Evaluation Of siRNAs Research Articles

Evaluation of siRNA and cationic liposomes complexes as a model for in vitro siRNA delivery to cancer cells

Controlled release of genetic material such as small interfering RNA (siRNA) using lipid-based non-viral vectors has gained substantial importance in gene therapy applications. Therefore, the interaction between siRNA and these vectors must be well understood. This study aims to investigate the effect of different molar charge ratios (R+/-) between positive charges from microfluidics-produced cationic liposomes (CL) (egg phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and negative charges from siRNA and on physico-chemical and morphological properties of the lipoplexes (CL/siRNA) as well as their in vitro luciferase silencing effect in HeLa cells. R+/- 3.27 was found to be the optimum point for complexation. This finding was confirmed by gel retardation and siRNA accessibility assays. According to Cryo-TEM analysis, the lipoplexes had multi-lamellarity. In vitro transfection efficiency of lipoplexes in HeLa cells was tested at three different siRNA concentrations (10, 25, and 35 nM). Significant knockdown of luciferase by siRNA lipoplexes was observed based on reduced luciferase activity of transfected HeLa cells. Our findings were comparable with the silencing effect of siRNA complexed with Lipofectamine®. No cytotoxicity of lipoplexes was detected at the tested concentrations. This study was essential for further complexation studies which will be performed using microfluidic systems to formulate next-generation lipid-based controlled release systems.

Evaluation of siRNAs that contain internal variable-length spacer linkages

The most widely accepted mechanism of RNAi-silencing involves the RNA-induced silencing complex (RISC) liberating the active antisense strand from the sense strand of an siRNA duplex to form an active RISC-antisense complex. This involves cleaving the sense strand between positions 9 and 10 from the 5′ end of the strand prior to dissociation. Destabilizing modifications near the center of the duplex in some cases can enhance the efficacy of the resultant construct and may trigger an alternative mechanism through which the sense strand is removed. By introducing alkyl spacers of varying lengths near or within the sense strand’s cleavage site, this study illustrates that siRNAs, in most cases, retained potent RNAi-silencing activity. Our results highlight that by substituting the scissile phosphodiester linkage on the sense strand with non-cleavable alkyl chains provides a novel and alternative method to destabilize the central region of siRNAs.

442. Evaluation of siRNA for Induction of In Vitro Resistance in HIV-1

Recent work has shown that small interfering RNA (siRNA) effectively inhibits HIV-1 replication. In this study, we tested the efficacy of three siRNAs, specifically targeted to the p24, vif and a shared common exon between the tat and rev genes, for their ability to inhibit HIV-1 replication. Each siRNA gene was placed under the control of the H1 promoter and co-transfected with an HIV-1 expression plasmid into 293T cells. All three siRNAs inhibited p24 production from the transfected cells although the p24 and the tat/rev siRNAs were at least 4-5 fold more efficient than the vif siRNA. Similar results were observed when the titer of the infectious HIV-1 was determined by infecting HeLa-CD4-LTR-|[beta]|-gal cells followed by |[beta]|-gal staining. Based on these results, we asked whether there was a relative difference in HIV resistance induction. We first inserted the hairpin p24 siRNA gene into a GFP-containing HIV vector, transduced H9 cells, and challenged with HIV-1NL4|[ndash]|3. At low challenge doses, the result showed a 2 to 6 log reduction in HIV-1 replication for up to 60 days after challenge when compared with control H9 cells. However, at higher challenge doses, |[ldquo]|outbreak|[rdquo]| virus emerged after 17 days. Sequence analysis of these outbreaks confirmed the presence of mutations in the siRNA target site. Thus, like HIV-1 drug therapy, the antiviral effect of siRNA permits the selection of mutant strains that can no longer be recognized by the siRNA. Using a similar approach to study the efficacy of the tat/rev siRNA, we showed that under similar HIV-1 challenge conditions, the tat/rev siRNA was more effective than other siRNAs in preventing the emergence of these HIV-1 outbreaks. Our study thus suggests that the single-targeted siRNAp approach is not sufficient to inhibit HIV-1 replication. If siRNA is to be used to constitute an effective gene therapy approach to treat HIV infection, multi-targeted siRNA designs need to be implemented.

SiRNA-mediated knockdown of the Nav1.8 sodium channel

Specific knockdown of Nav1.8 protein in the DRG by antisense oligonuclotides has been shown to prevent hyperalgesia and allodynia in chronic nerve injury models. To determine whether small interfering RNAs (siRNAs) may be used in treatment of chronic pain, we tested several siRNAs for their ability to block Nav1.8 expression and function. Knockdown of sodium channel expression and function was monitored in a DRG and neuroblastoma fusion cell line (ND7/23) that was stably transfected with the rat Nav1.8 gene. Using quantative-RTPCR analysis, we have identified four siRNAs capable of strongly downregulating Nav1.8 expression. Dose-dependent downregulation of the Nav1.8 protein by selected siRNAs was confirmed in HEK293 cells transiently co-transfected with a Nav1.8 expressing plasmid and siRNAs. Functional knockdown of the Nav1.8 current was confirmed by the membrane potential assay as well as whole-cell voltage clamping of siRNA-transfected ND7/23 cells. Down regulation of sodium currents was maintained for at least three days following siRNA administration. To test whether siRNA could be delivered for longer periods of time, we constructed an adenoviral vector expressing the Nav1.8 siRNA sequence under the control of an U6 promoter cassette. Infection by this virus induced expression of siRNA in ND7/23 cells. Virally-expressed siRNA was capable of knocking down both, Nav1.8 mRNA as well as Na+ current in ND7/23 cells. This knockdown lasted for at least ten days post infection. Specific and long term knockdown of Nav1.8 channel function using naked siRNA and virally-expressed siRNA will allow the evaluation of siRNA as a therapeutic agent in the treatment of chronic pain syndromes. Specific knockdown of Nav1.8 protein in the DRG by antisense oligonuclotides has been shown to prevent hyperalgesia and allodynia in chronic nerve injury models. To determine whether small interfering RNAs (siRNAs) may be used in treatment of chronic pain, we tested several siRNAs for their ability to block Nav1.8 expression and function. Knockdown of sodium channel expression and function was monitored in a DRG and neuroblastoma fusion cell line (ND7/23) that was stably transfected with the rat Nav1.8 gene. Using quantative-RTPCR analysis, we have identified four siRNAs capable of strongly downregulating Nav1.8 expression. Dose-dependent downregulation of the Nav1.8 protein by selected siRNAs was confirmed in HEK293 cells transiently co-transfected with a Nav1.8 expressing plasmid and siRNAs. Functional knockdown of the Nav1.8 current was confirmed by the membrane potential assay as well as whole-cell voltage clamping of siRNA-transfected ND7/23 cells. Down regulation of sodium currents was maintained for at least three days following siRNA administration. To test whether siRNA could be delivered for longer periods of time, we constructed an adenoviral vector expressing the Nav1.8 siRNA sequence under the control of an U6 promoter cassette. Infection by this virus induced expression of siRNA in ND7/23 cells. Virally-expressed siRNA was capable of knocking down both, Nav1.8 mRNA as well as Na+ current in ND7/23 cells. This knockdown lasted for at least ten days post infection. Specific and long term knockdown of Nav1.8 channel function using naked siRNA and virally-expressed siRNA will allow the evaluation of siRNA as a therapeutic agent in the treatment of chronic pain syndromes.