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

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.

Topical Application of Peptide Nucleic Acid Antisense Oligonucleotide for MMP-1 and Its Potential Anti-Aging Properties

“…The binding of an antisense RNA with sequences complementary to the target mRNA provides a powerful tool to modulate artificial gene expression, called RNA silencing [ 16 ]. Antisense oligonucleotides (ASO) can also bind to a pre-mRNA in the nucleus and affect the splicing of the pre-mRNA, producing an altered mRNA sequence [ 17 , 18 ]. However, one of the main barriers in applying ASO technology is the rapid degradation of DNA-based oligonucleotides in cells by nucleases [ 19 ].…”

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

“…To synthesized 5 -N-TUDCA-ASO 51, the free amino group of ssH-ASO 51-S was armed with a succinic acid linker before adding N-TUDCA derivative 12 in presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU) as a coupling reagent. The synthesis of 5 ,3 -bis-UDC-ASO 51, in which two residues of UDCA were added one for each end of ASO 51, was achieved by using our ASO 51 3 -UDC prepared via the pre-synthetic approach previously reported which required the synthesis of a novel solid support functionalized with UDCA before assembling the oligonucleotide [27]. Thus, as depicted in Scheme 4, ASO 51 3 -UDC held on to solid support was converted into the desired 5 ,3 -bis-UDC-ASO 51 in the same conditions employed for 5 -UDC-ASO 51.…”

“…To the cooled mixture, taurine (0.257 mmol) dissolved in 1 M NaOH aqueous solution (0.3 mL), was added and after warming to 25 • C, the reaction was stirred for additional 24 h. After adjusting the pH to 1 by adding diluted H 2 SO 4 , the resulting 3-α-N-BOC-TUDCA was extracted with 1-butanol (3 × 10 mL), then the organic phase was dried over Na 2 SO 4 and concentrated in vacuo. Finally, the deprotection of BOC-group was carried out by using TFA (1.5 mL) in CH 2 Cl 2 /MeOH 2:1 at 0 All ASO targeting human DMD exon 51 were synthesized at 7 µmole scale following our protocol for the synthesis of 2 OMe PS oligonucleotides [27]; PO linkage were performed in classical conditions; ssH-Linker TM was inserted at 5 -end of ASO after addition of the last nucleotide of sequence, exploiting the phosphoramidite chemistry and the MMT group was removed by using the classical detritylation step.…”

“…Recently, we reported for the first time the synthesis and in vitro exon skipping properties of ASO 51 conjugated at 3 -end with ursodeoxycholic acid (ASO 51 3 -UDC, Figure 1) [27]. ASO 51 is a 20-mer RNA-based oligonucleotide full-length modified as 2 -OMe PS, containing the sequence of clinically evaluated Drisapersen, targeting the human DMD exon 51 [6,28,29].…”