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mRNA knockdown by single strand RNA is improved by chemical modifications.

Haringsma HJ, Li JJ, Soriano F, Kenski DM, Flanagan WM, Willingham AT - Nucleic Acids Res. (2012)

Bottom Line: We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo.The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity.While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

View Article: PubMed Central - PubMed

Affiliation: Sirna Therapeutics, 1700 Owens Street, Fourth Floor, San Francisco, CA 94158, USA.

ABSTRACT
While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

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In vivo knockdown of single strand versus duplex siRNA demonstrates ssRNAi activity in vivo. RNAs formulated in a lipid nanoparticle (LNP) delivery vehicle. Modified guide strands (2'F) are compared to unmodified (2'OH). All oligos contain 5' phosphorylation. Comparison of Day 3 ApoB mRNA knockdown after an LNP delivered 3 mg/kg dose of dsRNA or ssRNA for target sites (A) ApoB (8786) and (B) ApoB (6981). Knockdown of 60–75% was observed for phosphorylated single strands containing 2'F modifications. Duration of mRNA knockdown after an LNP delivered 6 mg/kg dose was measured over 2 weeks (Day 2, Day 7, Day 14 time points) for (C) ApoB (8786) and (D) ApoB (6981). Knockdown values shown in Supplementary Table S2.
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gkr1301-F4: In vivo knockdown of single strand versus duplex siRNA demonstrates ssRNAi activity in vivo. RNAs formulated in a lipid nanoparticle (LNP) delivery vehicle. Modified guide strands (2'F) are compared to unmodified (2'OH). All oligos contain 5' phosphorylation. Comparison of Day 3 ApoB mRNA knockdown after an LNP delivered 3 mg/kg dose of dsRNA or ssRNA for target sites (A) ApoB (8786) and (B) ApoB (6981). Knockdown of 60–75% was observed for phosphorylated single strands containing 2'F modifications. Duration of mRNA knockdown after an LNP delivered 6 mg/kg dose was measured over 2 weeks (Day 2, Day 7, Day 14 time points) for (C) ApoB (8786) and (D) ApoB (6981). Knockdown values shown in Supplementary Table S2.

Mentions: We extended our comparison of ssRNA and dsRNA into mice to identify whether ssRNAs were competent for knockdown in vivo. Single strand and duplex RNAs were formulated in lipid nanoparticles (LNP) which permitted side by side comparison of these RNAs while using a well-established delivery vehicle with proven capabilities (30,32). Since the nanoparticle encapsulates the siRNAs, it is likely shielded from serum nucleases which eliminated an additional round of siRNA chemical optimization for serum stability. The same ApoB siRNAs assayed for cell-based potency (Figure 3) were formulated in LNPs and intravenously dosed at 3 mg/kg into mice. Three days later the mice were sacrificed and ApoB mRNA knockdown was measured from harvested livers. Compared to buffer control, the duplex siRNAs reduced ApoB expression by 85–98% (Figure 4A and B and Supplementary Table S2). Unmodified ssRNAs had no significant knockdown which was consistent with in vitro observations. However 2′F-modified single strands were quite effective, reducing ApoB expression levels by 61–74% and demonstrating that ssRNA functions in vivo.Figure 4.


mRNA knockdown by single strand RNA is improved by chemical modifications.

Haringsma HJ, Li JJ, Soriano F, Kenski DM, Flanagan WM, Willingham AT - Nucleic Acids Res. (2012)

In vivo knockdown of single strand versus duplex siRNA demonstrates ssRNAi activity in vivo. RNAs formulated in a lipid nanoparticle (LNP) delivery vehicle. Modified guide strands (2'F) are compared to unmodified (2'OH). All oligos contain 5' phosphorylation. Comparison of Day 3 ApoB mRNA knockdown after an LNP delivered 3 mg/kg dose of dsRNA or ssRNA for target sites (A) ApoB (8786) and (B) ApoB (6981). Knockdown of 60–75% was observed for phosphorylated single strands containing 2'F modifications. Duration of mRNA knockdown after an LNP delivered 6 mg/kg dose was measured over 2 weeks (Day 2, Day 7, Day 14 time points) for (C) ApoB (8786) and (D) ApoB (6981). Knockdown values shown in Supplementary Table S2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351186&req=5

gkr1301-F4: In vivo knockdown of single strand versus duplex siRNA demonstrates ssRNAi activity in vivo. RNAs formulated in a lipid nanoparticle (LNP) delivery vehicle. Modified guide strands (2'F) are compared to unmodified (2'OH). All oligos contain 5' phosphorylation. Comparison of Day 3 ApoB mRNA knockdown after an LNP delivered 3 mg/kg dose of dsRNA or ssRNA for target sites (A) ApoB (8786) and (B) ApoB (6981). Knockdown of 60–75% was observed for phosphorylated single strands containing 2'F modifications. Duration of mRNA knockdown after an LNP delivered 6 mg/kg dose was measured over 2 weeks (Day 2, Day 7, Day 14 time points) for (C) ApoB (8786) and (D) ApoB (6981). Knockdown values shown in Supplementary Table S2.
Mentions: We extended our comparison of ssRNA and dsRNA into mice to identify whether ssRNAs were competent for knockdown in vivo. Single strand and duplex RNAs were formulated in lipid nanoparticles (LNP) which permitted side by side comparison of these RNAs while using a well-established delivery vehicle with proven capabilities (30,32). Since the nanoparticle encapsulates the siRNAs, it is likely shielded from serum nucleases which eliminated an additional round of siRNA chemical optimization for serum stability. The same ApoB siRNAs assayed for cell-based potency (Figure 3) were formulated in LNPs and intravenously dosed at 3 mg/kg into mice. Three days later the mice were sacrificed and ApoB mRNA knockdown was measured from harvested livers. Compared to buffer control, the duplex siRNAs reduced ApoB expression by 85–98% (Figure 4A and B and Supplementary Table S2). Unmodified ssRNAs had no significant knockdown which was consistent with in vitro observations. However 2′F-modified single strands were quite effective, reducing ApoB expression levels by 61–74% and demonstrating that ssRNA functions in vivo.Figure 4.

Bottom Line: We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo.The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity.While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

View Article: PubMed Central - PubMed

Affiliation: Sirna Therapeutics, 1700 Owens Street, Fourth Floor, San Francisco, CA 94158, USA.

ABSTRACT
While RNAi has traditionally relied on RNA duplexes, early evaluation of siRNAs demonstrated activity of the guide strand in the absence of the passenger strand. However, these single strands lacked the activity of duplex RNAs. Here, we report the systematic use of chemical modifications to optimize single-strand RNA (ssRNA)-mediated mRNA knockdown. We identify that 2'F ribose modifications coupled with 5'-end phosphorylation vastly improves ssRNA activity both in vitro and in vivo. The impact of specific chemical modifications on ssRNA activity implies an Ago-mediated mechanism but the hallmark mRNA cleavage sites were not observed which suggests ssRNA may operate through a mechanism beyond conventional Ago2 slicer activity. While currently less potent than duplex siRNAs, with additional chemical optimization and alternative routes of delivery, chemically modified ssRNAs could represent a powerful RNAi platform.

Show MeSH
Related in: MedlinePlus