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PubMed2006

Development and validation of vectors containing multiple siRNA expression cassettes for maximizing the efficiency of gene silencing.

BACKGROUND: RNA interference (RNAi) was originally identified as a biological process in which short double-stranded RNA (dsRNA) suppress the expression of genes complimentary to the dsRNA. This cellular intrinsic gene silencing mechanism has subsequently been developed as a useful tool for studies of gene function. A major strategy for producing small interfering RNA (siRNA) in cultured cells involves the use of siRNA expression vectors in which a RNA polymerase III (Pol III) promoter and transcription stop signal are designed to constitute a functional siRNA expression cassette for production of siRNA. However, most of the available vectors contain only one siRNA expression cassette. RESULTS: In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed vectors containing multiple (up to six) tandem siRNA expression cassettes. Moreover, we demonstrated that these vectors can be used not only to produce different siRNA to simultaneously suppress the expression of multiple genes but also to maximize the silencing of a single gene. CONCLUSION: The vectors containing multiple siRNA expression cassettes can serve as useful tools for maximizing the efficiency of gene silencing.

Base Sequence↗

PubMed2006

Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.

Animals↗

PubMed2004

[Inhibitory effects of siRNA targeting epidermal growth factor receptor on proliferation and invasion of human glioblastoma cells].

OBJECTIVE: To study the inhibitory effects of siRNA targeting epidermal growth factor receptor (EGFR) on the proliferation and invasion of human glioblastoma cells. METHOD: Two siRNA expression constructs using psiRNA-NeoG2 vector, that targeted sequences of human EGFR receptor L domain (516 - 536) and catalytic domain (2400 - 2420) respectively, were constructed. Human malignant glioma cells of the line TJ905 were cultured in vitro and transfected with pcDNA3-hEGFR, anti-sense RNA, blank vector psiRNA-NeoG2 (as negative control), psiRNA-NeoG2-516, and psiRNA-NeoG2-2400 respectively mediated by LipofectAMINE. Immunofluorescence assay and Western blotting were used to detect the EGFR expression. Cell apoptosis was detected by apoptotic index (AI) using TUNEL method. Cell cycle was analyzed by flow cytometry, and cell proliferative activities were measured by MTT. The expression and enzymatic activities of matrix metalloproteinase 9 (MMP-9) were measured by Western blotting and gelatin zymography, and cell invasive capabilities were evaluated by Transwell-ECM method. RESULT: Immunofluorescence assay and Western blotting showed that the expression of EGFR was down-regulated by 90% and 92% respectively in the siRNA constructs transfected groups, while down-regulated by 82% in the antisense EGFR RNA transfected cells in comparison with the TJ905 cells and the cells transfected with blank vector. TUNEL assay showed that almost no apoptotic cell was found in the parental cells or the cells transfected with blank vector, however, apoptosis was increased in antisense EGFR transfected cells (AI = 7.2) and siRNA constructs transfected cells (AI = 13.7 and 14.7; chi(2) = 31.549, P < 0.001). Flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNA constructs transfected cells than that in the parental cells, the cells transfected with blank vector, and the antisense EGFR transfected cells. MTT assay indicated that compared to the parental cells and the cells transfected with blank vector, the survival rates of transfected cells dramatically dropped down from the first day after implantation (P < 0.05), the siRNA transfected cells demonstrated much lower survival rate than the antisense EGFR transfected cells. Meanwhile, the expression and enzymatic activities of MMP-9 decreased significantly after the transfection of antisense EGFR into the TJ905 cells compared to the TJ905 cells and the cells transfected with blank vector, and were much lower in the siRNA groups than that in the antisense groups (P < 0.05). Cell invasive capability assay demonstrated the similar inhibitory results in the Transwell ECM-Matrigel study. CONCLUSION: Compared with antisense approach, siRNA expression constructs targeting EGFR specifically suppresses the EGFR expression, induces gene silencing, and inhibits cell growth and invasion. The plasmid-based siRNA approach should be a new strategy in glioma gene therapy targeting EGFR.

Brain Neoplasms↗

PubMedDate not supplied

siRNA is not more effective than a first generation antisense oligonucleotide when directed against EGFR in the treatment of PC-3 prostate cancer.

siRNA specifically directed against the epidermal growth factor receptor (EGFR) was compared to the previously described and effective MR2 oligo (also specific for EGFR) in a total of six comparative studies utilizing the chemotherapeutic agents Taxol, cisplatin and Cytoxan and the PC-3 prostate tumor line. When Taxol was administered in combination with either MR2 or siRNA, the MR2 was significantly more effective (p = 0.000277) against PC-3 cells incubated for 24 h in their presence. In a sequential study in which a 24-h Taxol treatment was followed with either MR2 or siRNA for an additional 24 h, or in the reverse order where MR2 or siRNA was followed by Taxol, no significant differences were found. When cisplatin was similarly administered in combination with either MR2 or the siRNA, no significant differences in inhibition were found. In a subsequent study, in which a 24-h treatment with either MR2 or siRNA was followed by cisplatin for an additional 24 h, again no significant differences were found. Lastly, in a series of sequential administrations including Cytoxan the following was found. PC-3 cells treated for 24 h with Cytoxan followed by either MR2 or siRNA produced similar inhibition. When the cycle was reversed, with MR2 or siRNA treatment followed by Cytoxan, both treatments were again inhibitory, however the initial treatment with MR2 was significantly more effective (p = 0.026095). We conclude that, although siRNA against EGFR has efficacy against the PC-3 line when administered either alone vs untreated controls, with Taxol vs Taxol alone, or with cisplatin vs cisplatin alone, it is not more effective than the better characterized MR2 oligo.

Adenocarcinoma↗

PubMed2005

[Specific silencing of PCNA gene expression in leukemic cell lines using siRNA].

BACKGROUND: Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression. METHODS AND RESULTS: Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1 x 10(6) cells/ml and amount of Oligofectamine 4 microl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h. CONCLUSIONS: Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.

Cell Line, Tumor↗

PubMed2005

[Inhibitory effect of siRNA targeting HIF-1alpha on differentiation of peripheral blood endothelial progenitor cells].

BACKGROUND & OBJECTIVE: Transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and hypoxia-induced pathophysiologic events. This study applied RNA interference targeting HIF-1alpha to human peripheral blood endothelial progenitor cells (EPCs) for therapeutic antineovascularization in vitro. METHODS: Small interference RNA (siRNA) targeting HIF-1alpha was constructed and transfected into human EPCs. Transfection efficiency of siRNA-HIF-1alpha was measured by reverse transcription-polymerase chain reaction (RT-PCR), immunocytochemistry, and flow cytometry. The content of endothelial nitric oxide synthase (eNOS) was measured by ELISA. The morphology of EPCs/endothelial cells (ECs) cultured in normoxia or hypoxia was observed after siRNA-HIF-1alpha transfection. RESULTS: SiRNA-HIF-1alpha was successfully constructed with the transfection efficiency of about 20%. After transfected for 6 h, HIF-1alpha mRNA was detected in EPCs cultured in normoxia or hypoxia with suppression; the expression of vascular endothelial growth factor (VEGF) was significantly attenuated (P < 0.05); the expression of HIF-1beta had no obvious change (P > 0.05). Stable expression of HIF-1alpha protein was induced by hypoxia, and was inhibited by siRNA-HIF-1alpha (P < 0.05). When cultured in 1% or 21% oxygen pressure for 3 days, CD31+ EPCs were inhibited more efficiently by siRNA-HIF-1alpha than by pEGFP (P < 0.05). The content of eNOS was significantly attenuated by siRNA-HIF-1alpha in time- and VEGF concentration-dependent manners (P < 0.01). EPCs differentiation and proliferation were induced by hypoxic culture, and suppressed by siRNA-HIF-1alpha. CONCLUSIONS: The constitutive or hypoxia-induced expression of HIF-1alpha in EPCs is sufficient to affect the expression of downstream target genes. SiRNA targeting HIF-1alpha could inhibit the expression of the genes which promote neovascularization, and suppress the differentiation of EPC to EC.

Aryl Hydrocarbon Receptor Nuclear Translocator↗

PubMed2006

[Phosphodiesterase type 5 siRNA increases cGMP in the smooth muscle cells of human corpus cavernosum].

OBJECTIVE: To investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on the cGMP in the smooth muscle cells of human corpus cavernosum, and to provide an experimental groundwork for the gene therapy of erectile dysfunction (ED). METHODS: Small interfering RNAs targeting PDE5 gene were synthesized by using web design software provided by Ambion, three siRNAs and a control siRNA were synthesized by Ambion. siRNAs were transfected into the smooth muscle cells of human corpus cavernosum by using siPORT Lipid reagent. cGMP was detected by ELISA at different times (24, 48, 72 and 96 h) after transfection. RESULTS: The cGMP levels of the siRNA1, siRNA2 and siRNA3 groups were significantly higher than those of the siRNA control and blank control groups (P < 0.05), and so was it in the siRNA1 group than the siRNA2 and siRNA3 groups (P < 0.05), with significant difference between the siRNA control and the blank control group (P > 0.05). CONCLUSION: The synthesized siRNAs in vitro are capable of increasing the level of cGMP in the smooth muscle cells of human corpus cavernosum, different siRNAs with different capabilities. The siRNA technique could provide not only an extremely powerful tool for the functional analysis of genome but also a new approach to ED gene therapy.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2005

An in vitro study on the suppressive effect of glioma cell growth induced by plasmid-based small interference RNA (siRNA) targeting human epidermal growth factor receptor.

OBJECTIVES: To study the inhibitory effects of plasmid-based siRNA targeting human epidermal growth factor receptor (EGFR) on tumor proliferation and invasion of TJ905 glioblastoma cells. METHODS: Two siRNA expression constructs targeting human EGFR extracellular domain (516-536) and catalytic domain (2400-2420) were transfected into TJ905 cell as mediated by Lipofectamine. Immunofluorescence assay and Western blotting were used to detect EGFR expression. Cell cycle was analyzed by flow cytometry, cell proliferative activity was measured by MTT assay. The expression and enzymatic activity of MMP9 were measured by Western blotting and gelatin zymography. Cell invasive capability was evaluated by Transwell method. RESULTS: The expression of EGFR was knocked-down by 90 and 92, respectively in siRNA constructs transfected groups as indicated by immunofluorescence assay and Western blotting. The flow cytometric analysis showed that the S phase fraction (SPF) was lowered in both siRNAs transfected cells than that in parental cells and the cells transfected with empty vector. Compared to parental cells and the cells transfected with empty vector, the survival rates of glioma cells transfected with the siRNAs dramatically dropped down from the first day after implantation (P<0.05) as indicated by MTT assay. Meanwhile, the expression and enzymatic activity of MMP9 decreased significantly in siRNAs transfected in TJ905 cells, and cell invasive potential was also greatly inhibited in the Transwell study. CONCLUSION: The siRNA expression constructs targeting EGFR could specifically suppress EGFR expression, inhibit cell growth, induce cell cycle arrest and suppress invasion. The plasmid-based siRNA targeting human EGFR approach should be a new strategy for gene therapy of malignant gliomas.

Blotting, Western↗

PubMed2006

Ligand-targeted delivery of therapeutic siRNA.

RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon that is triggered by double-stranded RNA (dsRNA). Since many diseases are associated with the inappropriate production of specific proteins, attempts are being made to exploit RNAi in a clinical settings. However, before RNAi can be exploited as therapeutically, several obstacles must be overcome. For example, small interfering RNA (siRNA) is unstable in the blood stream so any effects of injected siRNA are only transient. Accordingly, methods must be developed to prolong its activity. Furthermore, the efficient and safe delivery of siRNA into target tissues and cells is critical for successful therapy. Any useful delivery method should be designed to target siRNA to specific cells and to promote gene-silencing activity once the siRNA is inside the cells. Recent chemical modifications of siRNA have overcome problems associated with the instability of siRNA, and various ligands, including glycosylated molecules, peptides, proteins, antibodies and engineered antibody fragments, appear to be very useful or have considerable potential for the targeted delivery of siRNA. The use of such ligands improves the efficiency, specificity and, as a consequence, the safety of the corresponding delivery systems.

Animals↗

PubMed2006

Light controllable siRNAs regulate gene suppression and phenotypes in cells.

Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.

Biological Assay↗

PubMed2003

Cationic liposome-mediated delivery of siRNAs in adult mice.

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-alpha and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.

Animals↗

PubMed2006

siRNA-mediated type 1 insulin-like growth factor receptor silencing induces chemosensitization of a human liver cancer cell line with mutant P53.

Overexpression of type 1 insulin-like growth factor receptor (IGF1R) contributes to the progression and metastasis of liver cancer, implying that IGF1R gene is a suitable target of RNA interference (RNAi) for liver cancer therapy. To investigate the possible regulation of IGF1R by P53, we examined the level of IGF1R expression in liver cancer cell lines in response to adriamycin. Levels of IGF1R mRNA and protein in cell lines with wild-type P53 decreased dramatically after P53 induction, but no such reduction of IGF1R was observed in cell lines with mutated P53. Inhibition of wild-type P53 in HEPG2 cells by small interfering RNA (siRNA) significantly upregulated the expression of IGF1R. IGF1R inhibition by siRNA in Huh7 cells with mutated P53 significantly depressed cell proliferation. To investigate the sensitivity of cancer cells to adriamycin after inhibition of IGF1R, we depressed IGF1R expression using siRNA, and then added adriamycin at an IC50 dose. After a further 48 h incubation with adriamycin, proliferation was significantly depressed in the cells treated with siRNA targeting IGF1R, in comparison with siRNA targeting scramble. Furthermore, both TUNEL and pro-caspase-3 expression assay showed a significant increase in apoptosis after combined treatment with adriamycin and siRNA targeting IGF1R. Our results demonstrate that IGF1R is downregulated by P53, and that siRNA targeting of IGF1R increases liver cancer cells sensitivity to adriamycin and promotes apoptosis. siRNA targeting of IGF1R could be potentially useful for increasing sensitivity to anti-cancer drugs, especially in drug-resistant cells with mutated P53.

Apoptosis↗

PubMed2005

Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs.

Arabidopsis encodes four DICER-like (DCL) proteins. DCL1 produces miRNAs, DCL2 produces some virus-derived siRNAs, and DCL3 produces endogenous RDR2-dependent siRNAs, but the role of DCL4 is unknown. We show that DCL4 is the primary processor of endogenous RDR6-dependent trans-acting siRNAs (tasiRNAs). Molecular and phenotypic analyses of all dcl double mutants also revealed partially compensatory functions among DCL proteins. In the absence of DCL4, some RDR6-dependent siRNAs were produced by DCL2 and DCL3, and in the absence of DCL3, some RDR2-dependent siRNAs were produced by DCL2 and DCL4. Consistent with partial redundancies, dcl2 and dcl3 mutants developed normally, whereas dcl4 and dcl3 dcl4 mutants had weak and severe rdr6 phenotypes, respectively, and increased tasiRNA target mRNA accumulation. After three generations, dcl3 dcl4 and dcl2 dcl3 mutants exhibited stochastic developmental phenotypes, some of which were lethal, likely owing to the accumulated loss of heterochromatic siRNA-directed marks. dcl1 dcl3 and dcl1 dcl4, but not dcl1 dcl2 mutants, had phenotypes more severe than dcl1 mutants, consistent with DCL1, DCL3, and DCL4 acting as the primary processors of the three respective classes of endogenous silencing RNAs and DCL2 acting to produce viral-derived siRNAs and as an alternative DCL for endogenous siRNA production.

Arabidopsis↗

PubMed2006

The effect of temperature on gene silencing by siRNAs: implications for silencing in the anterior chamber of the eye.

Gene silencing by siRNAs offers the potential for reducing the expression of mutated genes that cause disease. It has been shown that the folding or secondary structure of a specific mRNA was significantly correlated to the silencing observed. Since this base pairing is dependent on free energy, the temperature of the cells may influence the effectiveness of a particular siRNA. The aqueous humor of the human eye has been measured to be around 34 degrees C with the lens acting as a thermal barrier in the eye. The trabecular meshwork, bathed by the aqueous humor, probably has a temperature lower than body temperature under normal conditions. Mutated myocilin, that is associated with primary open angle glaucoma, would appear to be a candidate for silencing by siRNAs. Silencing of the mutated myocilin might prevent additional accumulation of this protein in the rough endoplasmic reticulum. These experiments were undertaken to determine the influence of lowered temperatures on the silencing of myocilin by five siRNAs. Three different patterns of silencing emerged when the silencings were compared with cells grown at 33, 35, and 37 degrees C. For one of the siRNAs, the silencing was increased at lower temperatures. For two siRNAs, no significant changes in silencing were observed with different temperatures. Two of the siRNAs were significantly influenced by temperature with little if any silencing occurring at the lowest temperature. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37 degrees C to determine their effectiveness.

Anterior Chamber↗

PubMed2005

Prediction of siRNA functionality using generalized string kernel and support vector machine.

Small interfering RNAs (siRNAs) are becoming widely used for sequence-specific gene silencing in mammalian cells, but designing an effective siRNA is still a challenging task. In this study, we developed an algorithm for predicting siRNA functionality by using generalized string kernel (GSK) combined with support vector machine (SVM). With GSK, siRNA sequences were represented as vectors in a multi-dimensional feature space according to the numbers of subsequences in each siRNA, and subsequently classified with SVM into effective or ineffective siRNAs. We applied this algorithm to published siRNAs, and could classify effective and ineffective siRNAs with 90.6%, 86.2% accuracy, respectively.

Algorithms↗

PubMed2006

Liposomes for phospholipase A2 triggered siRNA release: preparation and in vitro test.

Small interfering RNA (siRNA) is potent and highly specific for gene silencing. However, for therapeutic applications, delivery systems are required to protect siRNA from degradation, to enhance cellular uptake and for site-specific delivery. We used a double emulsion technique to encapsulate siRNA into stealth liposomes (SL) to increase entrapment efficiency compared to passive encapsulation. SL are designed for localized, active release of siRNA by secretory phosholipase A(2) (sPLA(2)). sPLA(2) acts as a site-specific enzymatic trigger that actively degrades the liposomal carrier in inflamed tissue releasing entrapped drug. Relatively good encapsulation efficiencies compared to passive encapsulation were demonstrated (7-9%) and SL size was appropriate for i.v. administration (60-90 nm). siRNA targeting enhanced green fluorescent protein (EGFP) entrapped in SL did not silence gene expression of HeLa-cells stably expressing EGFP. However, preliminary flow cytometry and confocal microscopy data showed that the SL siRNA formulation increased uptake of siRNA into vesicular compartments of HeLa-cells in a concentration-dependent manner that could be augmented by exogenuos sPLA(2). We hypothesize that the SL can be used to target siRNA to inflammed tissue for silencing of cytokine expression in rheumatoid arthritis.

Arthritis, Rheumatoid↗

PubMed2006

Development and characterisation of chitosan nanoparticles for siRNA delivery.

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for diseases with genetic defects. The use of siRNA, however, is hampered by its rapid degradation and poor cellular uptake into cells in vitro or in vivo. Therefore, we have explored chitosan as a siRNA vector due to its advantages such as low toxicity, biodegradability and biocompatibility. Chitosan nanoparticles were prepared by two methods of ionic cross-linking, simple complexation and ionic gelation using sodium tripolyphosphate (TPP). Both methods produced nanosize particles, less than 500 nm depending on type, molecular weight as well as concentration of chitosan. In the case of ionic gelation, two further factors, namely chitosan to TPP weight ratio and pH, affected the particle size. In vitro studies in two types of cells lines, CHO K1 and HEK 293, have revealed that preparation method of siRNA association to the chitosan plays an important role on the silencing effect. Chitosan-TPP nanoparticles with entrapped siRNA are shown to be better vectors as siRNA delivery vehicles compared to chitosan-siRNA complexes possibly due to their high binding capacity and loading efficiency. Therefore, chitosan-TPP nanoparticles show much potential as viable vector candidates for safer and cost-effective siRNA delivery.

Adsorption↗

PubMed2005

Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization.

The potential induction of inflammatory cytokines and interferon responses by small-interfering RNAs (siRNAs) represents a major obstacle for their use as inhibitors of gene expression. Therapeutic applications of siRNAs will require a better understanding of the mechanisms that trigger such unwanted effects, especially in freshly isolated human cells. Surprisingly, the induction of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6) in adherent peripheral blood mononuclear cells (PBMC) was not restricted to double-stranded siRNAs, because induction was also obtained with single-stranded siRNAs (sense or antisense strands). The immunostimulatory effects were sequence-dependent, since only certain sequences are prone to induce inflammatory responses while others are not. The induction of TNF-alpha, IL-6 and interferon alpha (IFN-alpha) was chloroquine-sensitive and dependent more likely on endosomal Toll-like receptor signaling in particular TLR8. Indeed, no significant immunostimulatory effects were detected when either double or single-stranded siRNAs were delivered directly to cytoplasm via electroporation. Both RNA types activated a NF-kappaB promoter-driven luciferase gene in transiently transfected human adherent PBMC. Moreover, culture of immature dendritic cells with either double or single-stranded siRNAs stimulated interleukin-12 production and induced the expression of CD83, an activation marker. Interestingly, several double-stranded siRNAs did not induce TNF-alpha, IL-6 and IFN-alpha production, however, their single-stranded sense or antisense did. Taken together, the present data indicate for the first time that the induction of inflammatory cytokines and IFN-alpha responses by either double-stranded or single-stranded siRNAs in adherent PBMC is sequence-dependent and requires endosomal intracellular signaling. The finding that endosomal localization of self-RNAs (sense strands) can trigger Toll-like receptor signaling in adherent human PBMC is intriguing because it indicates that endosomal self-RNAs can display a molecular pattern capable for activating innate immunity.

Cells, Cultured↗