Drug testing and analysis2020; 12(6); 743-751; doi: 10.1002/dta.2785

Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma.

Abstract: Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine IL-10 (rAAV-IL10). Twelve horses were randomly assigned to receive an intra-articular injection of rAAV-IL10 or phosphate buffered saline (vehicle) into a middle carpal joint. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Primer pairs were designed to detect two unique regions of rAAV. Using quantitative real time PCR, both sets of primers detected rAAV for 14-28 days in joints and up to 4 days in plasma, in all six treated horses. In synovial fluid, rAAV was detected on day 56 in 4/6 horses by both primer sets, and on day 84 in 1/6 horses by one primer set. In plasma, rAAV was detected for 7 days in 5/6 horses, 14 days in 2/6 horses, and 28 days in 1/6 horses by one primer set, and was detected for up to 14 days in 1/6 horses by the other primer set. This study is the first to validate that quantitative real time PCR can be used to systemically detect the local administration of a gene therapy product to horses.
Publication Date: 2020-03-16 PubMed ID: 32133745DOI: 10.1002/dta.2785Google Scholar: Lookup
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  • Journal Article

Summary

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The study focuses on developing a method to detect the administration of an experimental gene therapy to horses, through the sampling of synovial fluid and plasma. It showcases the use of quantitative real time PCR in detecting the local administration of a gene therapy product.

Overview of the Research

  • In experiments relating to gene therapy, a recombinant adeno-associated viral vector (rAAV) carrying a transgene for the anti-inflammatory cytokine IL-10 (rAAV-IL10) was used.
  • 12 horses were injected with either the rAAV-IL10 or a phosphate-buffered saline (as a control) in a middle carpal joint.
  • Samples of synovial fluid and plasma were collected from the animals at specified time intervals, ranging from the day of injection – day 0, to 84 days post-injection.

Methodology and Findings

  • Two unique antigenic regions on the rAAV were targeted in the detection process, using primer pairs in a process known as quantitative real time PCR (Polymerase Chain Reaction).
  • The rAAV was detected in the joint fluid for a period of 14-28 days post injection, and in the plasma for up to 4 days, in all six horses which received the gene therapy.
  • rAAV was still detectable in synovial fluid on day 56 in 4/6 of these horses, and on day 84 in 1/6 of the horses, by at least one of the primer sets.
  • In plasma, rAAV was detected for 7 days in 5/6 horses, 14 days in 2/6 horses, and 28 days in 1/6 horses by one primer set, and it was detected for up to 14 days in 1/6 of the horses by the other primer set.

Significance of the Study

  • The study shows that quantitative real time PCR can be used effectively and accurately to detect the presence and monitor the persistence of gene therapy vectors in equine subjects.
  • The technique can help in enforcing the regulations prohibiting the use of gene therapy in equine athletes.
  • This is the first study to validate the use of quantitative real time PCR in such a way, laying the groundwork for future research and development in this field.

Cite This Article

APA
Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA. (2020). Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma. Drug Test Anal, 12(6), 743-751. https://doi.org/10.1002/dta.2785

Publication

ISSN: 1942-7611
NlmUniqueID: 101483449
Country: England
Language: English
Volume: 12
Issue: 6
Pages: 743-751

Researcher Affiliations

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

MeSH Terms

  • Animals
  • DNA Primers / blood
  • Dependovirus / genetics
  • Doping in Sports / methods
  • Genetic Therapy / methods
  • Horses / metabolism
  • Injections, Intra-Articular
  • Interleukin-10 / blood
  • Limit of Detection
  • Real-Time Polymerase Chain Reaction / methods
  • Reproducibility of Results
  • Synovial Fluid / chemistry

Grant Funding

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

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