Biodistribution of lipid nanoparticle, eGFP mRNA and translated protein following subcutaneous administration in mouse

Abstract

Aim: Increased knowledge of biodistribution and pharmacokinetics of lipid nanoparticle (LNP)-encapsulated mRNA drug components may aid efficacy and safety evaluation. Methods: Mice were subcutaneously administrated LNP encapsulated enhanced green fluorescent protein mRNA and sampled up to 72 h after dosing. LNP, mRNA and translated protein were quantified by LC-MS, branched DNA and ELISA. Results: Highest levels of LNP and mRNA were detected in skin, followed by spleen, but also rapidly distributed to circulation. Translated protein showed high concentration in skin and spleen, but also in liver and kidney across 24 h where the LNP was cleared at 4 h. Conclusion: Subcutaneously dosing LNP encapsulated mRNA in mice resulted in a nonlinear relationship of LNP, mRNA and protein concentration across multiple tissues.

Article highlights

Background

• Quantitative biodistribution data of lipid nanoparticle (LNP) encapsulated mRNA drug components and translated protein after subcutaneous injection in mice is currently not available in the literature.

• As a result, the present study was intended to determine the exposure of LNP, mRNA and translated protein in multiple tissue and circulation after subcutaneous injection in mice, thereby bridging a necessary gap in the research literature.

Methods

• Branched DNA performance in biodistribution settings was evaluated and qualified for mRNA quantification in blood, bone marrow, brain, liver, lung, kidney, heart, spleen and skin.

• Quantification of LNP and protein were performed using LC-MS/MS and a ligand-binding assay respectively.

Results & discussion

• Nonlinear pharmacokinetics and tissue-specific associations were detected between mRNA, LNP and protein concentrations.

• Results show a rapid distribution to the liver, spleen and injection site. LNP and mRNA was rapidly cleared and protein expression was delayed and tissue dependent.

• Branched DNA was fit for purpose of mRNA quantification for biodistribution in blood and tissues analyzed.

• Significant findings include the importance of early sampling and careful tissue dissection to avoid blood contamination.

Conclusion

• Circulating levels of LNP, mRNA and protein is not representative of tissue concentrations.

• This study provides valuable insights into mRNA-LNP formulations by increasing our understanding of in vivo distribution relationships. Nonlinear pharmacokinetics observed in both rodents and nonhuman primates underscores the need for robust preclinical disposition data in translating findings to the clinic.

Keywords: :

• bioanalytical method validation
• branched DNA
• enhanced GFP
• LC–MS/MS
• lipid nanoparticle
• mRNA
• pharmacokinetics

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/17576180.2024.2360361

Acknowledgments

The authors would like to acknowledge Sima Kahn for assisting with LC-MS analysis of the LNP; Lynne Neveras, Frank McGrath and Mark Pietras for efforts in study planning and sampling; and Moderna for providing LNP material and encapsulating mRNA.

Author contributions

NH and AW were responsible for study conception and design. ÅS, NH, JL and HN were responsible for acquisition and interpretation of data. ÅS, NH and AW were responsible for drafting and revision of the manuscript. All authors provided critical review and approval of the final version to be published.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

All authors are employees and shareholders of AstraZeneca and declare that this did not influence the study design, analysis or interpretations and they have no other conflicts of interest with respect to the research, authorship and/or publication of this article. The authors have no other competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript apart from those disclosed.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval and followed the principles outlined in the Declaration of Helsinki for the animal experimental investigations.