FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2809145613> ?p ?o ?g. }

• W2809145613 endingPage "1982" @default.
• W2809145613 startingPage "1973" @default.
• W2809145613 abstract "Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5′-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5′-(E)-vinylphosphonate and alternating 2′-fluoro and 2′-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles. Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5′-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5′-(E)-vinylphosphonate and alternating 2′-fluoro and 2′-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles. Extracellular vesicles are being explored for therapeutic RNA delivery due to (1) their small size (50–150 nm) allowing penetration through some biological barriers,1Grapp M. Wrede A. Schweizer M. Hüwel S. Galla H.J. Snaidero N. Simons M. Bückers J. Low P.S. Urlaub H. et al.Choroid plexus transcytosis and exosome shuttling deliver folate into brain parenchyma.Nat. Commun. 2013; 4: 2123Crossref PubMed Scopus (212) Google Scholar, 2Yang T. Martin P. Fogarty B. Brown A. Schurman K. Phipps R. Yin V.P. Lockman P. Bai S. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.Pharm. Res. 2015; 32: 2003-2014Crossref PubMed Scopus (644) Google Scholar (2) their unique protein composition enabling target cell specificity,3Hoshino A. Costa-Silva B. Shen T.L. Rodrigues G. Hashimoto A. Tesic Mark M. Molina H. Kohsaka S. Di Giannatale A. Ceder S. et al.Tumour exosome integrins determine organotropic metastasis.Nature. 2015; 527: 329-335Crossref PubMed Scopus (2928) Google Scholar, 4Haraszti R.A. Didiot M.C. Sapp E. Leszyk J. Shaffer S.A. Rockwell H.E. Gao F. Narain N.R. DiFiglia M. Kiebish M.A. et al.High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources.J. Extracell. Vesicles. 2016; 5: 32570Crossref PubMed Scopus (406) Google Scholar and (3) their natural capacity to transfer RNA between cells.5Valadi H. Ekström K. Bossios A. Sjöstrand M. Lee J.J. Lötvall J.O. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat. Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8923) Google Scholar, 6Zomer A. Maynard C. Verweij F.J. Kamermans A. Schäfer R. Beerling E. Schiffelers R.M. de Wit E. Berenguer J. Ellenbroek S.I.J. et al.In Vivo imaging reveals extracellular vesicle-mediated phenocopying of metastatic behavior.Cell. 2015; 161: 1046-1057Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar Mesenchymal stem cell-derived extracellular vesicles can deliver therapeutic miRNAs to brain.7Xin H. Li Y. Buller B. Katakowski M. Zhang Y. Wang X. Shang X. Zhang Z.G. Chopp M. Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth.Stem Cells. 2012; 30: 1556-1564Crossref PubMed Scopus (657) Google Scholar, 8Xin H. Li Y. Liu Z. Wang X. Shang X. Cui Y. Zhang Z.G. Chopp M. MiR-133b promotes neural plasticity and functional recovery after treatment of stroke with multipotent mesenchymal stromal cells in rats via transfer of exosome-enriched extracellular particles.Stem Cells. 2013; 31: 2737-2746Crossref PubMed Scopus (519) Google Scholar, 9Lee H.K. Finniss S. Cazacu S. Xiang C. Brodie C. Mesenchymal stem cells deliver exogenous miRNAs to neural cells and induce their differentiation and glutamate transporter expression.Stem Cells Dev. 2014; 23: 2851-2861Crossref PubMed Scopus (91) Google Scholar, 10Cui C. Ye X. Chopp M. Venkat P. Zacharek A. Yan T. Ning R. Yu P. Cui G. Chen J. miR-145 Regulates Diabetes-Bone Marrow Stromal Cell-Induced Neurorestorative Effects in Diabetes Stroke Rats.Stem Cells Transl. Med. 2016; 5: 1656-1667Crossref PubMed Scopus (48) Google Scholar, 11Xin H. Wang F. Li Y. Lu Q.E. Cheung W.L. Zhang Y. Zhang Z.G. Chopp M. Secondary Release of Exosomes From Astrocytes Contributes to the Increase in Neural Plasticity and Improvement of Functional Recovery After Stroke in Rats Treated With Exosomes Harvested From MicroRNA 133b-Overexpressing Multipotent Mesenchymal Stromal Cells.Cell Transplant. 2017; 26: 243-257PubMed Google Scholar, 12Zhang Y. Chopp M. Liu X.S. Katakowski M. Wang X. Tian X. Wu D. Zhang Z.G. Exosomes Derived from Mesenchymal Stromal Cells Promote Axonal Growth of Cortical Neurons.Mol. Neurobiol. 2017; 54: 2659-2673Crossref PubMed Scopus (180) Google Scholar Small interfering RNAs (siRNAs), similar to therapeutic microRNAs (miRNAs), are capable of selective gene silencing.13Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature. 1998; 391: 806-811Crossref PubMed Scopus (11769) Google Scholar Thus, siRNAs offer a therapeutic option for genetically defined diseases, such as Huntington’s disease. However, delivery to target tissues remains the bottleneck for clinical application of therapeutic RNAs, including siRNAs. Extracellular vesicles represent a strategy to overcome the delivery challenge.14Didiot M.-C. Hall L.M. Coles A.H. Haraszti R.A. Godinho B.M.D.C. Chase K. Sapp E. Ly S. Alterman J.F. Hassler M.R. et al.Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.Mol. Ther. 2016; 24: 1836-1847Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 15Kamerkar S. LeBleu V.S. Sugimoto H. Yang S. Ruivo C.F. Melo S.A. Lee J.J. Kalluri R. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.Nature. 2017; 546: 498-503Crossref PubMed Scopus (1305) Google Scholar Cholesterol conjugation-mediated loading of siRNAs onto extracellular vesicles is among the most reproducible and scalable loading strategies,14Didiot M.-C. Hall L.M. Coles A.H. Haraszti R.A. Godinho B.M.D.C. Chase K. Sapp E. Ly S. Alterman J.F. Hassler M.R. et al.Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.Mol. Ther. 2016; 24: 1836-1847Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16O’Loughlin A.J. Mäger I. de Jong O.G. Varela M.A. Schiffelers R.M. El Andaloussi S. Wood M.J.A. Vader P. Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.Mol. Ther. 2017; 25: 1580-1587Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Stremersch S. Vandenbroucke R.E. Van Wonterghem E. Hendrix A. De Smedt S.C. Raemdonck K. Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs.J. Control. Release. 2016; 232: 51-61Crossref PubMed Scopus (92) Google Scholar characterized by efficient transfer of the loaded cholesterol-siRNA to target cells. However, productive gene silencing induced by the transferred cholesterol-siRNA was variable. We speculate that these variations are due to differences in siRNA chemical modification patterns (45%–71% of riboses modified), cholesterol placement position (5′ or 3′ of the sense strand), siRNA-to-extracellular vesicle loading ratio (100s versus 1,000s of siRNAs per extracellular vesicle), and siRNA concentrations used in silencing studies (∼50–1,500 nM).14Didiot M.-C. Hall L.M. Coles A.H. Haraszti R.A. Godinho B.M.D.C. Chase K. Sapp E. Ly S. Alterman J.F. Hassler M.R. et al.Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.Mol. Ther. 2016; 24: 1836-1847Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16O’Loughlin A.J. Mäger I. de Jong O.G. Varela M.A. Schiffelers R.M. El Andaloussi S. Wood M.J.A. Vader P. Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.Mol. Ther. 2017; 25: 1580-1587Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Stremersch S. Vandenbroucke R.E. Van Wonterghem E. Hendrix A. De Smedt S.C. Raemdonck K. Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs.J. Control. Release. 2016; 232: 51-61Crossref PubMed Scopus (92) Google Scholar All studies used a version of pyrimidine-modified siRNAs, which have been shown to provide stabilization against nucleases in vitro in serum.18Byrne M. Tzekov R. Wang Y. Rodgers A. Cardia J. Ford G. Holton K. Pandarinathan L. Lapierre J. Stanney W. et al.Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.J. Ocul. Pharmacol. Ther. 2013; 29: 855-864Crossref PubMed Scopus (52) Google Scholar, 19Leake, D., Reynolds, A., Khvorova, A., Marshall, W., and Scaringe, S. (2004). Stabilized polynucleotides for use in RNA interference. U.S. patent, WO/2004/090105.Google Scholar, 20Soutschek J. Akinc A. Bramlage B. Charisse K. Constien R. Donoghue M. Elbashir S. Geick A. Hadwiger P. Harborth J. et al.Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.Nature. 2004; 432: 173-178Crossref PubMed Scopus (1922) Google Scholar Cholesterol-conjugated, but not chemically modified, siRNAs have been shown to degrade in the presence of extracellular vesicles due to endogenous RNase activity.21Stremersch S. Brans T. Braeckmans K. De Smedt S. Raemdonck K. Nucleic acid loading and fluorescent labeling of isolated extracellular vesicles requires adequate purification.Int. J. Pharm. 2017; (Published online October 12, 2017)https://doi.org/10.1016/j.ijpharm.2017.10.022Crossref PubMed Scopus (17) Google Scholar Advances in oligonucleotide chemistry have enabled the expansion of siRNA use from in vitro serum-rich environments to systemic delivery in vivo.18Byrne M. Tzekov R. Wang Y. Rodgers A. Cardia J. Ford G. Holton K. Pandarinathan L. Lapierre J. Stanney W. et al.Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.J. Ocul. Pharmacol. Ther. 2013; 29: 855-864Crossref PubMed Scopus (52) Google Scholar, 22Nair J.K. Willoughby J.L. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (672) Google Scholar, 23Allerson C.R. Sioufi N. Jarres R. Prakash T.P. Naik N. Berdeja A. Wanders L. Griffey R.H. Swayze E.E. Bhat B. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.J. Med. Chem. 2005; 48: 901-904Crossref PubMed Scopus (371) Google Scholar, 24Choung S. Kim Y.J. Kim S. Park H.O. Choi Y.C. Chemical modification of siRNAs to improve serum stability without loss of efficacy.Biochem. Biophys. Res. Commun. 2006; 342: 919-927Crossref PubMed Scopus (272) Google Scholar, 25Czauderna F. Fechtner M. Dames S. Aygün H. Klippel A. Pronk G.J. Giese K. Kaufmann J. Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells.Nucleic Acids Res. 2003; 31: 2705-2716Crossref PubMed Scopus (543) Google Scholar In particular, siRNAs with modification of all riboses22Nair J.K. Willoughby J.L. Chan A. Charisse K. Alam M.R. Wang Q. Hoekstra M. Kandasamy P. Kel’in A.V. Milstein S. et al.Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing.J. Am. Chem. Soc. 2014; 136: 16958-16961Crossref PubMed Scopus (672) Google Scholar, 23Allerson C.R. Sioufi N. Jarres R. Prakash T.P. Naik N. Berdeja A. Wanders L. Griffey R.H. Swayze E.E. Bhat B. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.J. Med. Chem. 2005; 48: 901-904Crossref PubMed Scopus (371) Google Scholar, 26Coelho T. Adams D. Silva A. Lozeron P. Hawkins P.N. Mant T. Perez J. Chiesa J. Warrington S. Tranter E. et al.Safety and efficacy of RNAi therapy for transthyretin amyloidosis.N. Engl. J. Med. 2013; 369: 819-829Crossref PubMed Scopus (734) Google Scholar, 27Fitzgerald K. White S. Borodovsky A. Bettencourt B.R. Strahs A. Clausen V. Wijngaard P. Horton J.D. Taubel J. Brooks A. et al.A Highly Durable RNAi Therapeutic Inhibitor of PCSK9.N. Engl. J. Med. 2017; 376: 41-51Crossref PubMed Scopus (235) Google Scholar, 28Nikan M. Osborn M.F. Coles A.H. Godinho B.M. Hall L.M. Haraszti R.A. Hassler M.R. Echeverria D. Aronin N. Khvorova A. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain.Mol. Ther. Nucleic Acids. 2016; 5: e344Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 29Alterman J.F. Hall L.M. Coles A.H. Hassler M.R. Didiot M.C. Chase K. Abraham J. Sottosanti E. Johnson E. Sapp E. et al.Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain.Mol. Ther. Nucleic Acids. 2015; 4: e266Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar (with 2′-fluoro and 2′-O-methyl) were 10,000-fold more active in vivo30Hassler M.R. Turanov A.A. Alterman J.F. Haraszti R.A. Coles A.H. Osborn M.F. Echeverria D. Nikan M. Salomon W.E. Roux L. et al.Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo.Nucleic Acids Res. 2018; 46: 2185-2196Crossref PubMed Scopus (86) Google Scholar than partially modified siRNAs similar to siRNAs originally used for extracellular vesicle loading.14Didiot M.-C. Hall L.M. Coles A.H. Haraszti R.A. Godinho B.M.D.C. Chase K. Sapp E. Ly S. Alterman J.F. Hassler M.R. et al.Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.Mol. Ther. 2016; 24: 1836-1847Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16O’Loughlin A.J. Mäger I. de Jong O.G. Varela M.A. Schiffelers R.M. El Andaloussi S. Wood M.J.A. Vader P. Functional Delivery of Lipid-Conjugated siRNA by Extracellular Vesicles.Mol. Ther. 2017; 25: 1580-1587Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Stremersch S. Vandenbroucke R.E. Van Wonterghem E. Hendrix A. De Smedt S.C. Raemdonck K. Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs.J. Control. Release. 2016; 232: 51-61Crossref PubMed Scopus (92) Google Scholar A second type of modification, 5′-(E)-vinylphosphonate, also improved the activity of systemically administered conjugated siRNAs.31Prakash T.P. Kinberger G.A. Murray H.M. Chappell A. Riney S. Graham M.J. Lima W.F. Swayze E.E. Seth P.P. Synergistic effect of phosphorothioate, 5′-vinylphosphonate and GalNAc modifications for enhancing activity of synthetic siRNA.Bioorg. Med. Chem. Lett. 2016; 26: 2817-2820Crossref PubMed Scopus (42) Google Scholar, 32Parmar R. Willoughby J.L. Liu J. Foster D.J. Brigham B. Theile C.S. Charisse K. Akinc A. Guidry E. Pei Y. et al.5′-(E)-Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA-GalNAc Conjugates.ChemBioChem. 2016; 17: 985-989Crossref PubMed Scopus (86) Google Scholar, 33Haraszti R.A. Roux L. Coles A.H. Turanov A.A. Alterman J.F. Echeverria D. Godinho B.M.D.C. Aronin N. Khvorova A. 5′-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.Nucleic Acids Res. 2017; 45: 7581-7592Crossref PubMed Scopus (58) Google Scholar However, full chemical modification did not affect the activity of non-conjugated siRNAs delivered in cationic liposomes.24Choung S. Kim Y.J. Kim S. Park H.O. Choi Y.C. Chemical modification of siRNAs to improve serum stability without loss of efficacy.Biochem. Biophys. Res. Commun. 2006; 342: 919-927Crossref PubMed Scopus (272) Google Scholar, 34Morrissey D.V. Lockridge J.A. Shaw L. Blanchard K. Jensen K. Breen W. Hartsough K. Machemer L. Radka S. Jadhav V. et al.Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat. Biotechnol. 2005; 23: 1002-1007Crossref PubMed Scopus (1009) Google Scholar, 35Olearczyk J. Gao S. Eybye M. Yendluri S. Andrews L. Bartz S. Cully D. Tadin-Strapps M. Targeting of hepatic angiotensinogen using chemically modified siRNAs results in significant and sustained blood pressure lowering in a rat model of hypertension.Hypertens. Res. 2014; 37: 405-412Crossref PubMed Scopus (26) Google Scholar Extracellular vesicle-mediated delivery of hydrophobically modified asymmetric siRNAs (hsiRNAs) combines principles of siRNA conjugation and lipid nanoparticle technology. The impact of siRNA chemical modifications on efficacy of extracellular vesicle-mediated delivery is, therefore, difficult to predict and remains unknown. Among many synthetic approaches on cholesterol attachment to the siRNA, TEG (triethyl glycol) and C7 (2-aminobutyl-1-3-propanediol) linkers are frequently used and commercially available. In the amino linker class, the C7 linker was optimal for siRNA passive uptake.36Petrova N.S. Chernikov I.V. Meschaninova M.I. Dovydenko I.S. Venyaminova A.G. Zenkova M.A. Vlassov V.V. Chernolovskaya E.L. Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group.Nucleic Acids Res. 2012; 40: 2330-2344Crossref PubMed Scopus (68) Google Scholar Despite the common use of both linkers, no systematic comparison has been published to date. Here we evaluated the impact of siRNA chemical modification patterns, cholesterol attachment via different linkers, and siRNA-to-extracellular vesicle loading ratio on functional extracellular vesicle-mediated delivery of siRNAs. We used siRNA concentrations ranging from 23 to 1,500 nM in all experiments. We used mesenchymal stem cells (derived from umbilical cord Wharton’s jelly) as extracellular vesicle producer cells, because they have been proven safe in numerous clinical trials,37Reiner A.T. Witwer K.W. van Balkom B.W.M. de Beer J. Brodie C. Corteling R.L. Gabrielsson S. Gimona M. Ibrahim A.G. de Kleijn D. et al.Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.Stem Cells Transl. Med. 2017; 6: 1730-1739Crossref PubMed Scopus (192) Google Scholar, 38Lener T. Gimona M. Aigner L. Börger V. Buzas E. Camussi G. Chaput N. Chatterjee D. Court F.A. Del Portillo H.A. et al.Applying extracellular vesicles based therapeutics in clinical trials - an ISEV position paper.J. Extracell. Vesicles. 2015; 4: 30087Crossref PubMed Scopus (809) Google Scholar provide higher extracellular vesicle yield compared to other origins of mesenchymal stem cells (R.A.H., unpublished data), and mesenchymal stem cells have been shown to be beneficial in Huntington’s disease,39Lee M. Liu T. Im W. Kim M. Exosomes from adipose-derived stem cells ameliorate phenotype of Huntington’s disease in vitro model.Eur. J. Neurosci. 2016; 44: 2114-2119Crossref PubMed Scopus (80) Google Scholar the disease model used in this study. To compare two commercially available strategies to conjugate cholesterol to siRNAs (TEG and C7 linkers), we used a previously developed asymmetric siRNA scaffold,18Byrne M. Tzekov R. Wang Y. Rodgers A. Cardia J. Ford G. Holton K. Pandarinathan L. Lapierre J. Stanney W. et al.Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.J. Ocul. Pharmacol. Ther. 2013; 29: 855-864Crossref PubMed Scopus (52) Google Scholar, 40Alterman J.F. Hall L.M. Coles A.H. Hassler M.R. Didiot M.C. Chase K. Abraham J. Sottosanti E. Johnson E. Sapp E. et al.Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain.Mol. Ther. Nucleic Acids. 2015; 4: e266Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar characterized by a short duplex region (15 bp) and a fully phosphorothioated tail assisting membrane association18Byrne M. Tzekov R. Wang Y. Rodgers A. Cardia J. Ford G. Holton K. Pandarinathan L. Lapierre J. Stanney W. et al.Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.J. Ocul. Pharmacol. Ther. 2013; 29: 855-864Crossref PubMed Scopus (52) Google Scholar, 41Ly S. Navaroli D.M. Didiot M.C. Cardia J. Pandarinathan L. Alterman J.F. Fogarty K. Standley C. Lifshitz L.M. Bellve K.D. et al.Visualization of self-delivering hydrophobically modified siRNA cellular internalization.Nucleic Acids Res. 2017; 45: 15-25Crossref PubMed Scopus (63) Google Scholar, 42Geary R.S. Norris D. Yu R. Bennett C.F. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.Adv. Drug Deliv. Rev. 2015; 87: 46-51Crossref PubMed Scopus (499) Google Scholar (hsiRNAs). hsiRNAs are either partially modified with 2′-fluoro pyrimidines on the antisense strand and 2′-O-methyl pyrimidines on the sense strand, or they are fully modified using alternating 2′-O-methyl and 2′-fluoro pattern providing endonuclease stability and protection from innate immune response.23Allerson C.R. Sioufi N. Jarres R. Prakash T.P. Naik N. Berdeja A. Wanders L. Griffey R.H. Swayze E.E. Bhat B. Fully 2′-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA.J. Med. Chem. 2005; 48: 901-904Crossref PubMed Scopus (371) Google Scholar, 43Nallagatla S.R. Bevilacqua P.C. Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner.RNA. 2008; 14: 1201-1213Crossref PubMed Scopus (106) Google Scholar, 44Jackson A.L. Burchard J. Leake D. Reynolds A. Schelter J. Guo J. Johnson J.M. Lim L. Karpilow J. Nichols K. et al.Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing.RNA. 2006; 12: 1197-1205Crossref PubMed Scopus (654) Google Scholar We synthesized fully modified cholesterol-hsiRNAs targeting Huntingtin mRNA40Alterman J.F. Hall L.M. Coles A.H. Hassler M.R. Didiot M.C. Chase K. Abraham J. Sottosanti E. Johnson E. Sapp E. et al.Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain.Mol. Ther. Nucleic Acids. 2015; 4: e266Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar using either TEG or a C7 linkers (Figure 1). Cholesterol-hsiRNA variants were loaded onto extracellular vesicles at increasing hsiRNA-to-extracellular vesicle ratios (Figure 1A) Both variants showed efficient loading onto extracellular vesicles with saturation kinetics (Figure 1A). Cholesterol-TEG-hsiRNAs loaded more efficiently onto extracellular vesicles than cholesterol-C7-hsiRNAs at all ratios tested (Figure 1A; p = 0.0059). More efficient loading led to more potent Huntingtin mRNA silencing, when primary neurons were treated with extracellular vesicles loaded to saturation (cholesterol-TEG IC50 ∼8 × 106 extracellular vesicles, cholesterol-C7 IC50 ∼22 × 106 extracellular vesicles; p = 0.0008; Figure 1B). hsiRNA uptake into neurons was confirmed using peptide-nucleic acid (PNA) hybridization assay (Figure S3E). Normalization to hsiRNA content eliminated the observed differences (p > 0.05; Figure 1C) in silencing. Thus, silencing potency of the two hsiRNA variants was the same. Improved silencing activity upon extracellular vesicle-mediated delivery could be fully explained by better loading of cholesterol-TEG-hsiRNA onto extracellular vesicles. Therefore, cholesterol-TEG-hsiRNA was used for subsequent experiments. Contrary to conventional siRNA extracellular vesicle-loading approaches (i.e., electroporation or overexpression in parent cells), hydrophobic modifications of siRNA enable the association of a large number of RNA molecules per extracellular vesicle. To define an optimal hsiRNA-to-extracellular vesicle ratio, i.e., ratio supporting productive target mRNA silencing, we evaluated the effect of hsiRNA concentration during the loading process (1,000 to 100,000 hsiRNA copies per extracellular vesicle added to the loading mixture). The addition of 6,000 hsiRNAs per extracellular vesicle into the loading mixture resulted in ∼2,600 hsiRNAs associated per vesicle (Figure 2A), leading to 43% loading efficiency (Figure 2B). A further increase in the amount of hsiRNAs added to the loading mixture (9,000 and 12,000 per vesicle) did not support an increase in the amount of extracellular vesicle-associated hsiRNAs (Figure 2A), indicating a level of intermediate saturation at ∼2,500–3000 hsiRNAs per vesicle. As the amount of loaded hsiRNAs stayed constant, the estimated loading efficiency decreased from 43% to 23% (Figure 2B). Following this initial saturation phase, we observed a linear increase in the amount of hsiRNAs loaded per extracellular vesicle, starting at approximately 20,000 hsiRNA per extracellular vesicle added to the loading mixture, representing a constant loading efficiency of 18% (Figure 2B). Transmission electron microscopy showed similar lipid bilayer-surrounded vesicles post-loading at hsiRNA-to-extracellular vesicle ratios below the initial saturation phase (3,000), at the initial saturation phase (10,000), and in the linear increase phase (100,000) (Figure 2A). Increasing hsiRNA-to-extracellular vesicle loading ratio beyond 20,000 resulted in an increased total particle number (Figure 2C). Loading hsiRNAs onto extracellular vesicles did not alter the protein content (Figures S2A–S2C) or size (Figure S2D) of vesicles. The same phenomenon of initial saturation phase followed by a linear increase phase was observed when loading cholesterol-C7-hsiRNA to extracellular vesicles (Figure S3B) or both cholesterol-TEG-hsiRNA (Figure S3C) and cholesterol-C7-hsiRNA (Figure S3D) to conventional liposomes. During saturation phase, more hsiRNAs could be loaded onto liposomes than to extracellular vesicles (Figures S3C and S3D), suggesting that the protein content of extracellular vesicle membrane may interfere with hsiRNA loading. The formation of extra particles in the presence of vesicles may be explained by hsiRNA aggregation. Next, we evaluated how hsiRNA-to-extracellular vesicle ratio affected the ability of loaded extracellular vesicles to silence Huntingtin mRNA in primary neurons. 3,000, 10,000, and 100, 000 hsiRNAs (per vesicle) were added to the loading mixture, generating extracellular vesicles with 1,000, 3,000, and 18,000 RNA molecules loaded per vesicle, respectively. From three hsiRNA-to-extracellular vesicle ratios tested, extracellular vesicles containing 3,000 hsiRNAs per vesicle performed the best with an IC50 of 37 nM (Figure 2D). In contrast, extracellular vesicles underloaded (1,000 hsiRNAs per extracellular vesicle) or overloaded (18,000 hsiRNAs per extracellular vesicle) were less efficient in Huntingtin mRNA silencing (IC50 1,330 nM and 1,164 nM, respectively) (Figure 2D). As extracellular vesicles loaded with 3,000 hsiRNAs (saturation level) were 36-fold more potent than underloaded and 31-fold more potent than overloaded extracellular vesicles, extracellular vesicles loaded with 3,000 hsiRNAs were used for subsequent experiments. Extracellular vesicles were more efficient at delivering hsiRNA and inducing Huntingtin silencing than conventional liposomes or hsiRNA delivered carrier-free (p < 0.0001; Figure S3A). To evaluate the impact of chemical modifications on extracellular vesicle-mediated delivery of hsiRNAs, we synthesized four different hsiRNA variants: (1) partially modified (all pyrimidines modified, similar to commercially available siRNAs18Byrne M. Tzekov R. Wang Y." @default.
• W2809145613 created "2018-06-29" @default.
• W2809145613 creator A5001146694 @default.
• W2809145613 creator A5002641644 @default.
• W2809145613 creator A5028191953 @default.
• W2809145613 creator A5035448918 @default.
• W2809145613 creator A5037669890 @default.
• W2809145613 creator A5039890109 @default.
• W2809145613 creator A5055526480 @default.
• W2809145613 creator A5069125945 @default.
• W2809145613 creator A5076852749 @default.
• W2809145613 creator A5078887561 @default.
• W2809145613 creator A5084766520 @default.
• W2809145613 creator A5089607612 @default.
• W2809145613 date "2018-08-01" @default.
• W2809145613 modified "2023-10-10" @default.
• W2809145613 title "Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles" @default.
• W2809145613 cites W1522977578 @default.
• W2809145613 cites W1647075334 @default.
• W2809145613 cites W1652469027 @default.
• W2809145613 cites W1816689036 @default.
• W2809145613 cites W1832220924 @default.
• W2809145613 cites W1950118853 @default.
• W2809145613 cites W1979034566 @default.
• W2809145613 cites W1984085632 @default.
• W2809145613 cites W2003555906 @default.
• W2809145613 cites W2019631541 @default.
• W2809145613 cites W2021260035 @default.
• W2809145613 cites W2033425827 @default.
• W2809145613 cites W2055313665 @default.
• W2809145613 cites W2058180357 @default.
• W2809145613 cites W2062530126 @default.
• W2809145613 cites W2067845253 @default.
• W2809145613 cites W2076011658 @default.
• W2809145613 cites W2078890343 @default.
• W2809145613 cites W2080718344 @default.
• W2809145613 cites W2085898732 @default.
• W2809145613 cites W2094711438 @default.
• W2809145613 cites W2107762071 @default.
• W2809145613 cites W2111560326 @default.
• W2809145613 cites W2116424844 @default.
• W2809145613 cites W2136537310 @default.
• W2809145613 cites W2143313350 @default.
• W2809145613 cites W2166608208 @default.
• W2809145613 cites W2175659131 @default.
• W2809145613 cites W2207291742 @default.
• W2809145613 cites W2221400692 @default.
• W2809145613 cites W2295697145 @default.
• W2809145613 cites W2297436559 @default.
• W2809145613 cites W2330710978 @default.
• W2809145613 cites W2342982379 @default.
• W2809145613 cites W2343869769 @default.
• W2809145613 cites W2344343981 @default.
• W2809145613 cites W2419842412 @default.
• W2809145613 cites W2476696777 @default.
• W2809145613 cites W2502873589 @default.
• W2809145613 cites W2511735406 @default.
• W2809145613 cites W2527897220 @default.
• W2809145613 cites W2553886049 @default.
• W2809145613 cites W2555483953 @default.
• W2809145613 cites W2557242880 @default.
• W2809145613 cites W2559331577 @default.
• W2809145613 cites W2603213989 @default.
• W2809145613 cites W2610933455 @default.
• W2809145613 cites W2624053477 @default.
• W2809145613 cites W2624616322 @default.
• W2809145613 cites W2735330142 @default.
• W2809145613 cites W2762010667 @default.
• W2809145613 cites W2763915430 @default.
• W2809145613 cites W2774123716 @default.
• W2809145613 cites W2792194554 @default.
• W2809145613 doi "https://doi.org/10.1016/j.ymthe.2018.05.024" @default.
• W2809145613 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6094392" @default.
• W2809145613 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/29937418" @default.
• W2809145613 hasPublicationYear "2018" @default.
• W2809145613 type Work @default.
• W2809145613 sameAs 2809145613 @default.
• W2809145613 citedByCount "61" @default.
• W2809145613 countsByYear W28091456132018 @default.
• W2809145613 countsByYear W28091456132019 @default.
• W2809145613 countsByYear W28091456132020 @default.
• W2809145613 countsByYear W28091456132021 @default.
• W2809145613 countsByYear W28091456132022 @default.
• W2809145613 countsByYear W28091456132023 @default.
• W2809145613 crossrefType "journal-article" @default.
• W2809145613 hasAuthorship W2809145613A5001146694 @default.
• W2809145613 hasAuthorship W2809145613A5002641644 @default.
• W2809145613 hasAuthorship W2809145613A5028191953 @default.
• W2809145613 hasAuthorship W2809145613A5035448918 @default.
• W2809145613 hasAuthorship W2809145613A5037669890 @default.
• W2809145613 hasAuthorship W2809145613A5039890109 @default.
• W2809145613 hasAuthorship W2809145613A5055526480 @default.
• W2809145613 hasAuthorship W2809145613A5069125945 @default.
• W2809145613 hasAuthorship W2809145613A5076852749 @default.
• W2809145613 hasAuthorship W2809145613A5078887561 @default.
• W2809145613 hasAuthorship W2809145613A5084766520 @default.
• W2809145613 hasAuthorship W2809145613A5089607612 @default.
• W2809145613 hasBestOaLocation W28091456131 @default.

• next