Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles
RNA Therapeutics Institute; Program in Molecular Medicine; Department of Medicine
Biochemistry | Cell Biology | Genetics and Genomics | Medicinal-Pharmaceutical Chemistry | Molecular Biology | Nucleic Acids, Nucleotides, and Nucleosides | Therapeutics | Translational Medical Research
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 approximately 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.
RNA therapy, chemical modification, exosomes, extracellular vesicles, nanovesicles, oligonucleotides, siRNA, UMCCTS funding
DOI of Published Version
Mol Ther. 2018 Aug 1;26(8):1973-1982. doi: 10.1016/j.ymthe.2018.05.024. Epub 2018 Jun 21. Link to article on publisher's site
Molecular therapy : the journal of the American Society of Gene Therapy
Haraszti, Reka A.; Miller, Rachael; Didiot, Marie C.; Biscans, Annabelle; Alterman, Julia F.; Hassler, Matthew R.; Roux, Loic; Echeverria, Dimas; Sapp, Ellen; DiFiglia, Marian; Aronin, Neil; and Khvorova, Anastasia, "Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles" (2018). UMass Center for Clinical and Translational Science Supported Publications. 168.