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Comparative Elimination Analysis of Lipid Nanoparticle-Encapsulated Human Erythropoietin mRNA by Multimodal
Kin-Sing Wong1, Bruce P N Yuen2, Coco H Y Kwok1
1Racing Laboratory, The Hong Kong Jockey Club Sha Tin Racecourse, Sha Tin, N. T, Hong Kong.
Abstract:
Illicit use of messenger ribonucleic acid (mRNA) agents represents an imminent threat to gene doping control in equine sports. The effectiveness and safety of lipid nanoparticle-encapsulated mRNA agents (LNP-mRNA), which have been well-proven during COVID-19 pandemic, have resulted in prominent growth of the interest on LNP-mRNA agents for expressing desired proteins in vivo. The vast number of potential performance-enhancing mRNAs has demanded cost-effective and reliable methods for their detections. This study employed a multimodal analysis approach to detect an LNP-mRNA product ("LNP-epo" hereafter) arising from encapsulation of a black-market product of human erythropoietin (hEPO) mRNA. Lipid nanoparticle (LNP) components were detected by liquid chromatography/high-resolution tandem mass spectrometry after supported liquid extraction; hEPO mRNA was detected by reverse transcription quantitative polymerase chain reaction after RNA extraction; hEPO protein was detected by sandwich enzyme-linked immunosorbent assay. Comparative elimination analysis of LNP-epo in blood samples collected from an administered horse showed the longest detection time achieved by LNP monitoring (at least 34-day postadministration), followed by hEPO mRNA detection (up to 30 days in whole blood; up to 17 days in plasma), and shortest by hEPO protein detection (up to 3 days). Together with its low analysis cost, broad coverage of LNP components, expandable scope and independence from mRNA sequence modifications and the need for specific reagents (e.g., primers, antibodies), the detection of LNP can be a fit-for-purpose screening method for monitoring the misuse of LNP-mRNA in equine sports. The other two approaches could provide insight on the intentional effect elicited by LNP-mRNA.
Insights
Detecting lipid nanoparticle (LNP) components offers a cost-effective screening method for illicit messenger ribonucleic acid (mRNA) doping in equine sports, with detection lasting longer than mRNA or protein. This approach aids gene doping control.
Area of Science:
- Equine Sports Science
- Biotechnology
- Analytical Chemistry
Background:
- Messenger ribonucleic acid (mRNA) agents, particularly lipid nanoparticle-encapsulated mRNA (LNP-mRNA), are gaining interest for in vivo protein expression.
- The potential for illicit use of LNP-mRNA for performance enhancement in equine sports poses a significant challenge to anti-doping efforts.
- Developing reliable and cost-effective detection methods for these agents is crucial for maintaining fair competition.
Purpose of the Study:
- To develop and evaluate a multimodal analytical approach for detecting a specific LNP-mRNA product (LNP-human erythropoietin [hEPO]) in equine samples.
- To compare the detection windows of LNP components, hEPO mRNA, and hEPO protein following administration of LNP-hEPO.
- To assess the suitability of LNP detection as a screening method for LNP-mRNA doping in horses.
Main Methods:
- Lipid nanoparticle (LNP) components were analyzed using liquid chromatography/high-resolution tandem mass spectrometry.
- Human erythropoietin (hEPO) mRNA was detected via reverse transcription quantitative polymerase chain reaction after RNA extraction.
- hEPO protein was quantified using a sandwich enzyme-linked immunosorbent assay.
Main Results:
- Comparative elimination analysis in a horse showed LNP detection persisted for at least 34 days post-administration.
- hEPO mRNA was detectable up to 30 days in whole blood and 17 days in plasma.
- hEPO protein had the shortest detection window, lasting up to 3 days.
Conclusions:
- Detection of LNP components presents a cost-effective, broadly applicable screening method for LNP-mRNA doping in equine sports.
- LNP monitoring offers a longer detection period compared to mRNA or protein analysis.
- This LNP detection approach is independent of mRNA sequence and specific reagent requirements, making it a practical tool for anti-doping agencies.
