Rat and rabbit whole embryo culture as a new approach method for unlabeled therapeutic antisense oligonucleotide hazard identification with no requirement for microinjection or assisted transfection

Abstract

The developmental hazard screening of oligonucleotide therapeutics (ONTs) presents challenges due to their frequent lack of pharmacology in nonclinical species and embryofetal exposure is presumed to be limited in vivo. This study demonstrates that direct culture in media containing mipomersen, a 2-O-methoxyethyl phosphorothioated antisense oligonucleotide (ASO), results in dose-responsive morphological changes in rat and rabbit whole embryo culture (WEC). Automated miRNAscope in situ hybridization was used to confirm dose-dependent embryonic exposure and visualize the distribution pattern of mipomersen in the embryo and extra-embryonic membranes, suggesting that ONTs may enter the umbilical vasculature and pass into embryo circulation. Neither microinjections nor assisted transfections were required to achieve embryonic exposure. These findings support the utility of WEC as a new approach method (NAM) for developmental hazard identification of ONTs. WEC could complement or partially replace in vivo studies, reducing animal use and required test material amounts, while enabling robust developmental hazard identification for ONTs. This work informs future safety assessment strategies and regulatory guidance for ONTs.