Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Morin, Aurélie; Gallou-Kabani, Catherine; Mathieu, Jacques; Cabon, Florence

doi:10.2174/156802609789630820

Cited by 16 publications

(23 citation statements)

References 122 publications

(161 reference statements)

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“…Because of this, the total siRNA in tissues does not always correlate with efficacy (Abrams et al 2010). Imaging analysis may yield relevant information on subcellular siRNA delivery, but it is not always precise in quantification and it cannot distinguish between intact siRNA and the free labels released from siRNA by metabolism (Morin et al 2009). …”

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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Section: Introductionmentioning

confidence: 99%

Quantitative evaluation of siRNA delivery in vivo

Pei

Hancock²,

Zhang³

et al. 2010

RNA

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“…Human clinical trials using synthetic siRNA started in 2004 (Soutschek et al 2004), and a list of ongoing clinical trials applying siRNA was summarized (Morin et al 2009). The first siRNA clinical trial that used a targeted nanoparticle delivery system was reported (Davis et al 2010) in which a human transferrin protein targeting ligand was displayed on the exterior of a polymer nanoparticle to engage transferrin receptors of patients with melanoma after systemic administration.…”

mentioning

confidence: 99%

Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

Shu

Keough

Matter

et al. 2011

J Histochem Cytochem.

106

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Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles.

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“…43 The mechanisms for greater uptake of siRNA into tissue cells in vivo, compared with ineffectiveness in cultured cells, are not known, but could involve serum proteins or components of the complex extracellular matrices in tissues. 43 However, peptide C6M3 and C6M6 as carrier for siRNA could promote the antitumor effect further and provide better protection against enzymatic degradation. On day 27, mice were sacrificed and tumor tissues were excised (Figures 5c and 5d).…”

Section: Resultsmentioning

confidence: 99%

Design and Evaluation of Endosomolytic Biocompatible Peptides as Carriers for siRNA Delivery

Ran

Zhao

et al. 2014

Mol. Pharmaceutics

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Gene therapy using RNA interference (RNAi) technology has been explored to treat cancers, by regulating the expression of oncogene. However, even though small interfering RNA (siRNA), which triggers RNAi, may have great therapeutic potential, efforts at using them in vivo have been hampered by the difficulty of effective and safe delivery into cells of interest. In this study, to develop a safe and efficient carrier for in vitro and in vivo siRNA delivery, we designed a peptide library. These peptides are improved variants of a known peptide based siRNA carrier C6. All the modifications improved the transfection efficiency of C6 to some degree. After completing prescreening for activity, several promising candidates were used for further evaluation. Selected peptides C6M3 and C6M6 could form stable complexes with siRNA. These complexes could be greatly uptaken by cells and showed a punctate perinuclear distribution. Moreover, peptide/siRNA complexes achieved high transfection efficiency in vitro without inducing substantial cytotoxicity. We have validated the therapeutic potential of this strategy for cancer treatment by targeting Bcl-2 gene in mouse tumor models, and demonstrated that tumor growth was inhibited. In order to address possible immune side effects of these peptide carriers, biocompatibility study in terms of complement activation and cytokine activation assay were carried out, whereas none of the peptides induced such effects. In conclusion, these results support the potential of these peptides as therapeutic siRNA carrier.

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Systemic Delivery and Quantification of Unformulated Interfering RNAs In Vivo

Cited by 16 publications

References 122 publications

Quantitative evaluation of siRNA delivery in vivo

Quantitative evaluation of siRNA delivery in vivo

Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

Design and Evaluation of Endosomolytic Biocompatible Peptides as Carriers for siRNA Delivery

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