Impact of Oligonucleotide Structure, Chemistry, and Delivery Method on In Vitro Cytotoxicity

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Current affiliation: Sage Therapeutics, Cambridge, Massachusetts.

Address correspondence to: Maja M. Janas, PhD, DABT, Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, E-mail: [email protected]

Introduction

Oligonucleotide (ON) therapeutics are a promising class of synthetic nucleic acids designed to modulate gene expression by targeting endogenous RNAs or proteins [1]. There are multiple families of single-stranded (ss) ONs, including catalytic gapmers/antisense oligonucleotides (ASOs) and stoichiometric steric-blocking ONs, aptamers, antagomirs, exon-skipping ONs, splice-switching ONs, U1 adaptors, and Toll-like receptor agonists and antagonists. In contrast, small interfering RNAs (siRNAs) and microRNA (miRNA) mimics belong to the double-stranded (ds) ON category [2].

To confer optimal drug-like properties (pharmacokinetics, pharmacodynamics, minimal toxicity), many of these therapeutic modalities use common chemical modifications of the phosphodiester backbone and/or of the 2[variant prime]-position of the ribose moiety [3,4]. Replacement of the phosphodiester linkage by a phosphorothioate (PS), where nonbridging oxygen is substituted with sulfur, is the most common backbone modification [5,6]. Due to charge-charge interactions and the greater polarity of a sulfur versus an oxygen atom [7], PS linkages promote protein binding [8,9] leading to increased plasma half-life, reduced renal excretion [10-12], and enhanced nuclear accumulation [13,14]. In addition, PS linkages increase resistance to endo- and exonucleases and hence enhance metabolic stability of ONs [15-17].

However, PS also introduces chirality to the phosphate linkage giving rise to a large number of diastereomers (2^N , where N = number of PS linkages) that may be difficult to characterize. Undesired protein binding mediated by the PS backbone may be associated with dose-dependent liver toxicity [18], degenerative changes due to accumulation in the kidney proximal tubules, antagonism of innate immunity receptors, immunostimulatory effects, coagulopathy, and complement activation [19]. In the context of improved pharmacokinetic and nuclease stability profiles, ss ONs typically have a high number of PS modifications, while ds ONs have fewer PS substitutions since PS may also decrease affinity for the target RNA and reduce thermal stability of ds ONs...