Synthesis, biological activity, and molecular dynamics simulations of LNA-charge neutral linkages for enhanced splice-switching antisense oligonucleotides

Abstract

Antisense oligonucleotides are promising therapeutic agents for a range of diseases, having found special clinical success for splice-switching genetic conditions such as spinal muscular atrophy and Duchenne muscular dystrophy. However, novel chemistries are still required to discover modifications which improve their druggable properties. For in vitro studies, thermal duplex stability, resistance to enzymatic degradation and gymnotic cellular activity are important, and biodistribution, toxicology and potency must be optimised for clinical progression. We investigate the combination of locked nucleic acids (LNA) and charge neutral backbones in chimeric ASOs containing 2′-O-methyl sugars and phosphorothioate backbones by evaluating their physical and biological properties. Backbones investigated are LNA-amide, LNA-carbamate, LNA-alkoxyamide, and LNA-sulfamate. Molecular dynamics simulations of these LNA-charge neutral backbones were conducted to explore the structural features which determine the experimentally observed thermal duplex stability and conformation. The LNA-sulfamate linkage is of particular interest, forming very stable duplexes with its RNA target and having comparable gymnotic activity to the previously investigated LNA-amide, while being synthetically more accessible. Together, our studies indicate that a multi-faceted approach to expanding the ASO chemical space, using a combination of computational and experimental methods, can build structure-activity relationships and discover novel promising backbones for future therapeutic use.

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Identifiers

ORCID for Ysobel Baker: orcid.org/0000-0002-0266-771X

ORCID for Afaf H. El-Sagheer: orcid.org/0000-0001-8706-1292

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