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Home / Equine Research Bank / Drug Test Anal / A quantitative PCR screening method for adeno-associated vir...
Drug testing and analysis2021; 14(5); 963-972; doi: 10.1002/dta.3152

A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping.

Abstract: Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra-articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2-4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses.
© 2021 John Wiley & Sons, Ltd.
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Publication Date: 2021-09-03 PubMed ID: 34412153DOI: 10.1002/dta.3152Google Scholar: Lookup
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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.

The researchers have developed a new technique using quantitative polymerase chain reaction (QPCR) to detect any form of gene doping, involving recombinant adeno-associated virus (rAAV) vectors, in racehorses. This method distinguishes between wild type AAV2 and gene therapy products in equine plasma and synovial fluid samples.

Objective and Methodology

  • The primary aim of this investigation was to devise a QPCR screening tool that zeroes in on conserved AAV gene sequences. This method theoretically provides coverage for all rAAV-based gene therapies, no matter what particular transgene is encoded, while excluding the possibility of wild type AAV existing in horses.
  • To test this, researchers created and validated primer sets. These primer sets are specifically designed to react to an AAV2 DNA sequence, that is conserved across various rAAV vectors, and a sequence exclusive to wild type AAV2.
  • In the study, six horses were given an intra-articular injection of rAAV. On a systematic timeline, plasma and synovial fluid were collected from the subjects.

Findings

  • Using their QPCR method, the study team found rAAV in each horse’s plasma for a span of 2-4 days. Synovial fluid samples, available from two equines, showed a trace of rAAV DNA for up to 28 days.
  • Importantly, this methodology did not yield any false-positive results, with no instances of WTAAV2 DNA identified in the collected samples.

Conclusion

  • This specific experimental method marks the first instance of a QPCR test, formulated to monitor any potential use of rAAV vectors in equine gene doping.
  • The successful implementation of this assay can act as a crucial step in ensuring the integrity of competitive equine events and advancing the frontier in detecting performance-enhancing methods in sports.

Cite This Article

APA
Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA. (2021). A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping. Drug Test Anal, 14(5), 963-972. https://doi.org/10.1002/dta.3152

Publication

Drug testing and analysis
ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 14
Issue: 5
Pages: 963-972

Researcher Affiliations

Jiang, Zibin
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Haughan, Joanne
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Moss, Kaitlyn L
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Stefanovski, Darko
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Ortved, Kyla F
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Robinson, Mary A
  • Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
  • Pennsylvania Equine Toxicology and Research Laboratory, West Chester University, West Chester, Pennsylvania, USA.

MeSH Terms

  • Animals
  • DNA, Viral / genetics
  • Dependovirus / genetics
  • Horses / genetics
  • Humans
  • Real-Time Polymerase Chain Reaction / methods

Grant Funding

  • McCabe Fund, University of Pennsylvania
  • Pennsylvania State Horse Racing Commission
  • Pennsylvania Horse Breeders Association

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Citations

This article has been cited 3 times.
  1. Lu Y, Yan J, Ou G, Fu L. A Review of Recent Progress in Drug Doping and Gene Doping Control Analysis.. Molecules 2023 Jul 18;28(14).
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  2. Tozaki T, Ohnuma A, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Takahashi Y, Nagata SI. Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests.. J Equine Sci 2023 Jun;34(2):21-27.
    doi: 10.1294/jes.34.21pubmed: 37405066google scholar: lookup
  3. Tozaki T, Ohnuma A, Nakamura K, Hano K, Takasu M, Takahashi Y, Tamura N, Sato F, Shimizu K, Kikuchi M, Ishige T, Kakoi H, Hirota KI, Hamilton NA, Nagata SI. Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control.. Genes (Basel) 2022 Sep 4;13(9).
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