Functional delivery of commercially-available microRNA mimics to the organs of developing mice
Sven Willen1, Despoina Myti2, Razan Obeidat2
1Justus-Liebig-Universitat Giessen.
Abstract:
MicroRNA are pivotal regulators of organ and tissue homeostasis and development, and pathogenic mediators of disease. While antagonism of specific microRNA species in vivo is easily accomplished in experimental animal models using locked nucleic acid (LNA)-stabilized antimiRs, microRNA mimicry -that is, increasing the abundance of specific microRNA species- in experimental animals is more challenging. In this study, commercially available naked LNA-stabilized oligonucleotides designed as microRNA mimics were assessed for efficacy in newborn mouse pups, which frequently serve as models for normal human development, or neonatal and pediatric disease. Mimics targeting miR-497a-5p commercially marketed for in vivo or in vitro use were examined for distribution to five organs in developing mouse pups after parenteral administration. Mimics were most effectively delivered to the liver, and in limited instances also to the heart and kidneys, but could not be detected in the brain or lungs. A combination of bulk RNA-Seq transcriptomic screening and proteomic screening of liver homogenates revealed that mimics delivered to the liver were functional, either reducing the abundance of the Tff3 mRNA transcript, or the abundance of N4bp1 or Mt2 proteins, all of which are cognate targets of miR 497a-5p. Furthermore, the data presented here demonstrate that LNA-stabilized mimics for miR-497a-5p were short-lived in vivo, yet impacted the abundance of both primary and secondary targets of miR-497a-5p albeit with demonstrable hepatotoxicity. This study concludes that while microRNA mimicry using commercially-available naked LNA stabilized oligonucleotides is possible, there remains tremendous scope -and need- for refinement of this technology.
Insights
MicroRNA mimicry in newborn mice using LNA-stabilized oligonucleotides showed functional delivery to the liver, impacting target gene and protein expression. However, mimics were short-lived and caused hepatotoxicity, indicating a need for technological refinement.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- MicroRNAs (miRNAs) are crucial for development and disease.
- Antagonizing miRNAs in vivo is established, but miRNA mimicry is challenging.
- Newborn mice models are vital for studying human development and disease.
Purpose of the Study:
- To assess the efficacy of commercial LNA-stabilized miRNA mimics in newborn mice.
- To determine the biodistribution and functional impact of miR-497a-5p mimics.
- To evaluate the potential of miRNA mimicry for therapeutic applications.
Main Methods:
- Parenteral administration of LNA-stabilized miR-497a-5p mimics to newborn mice.
- Organ distribution analysis via molecular detection.
- RNA-Seq and proteomic screening of liver homogenates.
- Assessment of target gene and protein modulation.
Main Results:
- Mimics were primarily delivered to the liver, with limited presence in the heart and kidneys.
- Functional mimicry was confirmed by reduced Tff3 mRNA and N4bp1/Mt2 protein levels.
- LNA-stabilized mimics were short-lived in vivo but affected primary and secondary miRNA targets.
- Demonstrable hepatotoxicity was observed.
Conclusions:
- Commercially available LNA-stabilized oligonucleotides can achieve functional miRNA mimicry in vivo.
- Significant limitations exist, including short half-life, limited tissue distribution, and hepatotoxicity.
- Further refinement of miRNA mimic technology is essential for therapeutic development.
