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1.
Immunol Lett ; 141(2): 169-80, 2012 Jan 30.
Article in English | MEDLINE | ID: mdl-22001011

ABSTRACT

Small interfering RNAs (siRNAs) induce robust degradation of homologous mRNAs. Highly specific silencing of target genes makes siRNA an interesting tool in drug development. However, several non-specific effects complicate the use of RNA interference (RNAi). One of the most prevalent unspecific effects is triggering the innate immune system in mammals. In parallel, activating the immune system may open the possibility to develop dual siRNAs for treatment of a variety of diseases including cancer. Here, we show that the best use of unmodified siRNAs for RNAi and immune activation depends on the delivery system, formulation condition, sequence and siRNA design concerning ORN motifs. Testing several commercial delivery systems identified that the optimal siRNAs for dual functions should contain TLR7/8 ORN motifs at least in the antisense strand and be delivered by either Dharmafect or HiPerfect. Superfect delivery system only activates TLR7 and opens new capabilities in RNAi and immune activation.


Subject(s)
Leukocytes, Mononuclear/metabolism , Nucleotide Motifs , RNA, Small Interfering/genetics , Toll-Like Receptor 7/genetics , Toll-Like Receptor 8/genetics , Base Sequence , Cell Line , Cytokines/metabolism , Genetic Vectors , Humans , Immunity, Innate/genetics , Immunization/methods , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/pathology , Molecular Sequence Data , NF-kappa B/genetics , NF-kappa B/metabolism , Nucleotide Motifs/genetics , RNA Interference
2.
Nucleic Acid Ther ; 21(6): 423-36, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22196370

ABSTRACT

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.


Subject(s)
Oligoribonucleotides/administration & dosage , Oligoribonucleotides/chemistry , Toll-Like Receptor 7/drug effects , Toll-Like Receptor 8/drug effects , Toll-Like Receptor 9/drug effects , Animals , Cells, Cultured , Chemokines/drug effects , Cytokines/drug effects , Female , HEK293 Cells/drug effects , HEK293 Cells/immunology , Humans , Immunity, Innate/drug effects , Interferon Type I/drug effects , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Male , Mice , Phosphorothioate Oligonucleotides/administration & dosage , Phosphorothioate Oligonucleotides/chemistry , Toll-Like Receptor 7/immunology , Toll-Like Receptor 8/immunology , Toll-Like Receptor 9/immunology
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