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Rat and Rabbit Whole-Embryo Culture as a New Approach Method for Unlabeled Therapeutic Antisense Oligonucleotide
Sara M Bender1, Sharon Chapman1, Sreenivas Nannapaneni1
1GSK, Collegeville, Pennsylvania, USA.
Abstract:
The developmental hazard screening of oligonucleotide therapeutics (ONTs) presents challenges due to their frequent lack of pharmacology in nonclinical species and embryo-fetal 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 extraembryonic 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.
Insights
Whole-embryo culture (WEC) effectively identifies developmental hazards of oligonucleotide therapeutics (ONTs). Direct exposure in WEC shows dose-dependent effects and embryonic uptake, supporting WEC as a new approach method (NAM) for safety assessment.
Area of Science:
- Pharmacology and Toxicology
- Developmental Biology
- Drug Safety Evaluation
Background:
- Developmental hazard screening of oligonucleotide therapeutics (ONTs) is challenging due to limited nonclinical pharmacology and presumed low embryo-fetal exposure.
- Current methods often require animal testing, raising ethical and efficiency concerns.
Purpose of the Study:
- To evaluate whole-embryo culture (WEC) as a new approach method (NAM) for assessing developmental toxicity of ONTs.
- To demonstrate embryonic exposure to ONTs using WEC without invasive techniques.
- To investigate the utility of WEC in identifying developmental hazards associated with ONTs.
Main Methods:
- Rat and rabbit embryos were cultured directly in media containing mipomersen, a 2'-O-methoxyethyl phosphorothioated antisense oligonucleotide (ASO).
- Dose-dependent morphological changes were assessed in the whole-embryo culture (WEC) system.
- Automated miRNAscope in situ hybridization was employed to confirm embryonic exposure and map mipomersen distribution.
Main Results:
- Direct culture in mipomersen-containing media induced dose-responsive morphological alterations in rat and rabbit embryos.
- In situ hybridization confirmed dose-dependent embryonic uptake of mipomersen, with distribution observed in embryonic and extraembryonic tissues.
- Embryonic exposure was achieved without the need for microinjections or assisted transfections.
Conclusions:
- Whole-embryo culture (WEC) serves as a valuable new approach method (NAM) for developmental hazard identification of oligonucleotide therapeutics (ONTs).
- WEC demonstrates direct embryonic exposure and dose-dependent effects, supporting its use to complement or replace traditional in vivo studies.
- This approach can reduce animal use and test material, enhancing safety assessment strategies and regulatory guidance for ONTs.
