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- Bramsen, Jesper B., et al.
(författare)
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A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 37:9, s. 2867-2881
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Tidskriftsartikel (refereegranskat)abstract
- The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3'-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.
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| 2. |
- Bramsen, Jesper B., et al.
(författare)
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A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects
- 2010
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 38:17, s. 5761-5773
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Tidskriftsartikel (refereegranskat)abstract
- Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.
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| 7. |
- Gagnon, Keith T., et al.
(författare)
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Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat
- 2010
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 49:47, s. 10166-10178
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Tidskriftsartikel (refereegranskat)abstract
- Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.
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| 9. |
- Opalinska, Joanna B., et al.
(författare)
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Nucleic acid therapeutics for hematologic malignancies--theoretical considerations
- 2006
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Ingår i: OLIGONUCLEOTIDE THERAPEUTICS. - : Wiley. - 9781573315876 ; , s. 124-136
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Konferensbidrag (refereegranskat)abstract
- Our work is motivated by the belief that RNA targeted gene silencing agents can be developed into effective drugs for treating hematologic malignancies. In many experimental systems, antisense nucleic acids of various composition, including antisense oligodeoxynucleotides (AS ODNs) and short interfering RNA (siRNA), have been shown to perturb gene expression in a sequence specific manner. Nevertheless, our clinical experience, and those of others, have led us to conclude that the antisense nucleic acids (ASNAs) we, and others, employ need to be optimized with regard to intracellular delivery, targeting, chemical composition, and efficiency of mRNA destruction. We have hypothesized that addressing these critical issues will lead to the development of practical and effective nucleic acid drugs. An overview of our recent work which seeks to addresses these core issues is contained within this review.
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