Efficient and specific gene knockdown by siRNAs produced in bacteria
Inventors: Judy Lieberman, Linfeng Huang
Invention Types: Research Tool, Therapeutics
Research Areas: Immunology
Keywords: Drug Delivery, siRNAFor More Information Contact: Dietz, Ryan
siRNAs are an indispensable tool to investigate gene function in eukaryotic cells and may be used for therapeutic purposes to knock down genes implicated in disease. Most siRNAs have been produced by chemical synthesis. Researchers at Boston Children’s have developed a simple method to produce highly potent recombinant siRNAs in Escherichia coli using methods accessible to any laboratory capable of producing recombinant proteins. This method relies on ectopic expression of p19, an siRNA-binding protein found in a plant virus. When expressed in E. coli, p19 stabilizes ~21-nt siRNAs produced by bacterial RNase III (“prokaryotic siRNAs”). siRNAs generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene contain multiple sequences from the target gene sequence. Prokaryotic siRNAs can be easily purified from bacterial lysates using methods that could be adapted for large scale fermentation. When mammalian cells are transfected with low nanomolar concentrations of prokaryotic siRNAs, target gene knockdown is reproducibly reduced by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.
• A renewable method to produce highly potent siRNAs in bacteria suitable for research or therapeutic use.
• Especially useful for knockdown of polymorphic cellular or viral genes.
• Research tool companies could produce kits to generate a p19-siRNA expression plasmid and/or to express, isolate and purify prokaryotic siRNAs.
• Research service companies could provide p19-siRNA plasmids reliable for knocking down any gene.
• Generate siRNA libraries based on gene expression of specific cell types or diseases for use in screening to identify drug targets.
• This method shows for the first time that highly potent and pure siRNAs can be produced in a living organism.
• Synthetic siRNAs often require testing multiple constructs to achieve variable degrees of knockdown with associated reagent, labor and time costs.
• Prokaryotic siRNAs can be readily engineered to knock down any eukaryotic gene efficiently and specifically in mammalian cells without appreciable off-target effects and without detectable immunostimulation.
• Scaled up production using recombinant techniques could be cost effective for in vivo uses.
Huang L, Jin J, Deighan P, Kiner E, McReynolds L, Lieberman J. Efficient and specific gene knockdown by small interfering RNAs produced in bacteria. Nat Biotechnol. 2013 Apr;31(4):350-6. doi: 10.1038/nbt.2537. Epub 2013 Mar 10. PubMed PMID: 23475073
Huang L, Lieberman J. Production of highly potent recombinant siRNA in Escherichia coli. Nat. Protoc. 2013 Dec;8(12).
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