Abstract
The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics.
Highlights
There is strong interest in the therapeutic potential of antisense oligonucleotides (ASO), siRNA and splice switching oligonucleotides (SSOs)(1–5)
The cellular endomembrane system is highly dynamic with numerous membrane fusion and disjunction events occurring as materials are trafficked throughout the cell [42,44]
Intracellular trafficking could potentially be influenced by small molecule drugs that affect endomembrane proteins; there has been relatively little work done on the chemical biology of the endomembrane system
Summary
There is strong interest in the therapeutic potential of antisense oligonucleotides (ASO), siRNA and splice switching oligonucleotides (SSOs)(1–5). A third approach has been to covalently link oligonucleotides to ligands that interact with specific cell surface receptors promoting receptor-mediated endocytosis [24,25,26,27,28,29,30,31,32,33,34]. Unmodified ‘free’ oligonucleotides, as well as ligand-oligonucleotide conjugates, are taken up by cells via endocytosis and accumulate in various endomembrane compartments where they are pharmacologically inert [38,39]. Recent studies have shown that even in the case of lipid nanocarriers much of the oligonucleotide accumulated by cells remains entrapped in endosomes [40]. The biological effects of oligonucleotides may primarily be due to a small amount of material that escapes from endosomes and reaches key cytosolic or nuclear compartments
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