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Abstract
We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences.
Highlights
Antisense oligonucleotides (ASOs) and small interfering RNA are both recognized therapeutic agents for the silencing of specific genes at the posttranscriptional level.[1]
The 7-mer second wing was composed of phosphodiester-bound unlocked nucleic acid (UNA) (Toc-20-mer ASO) or phosphorothioate-bound UNA (Toc-20-mer ASO PS)
We previously showed that conjugation of α-tocopherol to small interfering RNA (siRNA) (Toc-siRNA) improves the gene silencing effect of this construct in vivo;[17] here, we found that the direct conjugation of α-tocopherol to ASO (Toc-13-mer ASO) abolished this ability (Figure 2a)
Summary
Antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) are both recognized therapeutic agents for the silencing of specific genes at the posttranscriptional level.[1] Chemical modifications, the use of locked nucleic acids (LNAs),2–4 2′-O-methoxyethyl (2′-O-MOE),[5,6] and constrained ethyl BNA (cEt),[7,8] markedly improve ASO binding affinity for the target mRNA, resulting in increased steric block efficiency. The FDA approved Kynamro (mipomersen sodium, Isis Pharmaceuticals, Carlsbad, CA) as a treatment for familial hypercholesterolemia.[11,12] Kynamro, a DNA 10-mer with 2′-O-MOE-modified-5-mers at both ends, targets Apolipoprotein B (ApoB). It has a strong target gene– silencing effect and greatly reduces serum low-density lipoprotein (LDL)-cholesterol in patients with familial hypercholesteremia. The inadequate delivery and poor cellular uptake of oligonucleotides, coupled with their inability to efficiently access the target mRNA during intracellular trafficking,[14] are major impediments to in vivo silencing.[15]
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