Chronic Toxicity Assessment of 2′-O-Methoxyethyl Antisense Oligonucleotides in Mice

Abstract

Advances in antisense oligonucleotide (ASO) chemistry and screening have enabled the design and selection of molecules that are optimized for a particular therapeutic application in terms of both potency and tolerability. The most-well studied of the chemically modified ASOs are single-stranded antisense inhibitors with phosphorothioate backbones and 2'-O-methoxyethyl modifications (2'-MOE ASO). The 2'-MOE chemical modification in the design of the ASO has conferred increased hybridization affinity, increased stability, and/or enhanced tissue residence time, resulting in better potency and pharmacokinetics. Compound screening and selection are also important in optimizing the tolerability of intended therapeutic antisense inhibitors. In this study, we report the chronic toxicity of multiple 2'-MOE ASOs in mice for several representative compounds that have progressed to later phases of clinical development. The results show that these 2'-MOE ASOs selected for development have consistent behavior between sequences, have tolerability profiles suitable for chronic administration, and exhibit a relative lack of progression of findings observed in subchronic studies in mice.

The Nonclinical Safety Profile of GalNAc-conjugated RNAi Therapeutics in Subacute Studies

“…Four ASO drugs have been approved, and four RNA interference (RNAi) therapeutics are currently in late-stage clinical trials, with multiple N -acetylgalactosamine (GalNAc)-conjugated siRNAs (GalNAc-siRNA) achieving proof of concept status in the clinic ( Fitzgerald, Kallend, and Simon 2017 ; Pasi et al 2017 ; Zimmermann et al 2017 ). Recent review articles have discussed the nonclinical safety profiles of ASOs ( Engelhardt et al 2015 ; Frazier 2015 ; Zanardi et al 2018 ), but differences in structure, chemical modifications, and modes of action result in distinct safety profiles for siRNAs. Here, we review the platform-wide nonclinical safety profiles of Enhanced Stabilization Chemistry (ESC) RNAi therapeutics conjugated to GalNAc ( Nair et al 2014 ; Foster et al 2018 ), which are currently in clinical development (givosiran, fitusiran, inclisiran, lumasiran, cemdisiran, and ALN-TTRSC02).…”

Impurity Qualification Toxicology Study for a 2′-O-Methoxyethyl-Modified Antisense Inhibitor in Mice

“…The 30 mg/kg/week dose level produced a comparable level of changes in hematology, serum chemistry, organ weights, and histology between ISIS 304801 and the lots with impurity enrichment. These observed findings were attributed to a known class effect of ASO administration that have been well studied and are attributed to the full-length oligonucleotide in mice [3,6] Liver concentrations of oligonucleotide were dose-dependent at 10 and 30 mg/kg/week and comparable across parent and Impurity Mixture groups indicating comparable exposure. The analytical methods used to determine drug concentration are specific for the full-length oligonucleotide or degradation products, but cannot discriminate the concentration of specific impurities.…”

“…This compound completed all animal and clinical testing and was submitted for approval in the United States and Europe at a dose of 300 mg/week (*5 mg/kg/week). In general toxicology studies, ISIS 304801 was reasonably well tolerated in mice up to 100 mg/kg/week for 3 months, producing the expected dose-dependent class effects for compounds in this chemical class [3]. The present study compared a representative lot of active pharmaceutical ingredient (API) to different lots that were enriched for several specific impurities ( Fig.…”

Current Development of siRNA Bioconjugates: From Research to the Clinic

“…Since the size of the modification may contribute to an increase in the nuclease resistance of siRNA (Cummins et al, 1995), attempts have been made to introduce more voluminous substituents into the 2′ position of ribose (2′-O-methoxyethyl [2′O-MOE] Prakash et al, 2005; Zanardi et al, 2018, 2′O-allyl Amarzguioui et al, 2003, 2′O-benzyl Kenski et al, 2012, and other modifications); however, these substitutions more significantly inhibited RNAi than 2′O-Me.…”