Targeted delivery and enhanced gene-silencing activity of centrally modified folic acid–siRNA conjugates

Abstract

One of the major hurdles in RNAi research has been the development of safe and effective delivery systems for siRNAs. Although various chemical modifications have been proposed to improve their pharmacokinetic behaviour, their delivery to target cells and tissues presents many challenges. In this work, we implemented a receptor-targeting strategy to selectively deliver siRNAs to cancer cells using folic acid as a ligand. Folic acid is capable of binding to cell-surface folate receptors with high affinity. These receptors have become important molecular targets for cancer research as they are overexpressed in numerous cancers despite being expressed at low levels in normal tissues. Employing a post-column copper-catalyzed alkyne–azide cycloaddition (CuAAC), we report the synthesis of siRNAs bearing folic acid modifications at different positions within the sense strand. In the absence of a transfection carrier, these siRNAs were selectively taken up by cancer cells expressing folate receptors. We show that centrally modified folic acid–siRNAs display enhanced gene-silencing activity against an exogenous gene target (∼80% knockdown after 0.75 μM treatment) and low cytotoxicity. In addition, these siRNAs achieved potent dose-dependent knockdown of endogenous Bcl-2, an important anti-apoptotic gene.

Cellular Targeting of Oligonucleotides by Conjugation with Small Molecules

“…This therefore represents an adequate binding site for modifications and targeting ligands, further enhancing strand separation and altering the silencing of protein expression [ 49 , 50 ]. This is demonstrated in several examples [ 51 , 52 , 53 , 54 , 55 , 56 ]. A prominent representative of RNAi-triggering therapeutics is the first FDA-approved siRNA drug Patisiran [ 57 , 58 ] against hereditary transthyretin-mediated amyloidosis.…”

“…In analogy to the AEA-conjugate described above, Carell et al [ 222 ] proved silencing in another reporter gene assay to be 50% at 1 μM using a FolA conjugate, with the ligand attached (by CuAAC) to the nucleobase at the 3′-end. However, in recently published work by Desaulniers et al, a remarkable conjugate was applied in HeLa and HT-29 cells with a FolA unit at several positions within an siRNA, representing the first FolA conjugate ever constructed in this fashion ( Figure 4 ) [ 51 ]. An N- propargyl diethanolamine-derived unit was chosen as anchor point for subsequent FolA conjugation (via CuAAC) and inserted between two nucleotides at position 10, thus replacing a single nucleotide.…”

Transfection types, methods and strategies: a technical review

“…Small, non-coding RNAs such as siRNAs are well known for their epigenetic regulation capabilities or, more specifically, regulation of specific gene expression post-transcriptionally (Zhao & Zhang, 2018). siRNA mimics and a plasmid DNA carrying the specific gene linked to a luciferase reporting system can be simultaneously co-transfected into a target cell (Salim, Islam & Desaulniers, 2020). Successful knockdown of specific gene expression by the siRNA mimic will lead to a measurable decrease in luciferase activity (Peralta-Zaragoza et al, 2016;Shyamasundar, Lim & Bay, 2016).…”

Folate Receptor-Mediated siRNA Delivery: Recent Developments and Future Directions for RNAi Therapeutics

“…In 2020, Salim et al reported FA-conjugated siRNAs through a copper-catalyzed cycloaddition reaction (Fig. 10b) [168]. The sense strand of siRNAs was modified with FA at certain places, including 5¢-end, 3¢-ends, and central regions.…”