Design and storage stability of reference materials for microfluidic quantitative PCR-based equine gene doping tests

In this 16th edition of the annual banned‐substance review on analytical approaches in human sports drug testing, literature on recent developments in this particular section of global anti‐doping efforts that was published between October 2022 and September 2023 is summarized and discussed. Most recent additions to the continuously growing portfolio of doping control analytical approaches and investigations into analytical challenges in the context of adverse analytical findings are presented, taking into account existing as well as emerging challenges in anti‐doping, with specific focus on substances and methods of doping recognized in the World Anti‐Doping Agency's 2023 Prohibited List. As in previous years, focus is put particularly on new or enhanced analytical options in human doping controls, appreciating the exigence and core mission of anti‐doping and, equally, the conflict arising from the opposingly trending extent of the athlete's exposome and the sensitivity of instruments nowadays commonly available in anti‐doping laboratories.

Section: Discussionmentioning

confidence: 99%

Annual banned‐substance review 16^th edition—Analytical approaches in human sports drug testing 2022/2023

Thevis,

Kuuranne,

Geyer

2023

“…89,90 Microfluidics QPCR (MFQPCR) has been used for simultaneous detection of 12 equine transgenes 91 spiked into equine plasma, and a custom reference material for multiplex assays was developed as a positive control. 92 Digital droplet PCR (ddPCR) has been shown to detect very low copy numbers of transgenes spiked into equine plasma 93 particularly if a preamplification step is used in a nested PCR method. 94 Combining PCR with NGS enables verification that the amplified PCR products correspond to the targeted transgene and has been validated using equine actinin alpha 3 (ACTN3), erythropoietin (EPO), hypoxia-inducible factor 1 subunit alpha (HIF1A), peroxisome proliferator-activated receptor delta (PPARD), and vascular endothelial growth factor A (VEGFA) transgenes spiked into equine plasma.…”

Section: In Vivo Raav Gene Therapy In Horsesmentioning

confidence: 99%

“…Duplexed quantitative PCR (QPCR) methods can detect human erythropoietin (hEPO), human growth hormone (hGH), human insulin‐like growth factor 1 (hIGF‐1), and equine EPO (eEPO) transgenes spiked into equine plasma and whole blood using the equine tubulin α 4A (TUBA4A) gene as an endogenous control 89,90 . Microfluidics QPCR (MFQPCR) has been used for simultaneous detection of 12 equine transgenes 91 spiked into equine plasma, and a custom reference material for multiplex assays was developed as a positive control 92 . Digital droplet PCR (ddPCR) has been shown to detect very low copy numbers of transgenes spiked into equine plasma 93 particularly if a pre‐amplification step is used in a nested PCR method 94 .…”

Section: Introductionmentioning

confidence: 99%

Administration and detection of gene therapy in horses: A systematic review

Haughan

Ortved

Robinson

2022

Gene therapy uses genetic modification of cells to produce a therapeutic effect.Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression.Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses.Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

Gene doping detection in the era of genomics

Li,

Su,

Shi

et al. 2024

Recent progress in gene editing has enabled development of gene therapies for many genetic diseases, but also made gene doping an emerging risk in sports and competitions. By delivery of exogenous transgenes into human body, gene doping not only challenges competition fairness but also places health risk on athletes. World Anti‐Doping Agency (WADA) has clearly inhibited the use of gene and cell doping in sports, and many techniques have been developed for gene doping detection. In this review, we will summarize the main tools for gene doping detection at present, highlight the main challenges for current tools, and elaborate future utilizations of high‐throughput sequencing for unbiased, sensitive, economic and large‐scale gene doping detections. Quantitative real‐time PCR assays are the widely used detection methods at present, which are useful for detection of known targets but are vulnerable to codon optimization at exon–exon junction sites of the transgenes. High‐throughput sequencing has become a powerful tool for various applications in life and health research, and the era of genomics has made it possible for sensitive and large‐scale gene doping detections. Non‐biased genomic profiling could efficiently detect new doping targets, and low‐input genomics amplification and long‐read third‐generation sequencing also have application potentials for more efficient and straightforward gene doping detection. By closely monitoring scientific advancements in gene editing and sport genetics, high‐throughput sequencing could play a more and more important role in gene detection and hopefully contribute to doping‐free sports in the future.