Cholesterol Conjugates of Small Interfering RNA: Linkers and Patterns of Modification.

Cholesterol siRNA conjugates attract attention because they allow the delivery of siRNA into cells without the use of transfection agents. In this study, we compared the efficacy and duration of silencing induced by cholesterol conjugates of selectively and totally modified siRNAs and their heteroduplexes of the same sequence and explored the impact of linker length between the 3' end of the sense strand of siRNA and cholesterol on the silencing activity of "light" and "heavy" modified siRNAs. All 3'-cholesterol conjugates were equally active under transfection, but the conjugate with a C3 linker was less active than those with longer linkers (C8 and C15) in a carrier-free mode. At the same time, they were significantly inferior in activity to the 5'-cholesterol conjugate. Shortening the sense strand carrying cholesterol by two nucleotides from the 3'-end did not have a significant effect on the activity of the conjugate. Replacing the antisense strand or both strands with fully modified ones had a significant effect on silencing as well as improving the duration in transfection-mediated and carrier-free modes. A significant 78% suppression of MDR1 gene expression in KB-8-5 xenograft tumors developed in mice promises an advantage from the use of fully modified siRNA cholesterol conjugates in combination chemotherapy.

Cholesterol-Modified Anti-Il6 siRNA Reduces the Severity of Acute Lung Injury in Mice.

Small interfering RNA (siRNA) holds significant therapeutic potential by silencing target genes through RNA interference. Current clinical applications of siRNA have been primarily limited to liver diseases, while achievements in delivery methods are expanding their applications to various organs, including the lungs. Cholesterol-conjugated siRNA emerges as a promising delivery approach due to its low toxicity and high efficiency. This study focuses on developing a cholesterol-conjugated anti-Il6 siRNA and the evaluation of its potency for the potential treatment of inflammatory diseases using the example of acute lung injury (ALI). The biological activities of different Il6-targeted siRNAs containing chemical modifications were evaluated in J774 cells in vitro. The lead cholesterol-conjugated anti-Il6 siRNA after intranasal instillation demonstrated dose-dependent therapeutic effects in a mouse model of ALI induced by lipopolysaccharide (LPS). The treatment significantly reduced Il6 mRNA levels, inflammatory cell infiltration, and the severity of lung inflammation. IL6 silencing by cholesterol-conjugated siRNA proves to be a promising strategy for treating inflammatory diseases, with potential applications beyond the lungs.

Cholesterol-Conjugated Supramolecular Multimeric siRNAs: Effect of siRNA Length on Accumulation and Silencing In Vitro and In Vivo.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules is one of the most promising approaches for delivering siRNA in vivo. The rate of molecular weight-dependent siRNA renal clearance is critical for the efficiency of this process. In this study, we prepared cholesterol-containing supramolecular complexes containing from three to eight antisense strands and examined their accumulation and silencing activity in vitro and in vivo. We have shown for the first time that such complexes with 2'F, 2'OMe, and LNA modifications exhibit interfering activity both in carrier-mediated and carrier-free modes. Silencing data from a xenograft tumor model show that 4 days after intravenous injection of cholesterol-containing monomers and supramolecular trimers, the levels of MDR1 mRNA in the tumor decreased by 85% and 68%, respectively. The in vivo accumulation data demonstrated that the formation of supramolecular structures with three or four antisense strands enhanced their accumulation in the liver. After addition of two PS modifications at the ends of antisense strands, 47% and 67% reductions of Ttr mRNA levels in the liver tissue were detected 7 days after administration of monomers and supramolecular trimers, respectively. Thus, we have obtained a new type of RNAi inducer that is convenient for synthesis and provides opportunities for modifications.