Antisense Oligonucleotides Conjugated with Lipophilic Compounds: Synthesis and In Vitro Evaluation of Exon Skipping in Duchenne Muscular Dystrophy

Abstract

Our groups previously reported that conjugation at 3′-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5′-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5′-UDC- and 5′,3′-bis-UDC-ASO 51.

Lipid and Peptide-Oligonucleotide Conjugates for Therapeutic Purposes: From Simple Hybrids to Complex Multifunctional Assemblies

“…Moreover, palmitic acid-, tocopherol-, and cholesterol-conjugated ( Scheme 1 ) antisense oligonucleotides were reported to increase protein binding and enhance intracellular uptake [ 95 ]. These properties were explored by several groups for the development of lipid-antisense oligonucleotides targeting the exon 51 of human Duchene Muscular Distrophy gene [ 96 , 97 ].…”

Update for Duchenne Muscular Dystrophy - Hope for Causal Treatment of a Disorder Presenting in Early Childhood?

“…AON-mediated exon skipping therapy can be applied for the treatment of about 55% of DMD patients by targeting affected exons with predesigned AONs to produce a shorter but working version of dystrophin [22,23]. A new approach is the conjugation of AONs with lipophilic compounds to explore the influence of the lipophilic moiety on exon skipping efficiency in DMD [24].…”

Self-Penetrating Oligonucleotide Derivatives: Features of Self-Assembly and Interactions with Serum and Intracellular Proteins

“…The attachment of hydrophobic residues to the oligonucleotide backbone, e.g., cholesterol, fatty acids, lipids, and alkyl-containing groups, is a promising approach to improving the pharmacokinetic properties of synthetic oligonucleotides [ 17 , 18 , 19 , 20 , 21 , 22 ]. The hydrophobic groups enhance the efficiency of membrane penetration of such derivatives into cells and improve their pharmacokinetic properties [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ].…”