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siRNAs and PCR primers used in thisstudy.
Gaithersburg, MD /PRNewswire/ -- Sirnaomics, Inc. () announced today that the company has licensed a polypeptide nanoparticle technology invented by Professor A. James Mixson of University of Maryland Medical School. The licensing agreement grants Sirnaomics exclusive rights to a patent covering use of Histidine-Lysine polymer (HKP) for siRNA therapeutics in the areas of wound healing and ocular diseases (U.S. patent application 60/173,576 filed December 29, 1999). Sirnaomics, Inc., a biopharmaceutical company founded in early 2007, is dedicated to advancing RNA interference (RNAi) technology for novel targeted therapeutic development. The company's multi-targeted siRNA therapeutic programs utilizing its nanoparticle-enhanced delivery technologies represent a unique approach for truly realizing the advantages of small interfering RNA (siRNA)-based drugs to treat various critical human diseases.
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The above findings were further validated by examining the EGFP expression in homogenized cell lysates supplied with protease inhibitors using the western-blot method. Among the aforementioned six test samples, the LMWP-PEG-S-S-siRNA conjugate once again yielded the strongest gene silencing effect on MDA-MB-231-EGFP cells (marked as Sample #6 in Fig. D), with the EGFP down-regulating ratio being about 83.5%; consistent with the fluorescent intensity and FACS analysis data. However, the charge-associated LMWP/siRNA aggregates (Sample #5; Fig. D) and cationic lipofecter/siRNA complex (Sample #4; Fig. D) also displayed comparatively low gene down-regulating efficiency, at a level of 51.5% and 23.7%, respectively. For comparison, flow cytometry examination of the non-reducible LMWP-PEG-S-Mal-siRNA (anti-EGFP siRNA) conjugate (Conj-2) was also carried out. As depicted in Fig. E, Conj-2 yielded a much weaker down-regulation on EGFP expression, reducing its level to 68.3% of the baseline total; as compared to that (21.2%) by the cytosol-reducible LMWP-PEG-S-S-siRNA (anti-EGFP siRNA) conjugate (Sample #6 in Fig. B). This finding demonstrated the importance of retaining the cell-delivered siRNA in the cytosolic compartment, should one want to achieve the most potent gene-silencing efficacy.
RNAi: siRNA Oligo Synthesis_Shanghai Wansheng …
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Purification of the desired LMWP-PEG-S-S-siRNA conjugates was achieved via an anion exchange HiTrapTM DEAE FF (1ml, GE Healthcare, Sweden) chromatography using 20 mM sodium phosphate solution containning 10 mM EDTA at pH 7.2 (solution A) as the equilibrium buffer and the same solution containing 1M NaCl (solution B) as the elution buffer. The optimum elution conditions were as follows: Flow rate: 1 ml/min; Linear Gradient: 10-70% of solution B; Detection Wavelength: 260 nm. For comparison, the retention times on the DEAE column of LMWP-PEG-SH and siRNA were examined under identical conditions, with detection wavelength being set at 215nm for LMWP-PEG-SH.
The Lipofecter/siRNA complex was produced by blending 5μl Lipofecter with 2μg siRNA according to the protocol provided by the supplier. The physical mixtures of LMWP-PEG/siRNA and LMWP/siRNA were prepared by adding equal moles of either LMWP-PEG or unmodified LMWP to siRNA (100 nM) prepared in the DEPC buffer containing 0.15 M NaCl and 10 mM EDTA at pH 7.2. The reaction mixtures were incubated for 5 minutes and then centrifuges at 1200 rpm to collect the final products.
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The effect of PCSK9 on the inflammatory response ofmacrophages induced by oxLDL remains unknown. The present studyindicates that PCSK9 affects the biological effect induced by oxLDLin THP-1-derived macrophages. Therefore, we used THP-1-derivedmacrophages to examine the effect of oxLDL on PCSK9 expression. Therole of PCSK9 in the oxLDL-induced inflammatory response of thesemacrophages was examined using small interfering RNA (siRNA) toknockdown PCSK9 expression.
As known the PEG polymer chain, due to its dynamic spatial movement, could shield its attached cargo from recognition and interaction by the host immune system, we developed an innovative strategy to formulate an 1:1 monomeric LMWP (note: a well-established CPP )-siRNA covalent conjugate via a cytosol-cleavable disulfide linkage (Fig, 1A), without confronting the charge-induced neutralization and aggregation by the two oppositely charged substrates. Presented herein were cell culture results that offered a proof-of-concept demonstration of the plausibility of this chemical conjugation method. Comparing with the charge-complexed CPP-siRNA aggregated product, this chemical conjugate displayed far superior efficacy in delivering fully functional siRNA into the cytosolic compartment in executing presumably the most operational gene-silencing event (Fig. B).
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High-Yield Synthesis of Monomeric LMWP(CPP)-siRNA …
Human breast cancer cell line MDA-MB-231 cells were utilized for these studies. Briefly, cells were grown at 37 °C in humidified atmosphere in L15 medium supplemented with 1% antibiotics, 1% NEAA and 10 % FBS, according to a previously established protocol . Formulations containing FITC-labeled siRNA (including: PBS, native siRNA, Lipofecter/siRNA complex, LMWP-PEG/siRNA physical mixture, LMWP/siRNA physical mixture, the LMWP-PEG-S-S-siRNA (Conj-1) and LMWP-PEG-mal-S-siRNA (Conj-2) were then incubated the MDA-MB-231 cells with at 37 °C for 2 h. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, treated with the DAPI (2μg/ml) nucleic acid staining, and then subjected to confocal laser scanning microscopy analysis using an Olympus FV-300 laser scanning microscope operated with FLUOVIEW software (Olympus, Tokyo, Japan).
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The EGFP stable transfected cell line MDA-MB-231-EGFP cells were used for these studies. Briefly, the cells were grown at 37 °C in humidified atmosphere in L15 medium supplemented with 1% antibiotics, 1% NEAA and 10 % FBS. inhibition on EGFP expression was evaluated by treating the cells with formulations containing PBS, negative control siRNA, naked siRNA, lipofecter/siRNA complex, LMWP/siRNA physical mixture, the bio-reducible Conj-1, or the non-reducible Conj-2). The concentration of siRNA was maintained at 50 nM in all of these formulations. The treated cells were incubated for 12h, followed by the replacement with fresh culture media and further incubation for up to 3 days at 37 °C. The siRNA gene silencing down effect were assessed by the inhibition on EGFP expression, using different methodologies including confocal microscopy, FACS, fluorescence intensity and Western-blot analysis. Experimental details were described in each of the following sections.
PCSK9 siRNA suppresses the inflammatory response induced …
The particle sizes of the physical mixtures of LMWP/siRNA and LMWP-PEG/siRNA, as well as the LMWP-PEG-S-S-siRNA covalent conjugate were measured using Brookhaven Goniometer Light Scattering system, with deionized water as reference. Samples were prepared in light-scattering vials at the same concentration of siRNA. Dynamic light scattering was performed in high resistivity water as indicated below. All data points for dynamic light scattering were the average of three measurements performed on the same sample, and error bars represented the standard deviation.
While siRNA silencing requires an exact match to its target ..
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