Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles

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.

Exosomes From 3d Culture of Marrow Stem Cells Enhances Endothelial Cell Proliferation, Migration, and Angiogenesis via Activation of the Hmgb1/akt Pathway

“…Increasing evidence demonstrates that the therapeutic effect of BMSCs is not attributed to multi-lineage function but secreted exosomes, through which proteins, miRNAs and other molecular could be transferred to recipient cells (15,33,34).The 3D scaffolds have been proved to be a key material that modulates the paracrine function of cells (15). Haraszti et al have reported that 3D-exos show higher yield and improved activity than 2D-exos (35). In present study, we found that 3D-exos of BMSCs had a higher yield, and exerted a more signi cant effect on HUVEC proliferation, migration, angiogenesis.…”

Extracellular Vesicle-Based Nucleic Acid Delivery: Current Advances and Future Perspectives in Cancer Therapeutic Strategies

“…It has been shown that is possible not only to isolate exosomes from human serum but also to modify the cargo (e.g., binding phosphorodiamidate morpholino oligomer (PMO)) for a specific therapeutic use [ 82 ]. TEG and C7 (2-aminobutyl-1-3-propanediol) were applied to attach molecules on the EV surface due to their affinity for phospholipid membrane [ 87 , 88 ]. An outdated physical method was employed to add antibodies to the EV surface through sonication.…”

“…Binding efficiency depends on the time and power of sonication and on the lipidic composition carried by EVs [ 89 ]. It was also recently reported that the presence of nucleases inside EVs might degrade therapeutic nucleic acids and impair their action [ 88 ]. Another possibility could be loading therapeutics onto the EV surface, depending on their modality of action (i.e., binding/activating receptors on the cell surface).…”

Systemic Delivery of Small Interfering RNA Therapeutics: Obstacles and Advances

“…Immunostimulation is another barrier in the delivery of siRNA as it leads to degradation of siRNA after recognition as an alien moiety by the macrophages and kupffer cells (Whitehead et al, 2011;Khairuddin et al, 2012). In order to tackle this issue, 2'-position of the ribose sugar had been modified with 2'-O-methyl and 2'-fluoro groups to suppress the immune response and improve serum provide stability (Valenzuela et al, 2014;Haraszti et al, 2018).…”