Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing

Jackson, Aimee L.; Burchard, Julja; Leake, Devin; Reynolds, Angela; Schelter, Janell M.; Guo, Jie; Johnson, Jason M.; Lim, Lee P.; Karpilow, Jon; Nichols, Kim E.; Marshall, William S.; Khvorova, Anastasia; Linsley, Peter S.

doi:10.1261/rna.30706

Cited by 706 publications

(567 citation statements)

References 32 publications

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“…We chose to target the mouse apolipoprotein B (apoB) gene, a hepatocyte-expressed gene involved in cholesterol transport. All siRNAs used in this report contained modifications designed to increase resistance to nucleases and suppress off-target effects (22,35). We found that transfection of primary hepatocytes with apoB-1 siRNA polyconjugate was highly effective, resulting in nearly 80% knockdown of apoB mRNA (Fig.…”

Section: Resultsmentioning

confidence: 86%

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Rozema

Lewis

Wakefield

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

609

622

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Achieving efficient in vivo delivery of siRNA to the appropriate target cell would be a major advance in the use of RNAi in gene function studies and as a therapeutic modality. Hepatocytes, the key parenchymal cells of the liver, are a particularly attractive target cell type for siRNA delivery given their central role in several infectious and metabolic disorders. We have developed a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which we have named siRNA Dynamic PolyConjugates. Key features of the Dynamic PolyConjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. Using this delivery technology, we demonstrate effective knockdown of two endogenous genes in mouse liver: apolipoprotein B (apoB) and peroxisome proliferator-activated receptor alpha (ppara). Knockdown of apoB resulted in clear phenotypic changes that included a significant reduction in serum cholesterol and increased fat accumulation in the liver, consistent with the known functions of apoB. Knockdown of ppara also resulted in a phenotype consistent with its known function, although with less penetrance than observed in apoB knockdown mice. Analyses of serum liver enzyme and cytokine levels in treated mice indicated that the siRNA Dynamic PolyConjugate was nontoxic and well tolerated.pH labile bonds ͉ nonviral siRNA delivery ͉ siRNA-polymer conjugates ͉ endosomolytic polymers

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Section: Resultsmentioning

confidence: 86%

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Rozema

Lewis

Wakefield

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

609

622

View full text Add to dashboard Cite

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“…To eliminate the characterization of artifacts arising through off-target effects, 3 independent FEN1 silencing conditions were used-2 independent siRNA duplexes (FEN1-2 and FEN1-3) and a FEN1-pool comprised of 4 independent siRNA duplexes (effectively quartering the concentration of each duplex), that has been shown to greatly diminish off-target effects (42)(43)(44)(45)(46). Using fixed and live cell HC-DIM, we showed that diminished FEN1 expression adversely affects overall cell numbers, which presumably occurs through the corresponding increases in cellular cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

McManus

Barrett

Nouhi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

116

130

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Mutations that cause chromosome instability (CIN) in cancer cells produce ''sublethal'' deficiencies in an essential process (chromosome segregation) and, therefore, may represent a major untapped resource that could be exploited for therapeutic benefit in the treatment of cancer. If second-site unlinked genes can be identified, that when knocked down, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, novel candidate therapeutic targets will be identified. To test this idea, we took a cross species SL candidate gene approach by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockdown of the highly sequence-and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We show that knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes CIN in mammalian cells. Using high-content microscopy techniques, we demonstrate that RAD54B-deficient human colorectal cancer cells are sensitive to SL killing by reduced FEN1 expression, while isogenic RAD54B proficient cells are not. This conserved SL interaction suggests that extrapolating SL interactions observed in model organisms for homologous genes mutated in human cancers will aid in the identification of novel therapeutic targets for specific killing of cancerous cells exhibiting CIN.cancer therapeutics ͉ chromosome instability ͉ synthetic lethality

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“…To reduce off-target effects, these siRNAs have dual strand modification (18) and use an algorithm to minimize seed region matches (19). The Dharmafect 4 transfection reagent was used at final concentration of 0.1-0.2 mL per well (100 mL total volume) for MCF-7 and HC11 cells and 0.05 mL per well for MDA-MB-231 cells.…”

Section: Sirnamentioning

confidence: 99%

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

McAndrew

Grice

Peters

et al. 2011

Molecular Cancer Therapeutics

194

206

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The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced storeoperated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1 high /STIM2 low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448-60. Ó2011 AACR.

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Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing

Cited by 706 publications

References 32 publications

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes

Specific synthetic lethal killing of RAD54B-deficient human colorectal cancer cells by FEN1 silencing

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

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