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Home / Equine Research Bank / Gene Ther / Administration and detection of a multi-target rAAV gene dop...
Gene therapy2024; 31(9-10); 477-488; doi: 10.1038/s41434-024-00462-0

Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques.

Abstract: Gene doping, which includes the non-therapeutic use of genes or genetic elements that have the capacity to enhance athletic performance, is prohibited in horseracing and equestrian sports. To provide a comprehensive assessment of matrix and detection techniques, a custom adeno-associated virus serotype 8 vector was designed to include PCR binding sites for multiple target genes and assay types. The vector was injected via an intramuscular route into two Thoroughbred horses and matrices collected at defined timepoints. DNA was analysed using 3 detection methods: qPCR, digital PCR, and NGS. Overall, there was a strong correlation across the different detection methods employed, although digital PCR was less sensitive at lower concentrations. High concentrations of vector were detected at early timepoints in plasma and whole blood, which rapidly dropped after 0.5 d to trace levels by 4 d and 9 d post-administration respectively, following a similar pattern to previous studies. Vector was detected in dried blood spots at lower levels than whole blood, but with a similar detection time. Detection in hair root bulbs in one horse was observed at over a month post-administration, which opens new avenues for future gene doping testing in humans and animals.
© 2024. The Author(s), under exclusive licence to Springer Nature Limited.
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Publication Date: 2024-07-07 PubMed ID: 38972888PubMed Central: 5177998DOI: 10.1038/s41434-024-00462-0Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study evaluated the administration of a gene doping vector in horses and compared various biological samples and molecular techniques to detect the vector over time.
  • Researchers aimed to identify effective ways to detect gene doping using multiple matrices (sample types) and molecular assays, offering insights for enhancing anti-doping strategies in equine sports.

Background

  • Gene doping refers to the illicit use of genetic material to artificially enhance athletic performance, which is banned in horse racing and equestrian sports.
  • Adeno-associated virus (AAV) vectors are common tools used for gene delivery and can potentially be misused for gene doping.
  • Detecting these vectors in biological samples is crucial for enforcing doping regulations.

Study Design

  • A custom recombinant AAV serotype 8 vector was engineered to carry binding sites for multiple target genes, enabling simultaneous detection by several molecular techniques.
  • Two Thoroughbred horses were administered the vector intramuscularly to simulate gene doping.
  • Samples were collected at predetermined time points from various biological matrices including plasma, whole blood, dried blood spots, and hair root bulbs.

Molecular Detection Techniques Used

  • Quantitative PCR (qPCR): Amplifies and quantifies DNA sequences in real-time, effective for detecting moderate to high DNA concentrations.
  • Digital PCR (dPCR): Provides absolute quantification of DNA by partitioning the sample, highly sensitive but less effective at detecting very low concentrations according to this study.
  • Next-Generation Sequencing (NGS): Offers comprehensive sequencing of DNA, allowing detection of vector sequences and confirmation with high specificity.

Key Findings

  • All three methods showed strong correlation in detecting the vector DNA, validating their use in monitoring gene doping.
  • Digital PCR displayed lower sensitivity at very low vector concentrations compared to qPCR and NGS.
  • High levels of vector DNA were detected in plasma and whole blood shortly after administration, with a sharp decline seen within 12 hours (0.5 days).
  • Vector levels dropped further to trace amounts by day 4 in plasma and day 9 in whole blood, consistent with prior related studies.
  • Dried blood spot samples showed detectable vector DNA but at lower levels than whole blood, with similar detection windows.
  • Importantly, vector DNA was detected in hair root bulbs from one horse more than a month post-administration, demonstrating persistence in this matrix.

Implications

  • This study establishes multiple sample types and molecular methods for gene doping detection in horses, aiding regulatory enforcement.
  • The detection of vector DNA in hair root bulbs long after administration suggests a novel, non-invasive matrix for future anti-doping tests in both animals and humans.
  • The rapid decline of the vector in blood highlights the need for timely sampling post-suspicion to maximize detection chances.
  • Comparison of qPCR, dPCR, and NGS informs selection of optimal techniques depending on expected DNA concentration and testing context.

Conclusion

  • The comprehensive approach combining multiple molecular detection methods and sampling matrices improves the detection of AAV-mediated gene doping in horses.
  • Long-term detection potential in hair follicles encourages further research to develop more effective, less invasive anti-doping protocols.
  • This work supports equine sports authorities in combating gene doping, preserving fairness and animal welfare.

Cite This Article

APA
Maniego J, Harding C, Habershon-Butcher J, Hincks P, Ryder E. (2024). Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques. Gene Ther, 31(9-10), 477-488. https://doi.org/10.1038/s41434-024-00462-0

Publication

Gene therapy
ISSN: 1476-5462
NlmUniqueID: 9421525
Country: England
Language: English
Volume: 31
Issue: 9-10
Pages: 477-488

Researcher Affiliations

Maniego, Jillian
  • Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
Harding, Caitlin
  • Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
Habershon-Butcher, Jocelyn
  • British Horseracing Authority, Holborn Gate, 26 Southampton Buildings, London, WC2A 1AN, UK.
Hincks, Pamela
  • Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK.
Ryder, Edward
  • Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK. edward.ryder@lgcgroup.com.

MeSH Terms

  • Horses / genetics
  • Animals
  • Dependovirus / genetics
  • Doping in Sports / methods
  • Genetic Vectors / genetics

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Citations

This article has been cited 2 times.
  1. Marchand A, Roulland I, Ericsson M. Improvement of EPO Transgene Detection From Polymeric Dried Blood Spots for Antidoping Application.. Drug Test Anal 2026 Feb;18(2):230-238.
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