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

Zibin Jiang1, Joanne Haughan1, Kaitlyn L Moss1

  • 1Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.

Drug Testing and Analysis
|August 19, 2021
PubMed
Summary

A new quantitative polymerase chain reaction (QPCR) test can detect recombinant adeno-associated virus (rAAV) gene therapy in horses. This method aids in screening for gene doping, excluding wild-type AAV DNA.

Keywords:
AAVequinegene dopingprimers

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Area of Science:

  • Veterinary Medicine
  • Molecular Biology
  • Biotechnology

Background:

  • Gene therapy is prohibited in equine athletes, necessitating reliable detection methods.
  • Recombinant adeno-associated virus (rAAV) vectors are commonly used for in vivo gene therapy.
  • Conserved sequences in rAAV vectors present a target for detection, distinct from wild-type AAV (WTAAV).

Purpose of the Study:

  • To develop and validate a quantitative polymerase chain reaction (QPCR) screening test for detecting rAAV gene therapy products in horses.
  • To target conserved AAV sequences for broad detection of rAAV vectors, irrespective of transgene.
  • To ensure the test excludes the detection of WTAAV DNA.

Main Methods:

  • Development and validation of primer sets targeting conserved rAAV2 sequences and WTAAV2-specific sequences.
  • Administration of rAAV via intra-articular injection to six horses.
  • Collection and QPCR analysis of plasma and synovial fluid samples over 84 days.

Main Results:

  • rAAV DNA was detected in plasma of all horses for up to 2-4 days post-injection.
  • rAAV DNA was detected in synovial fluid for up to 28 days in some horses.
  • No WTAAV2 DNA was detected in any tested samples, confirming test specificity.

Conclusions:

  • This study presents the first QPCR test for screening rAAV vectors in horses, crucial for detecting potential gene doping.
  • The developed test is specific for rAAV and does not detect endogenous WTAAV.
  • This analytical method supports the integrity of equine sports by enabling detection of gene therapy use.