Synthesis and Exon-Skipping Properties of a 3′-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach

Elena Marchesi,Kamel Mamchaoui,Monica Bertoldo,Matteo Bovolenta,Massimo L Capobianco,Daniela Perrone,Lorenzo Preti

doi:10.3390/molecules26247662

Abstract

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2′-OMe PS oligonucleotide conjugated at 3′-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3′-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3′-UDC with an average 9.5-fold increase with respect to ASO 51.

Highlights

Antisense oligonucleotides (ASO) are therapeutically attractive short nucleic acid chains exploited for gene modulation by targeting specific sequences in pre-mRNA or mRNA [1]
We previously reported on the potential of certain cationic polymethylmethacrylate (PMMA) nanoparticles loaded with a 20 OMe PS ASO to induce dystrophin restoration in the muscles of mdx animal model [11]
Synthesis of ASO 51 30 -UDC via Post-Synthetic Approach

Summary

Introduction

Antisense oligonucleotides (ASO) are therapeutically attractive short nucleic acid chains exploited for gene modulation by targeting specific sequences in pre-mRNA or mRNA [1]. Their mode of action depends on the nature of target sequence and the chemistry used [2]. With two ASO chemistries evaluated in clinical trials for the targeting of DMD exon 45, 51, and 53: 20 -O-methyl-phosphorothioate (20 -OMe PS) and phosphorodiamidate morpholino oligomers (PMO) [6,7]. We previously reported on the potential of certain cationic polymethylmethacrylate (PMMA) nanoparticles loaded with a 20 OMe PS ASO to induce dystrophin restoration in the muscles of mdx animal model [11]. Slow biodegradability and tissue accumulation of PMMA nanoparticles limited their use in chronic treatments

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Discussion

Conclusion