Journal
MOLECULAR THERAPY
Volume 26, Issue 8, Pages 1973-1982Publisher
CELL PRESS
DOI: 10.1016/j.ymthe.2018.05.024
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Funding
- NIH UH3 grant [TR 000888 05]
- UMass grant [CCTS UL1 TR000161]
- NIH [RO1GM10880304, RO1NS10402201, S10 OD020012]
- CHDI Foundation [A-6119, JSC A6367]
- Huntington's Disease Society of America Postdoctoral Fellowship
- NIH Common Fund's exRNA Communication Program
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Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extra cellular 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 [C71) 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 similar to 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.
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