TNF-α Gene Silencing Using Polymerized siRNA/Thiolated Glycol Chitosan Nanoparticles for Rheumatoid Arthritis
So Jin LeeAeju LeeSeung Rim HwangJong Sung ParkJiyeon JangMyung Sook HuhDong‐Gyu JoSoo-Young YoonYoungro ByunSun Hwa KimIck Chan KwonInchan YounKwangmeyung Kim
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Abstract:
Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.Keywords:
Proinflammatory cytokine
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.
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Although the use of small interfering RNA (siRNA) is a promising technique for gene regulation, spatiotemporal control of the effects of siRNA must be achieved if the technique is to be safe and practical. Here, a method for spatiotemporal regulation of genes with nanoparticles containing siRNA is reported. The siRNA is encapsulated in photodegradable nanoparticles that are internalized to SKOV3-Luc cells, where the siRNA is released from the nanoparticles by UV irradiation for 30 s. The encapsulated siRNA only shows no gene-silencing effects, but release of the siRNA upon UV radiation leads to sequence-specific silencing of the luciferase gene in the cells. These results indicate that photodegradable siRNA-containing nanoparticles can be useful for time- and space-dependent regulation of gene expression in cells.
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Chemically synthesized small interfering RNAs (siRNAs) have been widely used to identify gene function and hold great potential in providing a new class of therapeutics. Chemical modifications are desired for therapeutic applications to improve siRNA efficacy. Appropriately protected ribonucleoside-3′-yl S-[β-(benzoylmercapto)ethyl]pyrrolidino-thiophosphoramidite monomers were prepared for the synthesis of siRNA containing phosphorodithioate (PS2) substitutions in which the two non-bridging oxygen atoms are replaced by sulfur atoms. A series of siRNAs containing PS2 substitutions have been strategically designed, synthesized, and evaluated for their gene silencing activities. These PS2-siRNA duplexes exhibit an A-form helical structure similar to unmodified siRNA. The effect of PS2 substitutions on gene silencing activity is position-dependent, with certain PS2-siRNAs showing activity significantly higher than that of unmodified siRNA. The relative gene silencing activities of siRNAs containing either PS2 or phosphoromonothioate (PS) linkages at identical positions are variable and depend on the sites of modification. 5′-Phosphorylation of PS2-siRNAs has little or no effect on gene silencing activity. Incorporation of PS2 substitutions into siRNA duplexes increases their serum stability. These results offer preliminary evidence of the potential value of PS2-modified siRNAs.
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Small interfering RNA (siRNA) and short hairpin RNA (shRNA) targeting different regions of transforming growth factor beta1 (TGF-beta1) mRNA were designed and the silencing effect was determined after transfection into immortalized rat liver stellate cells (HSC-T6). There was not only significant decrease in TGF-beta1, tissue inhibitor of metalloproteinase 1 (TIMP-1), alpha-smooth muscle actin (alpha-SMA) and type I collagen after transfection with TGF-beta1 siRNAs, but also synergism in gene silencing when siRNAs targeting two different start sites were used as a pool for transfection. The two siRNA sequences which efficiently inhibited TGF-beta1 gene expression were converted to shRNAs via cloning into the pSilencer1.0. There was significant decrease in TGF-beta1 and TIMP-1 when HSC-T6 cells were transfected with pshRNA targeting the same regions of TGF-beta1 mRNA as siRNAs. Furthermore, TGF-beta1 gene silencing in HSC-T6 cells significantly decreased the levels of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). In conclusion, both siRNA and shRNA showed sequence-specific and dose dependent TGF-beta1 gene silencing and have the potential to treat liver fibrosis.
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Although small interfering RNA (siRNA) can silence the expression of disease-related genes, delivery of these highly charged molecules is challenging. Delivery approaches for siRNAs are actively being pursued, and improved strategies are required for nontoxic and efficient delivery for gene knockdown. Low density lipoprotein (LDL) is a natural and endogenous nanoparticle that has a rich history as a delivery vehicle. Here, we examine purified LDL nanoparticles as carriers for siRNAs. When siRNA was covalently conjugated to cholesterol, over 25 chol-siRNA could be incorporated onto each LDL without changing nanoparticle morphology. The resulting LDL-chol-siRNA nanoparticles were selectively taken up into cells via LDL receptor mediated endocytosis, resulting in enhanced gene silencing compared to free chol-siRNA (38% gene knock down versus 0% knock down at 100 nM). However, silencing efficiency was limited by the receptor-mediated entrapment of the LDL-chol-siRNA nanoparticles in endolysosomes. Photochemical internalization demonstrated that endolysosome disruption strategies significantly enhance LDL-mediated gene silencing (78% at 100 nM).
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In RNA interference (RNAi), double-stranded short interfering RNA (ds-siRNA) inhibits expression from complementary mRNAs. Recently, it was demonstrated that short, single-stranded antisense RNA (ss-siRNA) can also induce RNAi. While ss-siRNA may offer several advantages in both clinical and research applications, its overall poor activity compared with ds-siRNA has prevented its widespread use. In contrast to the poor gene silencing activity of native ss-siRNA, we found that the silencing activity of boranophosphate-modified ss-siRNA is comparable with that of unmodified ds-siRNA. Boranophosphate ss-siRNA has excellent maximum silencing activity and is highly effective at low concentrations. The silencing activity of boranophosphate ss-siRNA is also durable, with significant silencing up to 1 week after transfection. Thus, we have demonstrated that boranophosphate-modified ss-siRNA can silence gene expression as well as native ds-siRNA, suggesting that boranophosphate-modified ss-siRNAs should be investigated as a potential new class of therapeutic agents.
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Simultaneous suppression of multiple oncogenes is an attractive strategy to treat cancers. Herein we present a series of long double-stranded multiplex small interfering RNAs (multi-siRNAs) that is suitable for dual genes silencing through a sequence-specific RNA interference process without inducing significant immune responses. A gap feature structurally designed in either of the nucleotide strands of the multi-siRNAs was proved to be essential toward silencing target genes and avoiding immune responses. Furthermore, the silencing effect of multi-siRNAs against SURVIVIN and BCL-2 genes was shown to be effective and resulted in up-regulation of caspase-3 related apoptosis and, in turn, inhibition of bladder cancer cell proliferation. Our observation suggested that the rationally designed multi-siRNAs would have great potential for therapeutic siRNA design.
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ABSTRACT Human gremlin-1 is a physiologically versatile signaling molecule that has been associated with several human diseases including cancer. The ability of gremlin-1 to induce fibrosis in organs and transduce angiogenesis makes it a target for cancer therapy. RNAi-based therapy has proven to be very efficient and specific in tumor growth inhibition. The efficacy and specificity of siRNA-mediated gene silencing depends on the designing approaches. Here, empirical guidelines for siRNA design and comprehensive target site availability analysis were used to select effective siRNA from a plethora of potential candidates designed using several computation algorithms. Then, the selected siRNA candidates were subjected to stringent similarity searches in order to obtain siRNA candidates with reduced off-target effects (high specificity). The best candidates were compared to experimentally successful gremlin-1 siRNAs in order to predict the silencing potency of the selected siRNAs. siRNA-6 (sense strand: 5’-CCAAGAAAUUCACUACCAU-3’), siRNA-7 (sense strand: 5’-CCAUGAUGGUCACACUCAA-3’) and siRNA-47 (sense strand: 5’-GGCCCAGCACAAUGACUCA-3’) were predicted to be highly effective siRNA candidates for gremlin-1 silencing. These siRNAs can be considered for RNAi-based therapy because off-target effects are predicted to be minimal.
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我们的以前的学习表明了引起 amyotrophic 的变异的基因的调停 siRNA 的、等位基因特定的 silencing。为了改进 siRNA ，为 RNA 干扰的更好治疗学的使用设计，我们系统地在不对称的 siRNA.Methods 的设计测试了基础配对的失配策略:指向人的 Cu 的自然地对称的 siRNA Zn 超级氧化物歧化酶 G85R 变异的等位基因被从在的位置 1～4 把 1 或 2 失配放在 siRNA 的结束修改每次。目标偏爱和修改 siRNA 的 silencing 功效用修改双酶 system.Results 被测量: 单个基础配对的失配的修正成功地完成了原来被赞成到变异的等位基因的反感觉到被赞成到基因的感觉海滨的那的 siRNA 的变换。比作错配单人赛的 siRNA，那些与在一结束的双失配表明了增加的不对称现象，和 thus，基因 silencing 的提高的特性和功效。另外，有在两结束的双失配的 siRNA 留在 symmetry.Conclusion :我们的结果建议由介绍失配进它的结构把对称的 siRNA 变换成不对称的有效性，并且到在生产源于 siRNA 的混乱的选择基因 silencing 的错配单人赛的 siRNA 的 双mismatched siRNA 的优势对称。双失配策略是单个失配的方法的改进并且能在为引起由的疾病的处理的有效 siRNAs 的设计有用主导， gain-of-function 基因变化，例如 ALS。
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