Compositions for inhibiting gene expression and uses thereof
a technology of compound and gene expression, applied in the direction of drug composition, antibacterial agent, immunologic disorder, etc., can solve the problems of inability to optimize antisense oligonucleotides that have true potential to inhibit gene expression and therefore be good clinical candidates, unstable in vivo, and potential to produce off-target effects, etc., to achieve the effect of improving stability, retaining specificity and biologic activity, and improving biological stability
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example 1
Preparation of Oligonucleotide-Based Compounds
[0136]The oligonucleotide-based compounds of the invention were chemically synthesized using phosphoramidite chemistry on automated DNA / RNA synthesizer. TAC protected (Except U) 2′-O-TBDMS RNA monomers, A, G, C and U, were purchased from Sigma-Aldrich. 7-deaza-G, inosine and loxoribine monomers were purchased from ChemGenes Corporation. 0.25M 5-ethylthio-1H-tetrazole, PAC— anhydride Cap A and Cap B were purchased from Glen Research. 3% trichloroacetic acid (TCA) in dichloromethane (DCM) and 5% 3H-1,2-Benzodithiole-3-one-1,1-dioxide (Beaucage reagent) were made in house.
[0137]Oligonucleotide-based compounds of the invention were synthesized at 1-2 μM scale using a standard RNA synthesis protocol.
Cleavage and Base Deprotection
[0138]Oligonucleotide-based compounds of the invention were cleaved from solid support and the solution was further heated at 65° C. to removing protecting groups of exo cyclic-amines. The resulting solution was dried...
example 2
Human PBMC Isolation
[0171]Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthy volunteer blood (CBR Laboratories, Boston, Mass.) were isolated by Ficoll density gradient centrifugation method (Histopaque-1077, Sigma).
Human pDC Isolation
[0172]Human plasmacytoid dendritic cells (pDCs) were isolated from freshly obtained healthy human volunteer's blood PBMCs by positive selection using the BDCA4 cell isolation kits (Miltenyi Biotec) according to the manufacturer's instructions.
Treatment of PBMCs and pDCs
[0173]Human PBMCs were plated in 48-well plates using 5×106 cells / ml. Human pDCs were plated in 96-well dishes using 1×106 cells / ml. The exemplary oligonucleotide-based compounds of the invention, dissolved in DPBS (pH 7.4; Mediatech), were added to the cell cultures at doses of 0, 0.01, 1.0 or 10.0 μg / ml. The cells were then incubated at 37° C. for 24 hours and subsequently stimulated with 10 μg / ml TLR9 agonist for 24 h. After treatment and stimulation, the supernatant...
example 3
In Vivo Activity of Oligonucleotide-Based Compositions
[0184]To assess the in vivo activity of antisense oligonucleotides according to the invention, Female C57BL / 6 mice of 5-6 weeks age (N=3 / group) were injected with exemplary oligonucleotide-based compositions according to the invention at 0.25, 2, or 5 mg / kg, or PBS, subcutaneously in the left flank. Twenty-four hours after administration of the oligonucleotide-based compositions, mice were injected with 0.25 mg / kg of a TLR agonist subcutaneously in the right flank. Two hours after administration of the TLR agonist, blood was collected and serum IL-12 concentration was determined by ELISA. Data are shown as absolute IL-12 concentrations or as a percentage of IL-12 production.
Duration of In Vivo Activity of Oligonucleotide-Based Compositions
[0185]To assess the duration of in vivo activity of antisense oligonucleotides according to the invention, Female C57BL / 6 mice of 5-6 weeks age (N=3 / group) were injected with exemplary oligonucl...
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