell. The silencing rate of pre-designed siRNAs that targeted VEGF and HER2 was detected by Real-time Quantitative PCR (RT-QPCR) analysis. Furthermore, the best silencing siRNA that targeted VEGF and HER2 was prepared as a cocktail to co-knockdown VEGF and HER2 expression at both mRNA and protein levels which were detected by RT-QPCR and Western blot analysis. Cell proliferation inhibition rates were determined by CCK8 assay. The effect of siRNA cocktail on cell apoptosis was determined by flow cytometry. The migration inhibition of siRNA cocktail was analyzed by wound-healing assay. The ability of VEGF to induce endothelial cells to proliferate was examined in HUVECs by the method of tube formation assay. The pre-designed siRNAs could inhibit VEGF and HER2 mRNA level. siRNA cocktail, and co-downregulation of VEGF and HER2 result in significant inhibition of gastric cancer growth and migration in vitro. The inhibition of VEGF and HER2 expressions can induce apoptosis of SGC-7901 cells.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.