2015
DOI: 10.1038/nbt.3298
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Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown

Abstract: A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA rel… Show more

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Cited by 461 publications
(579 citation statements)
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“…The mechanism of endosomal escape of lipid reagents like Lipofectamine 2000 is not fully established, but recent reports have shown that the vast majority of lipid nanoparticles still reach lysosomes and escape is a relatively rare event. 39,40 Overall, we detected only minimal differences in the calculated pH between DsRed + and DsRed À populations of cells transfected with Lipofectamine 2000 and did not find any correlation between nanoparticle uptake and transfection or nanoparticle uptake and pH. That being said, acidic pH (4.0-6.0) correlated with a slightly decreased frequency of successful transfection (DsRed À > DsRed + ), whereas higher pH (7.0-8.0) weakly correlated with a higher frequency of successful transfection (DsRed + > DsRed À ).…”
Section: Application To Non-polymeric Non-viral Nanoparticlesmentioning
confidence: 99%
See 1 more Smart Citation
“…The mechanism of endosomal escape of lipid reagents like Lipofectamine 2000 is not fully established, but recent reports have shown that the vast majority of lipid nanoparticles still reach lysosomes and escape is a relatively rare event. 39,40 Overall, we detected only minimal differences in the calculated pH between DsRed + and DsRed À populations of cells transfected with Lipofectamine 2000 and did not find any correlation between nanoparticle uptake and transfection or nanoparticle uptake and pH. That being said, acidic pH (4.0-6.0) correlated with a slightly decreased frequency of successful transfection (DsRed À > DsRed + ), whereas higher pH (7.0-8.0) weakly correlated with a higher frequency of successful transfection (DsRed + > DsRed À ).…”
Section: Application To Non-polymeric Non-viral Nanoparticlesmentioning
confidence: 99%
“…14 In recent studies, the rarity of endosomal escape of lipid nanoparticles as well as polymeric nanoparticles has been likewise well documented. 39,40 These studies in particular have noted the difficulty in using fluorescence-based imaging for tracking labeled molecules or particles that have successfully escaped the endosome, because the fraction of total fluorescence detectable in the cytosol is very low. With this in mind, we have chosen to focus on the specific barrier to transfection of endosomal escape in our creation of a new tool.…”
Section: Ymthe 4338mentioning
confidence: 99%
“…This principle was elegantly explored to develop an endosomal escape assay based on the (de)quenching of small oligonucleotide fragments 168 . By simultaneously labeling different types of endosomal vesicles, the specific type of endosomes from which complexes are preferentially released has been recently identified 169 . Apart from single color-labeling, both the carrier and nucleic acids can be labeled with spectrally separated fluorophores to obtain dual-colored complexes 170 .…”
Section: Lighting Up the Intracellular Delivery Path Of Pdna And Mrnamentioning
confidence: 99%
“…Consequently, since its discovery in the late 1990s, RNAi-based therapeutics have been used in more than 50 clinical trials involving 26 different siRNAs (Ling et al 2013;Lundin et al 2015;Wittrup et al 2015). Two phase III clinical trials are in progress to treat familial neurodegenerative and cardiac syndromes caused by transthyretin mutations (Singh and Peer 2016) while studies on the potential of miRNA replacements or miRNA inhibitions to reduce hypertrophic dermal scarring by targeting connective tissue growth factor or to treat pancreatic cancer have also started (Singh and Peer 2016).…”
Section: Substance-induced Alterations In Ncrna Expression Profilesmentioning
confidence: 99%
“…Two phase III clinical trials are in progress to treat familial neurodegenerative and cardiac syndromes caused by transthyretin mutations (Singh and Peer 2016) while studies on the potential of miRNA replacements or miRNA inhibitions to reduce hypertrophic dermal scarring by targeting connective tissue growth factor or to treat pancreatic cancer have also started (Singh and Peer 2016). Outstanding issues in ncRNA therapy, however, are targeted RNA-drug delivery to specific organs, specificity, efficacy and absence of side effects (Wittrup et al 2015). Northern blotting has very low throughput and sensitivity, it can often detect both mature and precursor forms of miRNA.…”
Section: Substance-induced Alterations In Ncrna Expression Profilesmentioning
confidence: 99%