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

Gene Therapy volume 31, pages 477–488 (2024)Cite this article

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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.

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Fig. 1: Amplification of target from DNA extracted from plasma of two horses.
Fig. 2: qPCR, NGS, and dPCR comparison by assay and time for detection of rAAV8-CV in hair root bulbs for horse H1.
Fig. 3: Mean altered reference sequence motif hits in plasma samples at defined timepoints (hr) from duplicate reactions for the full rAAV-CV8 target pool of 20 NGS assays multiplexed into one reaction.
Fig. 4: Robustness assessment of matrices and molecular techniques based on different criteria for assay and replicate amplification, showing detection time in hours.
Fig. 5: rAAV8-CV post-administration detection time comparison.

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Data availability

Sequence information, computer code and resources are available by permission from the British Horseracing Authority under an appropriate materials transfer agreement.

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Acknowledgements

The British Horseracing Authority for funding the analytical work carried out for this study. Staff and students at the Centre for Racehorse Studies (CRS), UK, are also acknowledged for their care and sampling of the horses involved in this work.

Funding

This work was funded by the British Horseracing Authority (BHA).

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Author notes

  1. These authors contributed equally: Jillian Maniego, Caitlin Harding.

Authors and Affiliations

  1. Sport and Specialised Analytical Services, LGC, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, UK

    Jillian Maniego, Caitlin Harding, Pamela Hincks & Edward Ryder

  2. British Horseracing Authority, Holborn Gate, 26 Southampton Buildings, London, WC2A 1AN, UK

    Jocelyn Habershon-Butcher

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Contributions

ER and PH conceived and secured funding for the study. Experimental data was generated and analysed by JM, CH, and ER. JH-B performed the administration of the rAAV8-CV vector and was responsible for the veterinary health of the animals during the study. All authors were involved in the writing and editing of the manuscript.

Corresponding author

Correspondence to Edward Ryder.

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Competing interests

Sport and Specialised Analytical Services, LGC Assure, have a commercial interest in developing tests based on gene doping detection.

Ethical approval

Ethical approval was obtained for this study from the BHA Ethics Board. The care and use of all animals in this study were in accordance with the UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986.

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

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