Assessing siRNA Pharmacodynamics in a Luciferase-expressing Mouse

Svensson, Robert; Shey, Michael R.; Ballas, Zuhair K.; Dorkin, J. Robert; Goldberg, Michael S.; Akinc, Akin; Langer, Robert; Anderson, Daniel G.; Bumcrot, David; Henry, Michael D.

doi:10.1038/mt.2008.187

Cited by 24 publications

(23 citation statements)

References 36 publications

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“…Using formulated siRNAs in vivo could thus provide a mechanism of slow release for siRNAs, ensuring their continuous loading into the RISC complex and avoiding peaks and valleys in the mRNA expression. Interestingly, formulating siRNAs can modify their biodistribution, providing a degree of targeting to a specific organ [5,107]. Some formulations were also used for rectal [54] or oral [108] delivery.…”

Section: Lessons From Naked Rnai In Vivomentioning

confidence: 99%

Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Morin¹,

Gallou-Kabani²,

Mathieu³

et al. 2009

CTMC

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Synthetic small interfering RNAs (siRNAs) open promising new therapeutic perspectives in acute and chronic pathologies. A number of experiments in mice demonstrated the ability of naked siRNAs injected under a normal pressure to trigger gene silencing in vivo, translating into a measurable phenotype. We focus in this review on the information that we can gain from these experiments, and discuss how the specificity of the gene silencing in vivo can be controlled. Because the activity of most drugs increases with the dosing, we are prone to consider that increasing the concentration of siRNAs within cells enhances the efficiency and the duration of the silencing. However, because RNAi is a saturable process, and because increasing the siRNA concentration into cells can induce undesirable side effects, this must be demonstrated. We compare in this review the methods used to quantify and study the biodistribution of siRNAs in living animals, and discuss how these methods can help in designing for each model and each siRNA the most adequate protocol to silence a cognate target gene in vivo.

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Section: Lessons From Naked Rnai In Vivomentioning

confidence: 99%

Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Morin¹,

Gallou-Kabani²,

Mathieu³

et al. 2009

CTMC

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“…48 Sense strand: 5'-cuuAcGcuGAGuAcuucGAT*T-3' in a white 96-well plate at 37°C under 5% CO 2 conditions. Culturing was initiated by the seeding of 2,000 cells per well.…”

Section: Knockdown Assay Using Sirna-aunp Conjugatesmentioning

confidence: 99%

Amphiphilic Gold Nanoparticles Displaying Flexible Bifurcated Ligands as a Carrier for siRNA Delivery into the Cell Cytosol

Niikura

Kobayashi

Takeuchi

et al. 2014

ACS Appl. Mater. Interfaces

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The nanoparticle-based delivery of siRNA with a noncationic outermost surface at a low particle concentration is greatly desired. We newly synthesized a bifurcated ligand (BL) possessing hydrophobic and hydrophilic arms as a surface ligand for gold nanoparticles (AuNPs) to allow siRNA delivery. The concept underlying the design of this ligand is that amphiphilic property should allow AuNPs to permeate the cell cytosol thorough the endosomal membrane. BLs and quaternary cationic ligands were codisplayed on 40 nm AuNPs, which were subsequently coated with siRNA via electrostatic interaction. The number of siRNAs immobilized on a single nanoparticle was 26, and the conjugate showed a negative zeta potential due to siRNAs on the outermost surface of the AuNPs. Apparent gene silencing of luciferase expression in HeLa cells was achieved at an AuNP concentration as low as 60 pM. Almost no gene silencing was observed for AuNPs not displaying BLs. To reveal the effect of the BL, we compared the number of AuNPs internalized into HeLa cells and the localization in the cytosol between AuNPs displaying and those not displaying BLs. These analyses indicated that the role of BLs is not only the simple promotion of cellular uptake but also involves the enhancement of AuNPs permeation into the cytosol from the endosomes, leading to effective gene silencing.

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“…Reduction in the target mRNA/protein level provides a critical measure of siRNA pharmacodynamics (Svensson et al 2008) but provides a relatively indirect measure of cellular delivery and may be complicated by unidentified target sequence polymorphism and off-target effects not related to RNAi (Kleinman et al 2008). In addition, because reduction of target expression can reach a steady state at elevated doses, it is often necessary to perform resource-intensive dose titrations to differentiate siRNAs or vehicles.…”

Section: Introductionmentioning

confidence: 99%

Quantitative evaluation of siRNA delivery in vivo

Pei

Hancock²,

Zhang³

et al. 2010

RNA

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Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.

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Assessing siRNA Pharmacodynamics in a Luciferase-expressing Mouse

Cited by 24 publications

References 36 publications

Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Amphiphilic Gold Nanoparticles Displaying Flexible Bifurcated Ligands as a Carrier for siRNA Delivery into the Cell Cytosol

Quantitative evaluation of siRNA delivery in vivo

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