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Gene Doping Control in Human and Animal Sports
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Gene Doping Control in Human and Animal Sports

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: 20 September 2024 | Viewed by 5227

Special Issue Editor

Dr. Teruaki Tozaki

E-Mail Website
Guest Editor
Genetic Analysis Department, Laboratory of Racing Chemistry, Utsunomiya 320-0851, Japan
Interests: gene doping; gene editing; gene therapy; cell doping; cell therapy; therapeutic oligonucleotide, horse, thoroughbred

Special Issue Information

Dear Colleagues,

Gene doping is prohibited in human and animal sports, including horseracing and equine competition, for fair competition and their welfare. Gene doping, in a narrow sense, is the method of introducing exogenous genes (called transgenes) into the body; however, with progression in science and technology, various methods, including gene silencing (therapeutic oligonucleotide), have emerged as potential gene doping methods. More recently, the emergence of genome editing (also known as CRISPR) as a more refined gene therapy technique has highlighted the potential for editing an egg to produce genetically modified animals. This type of gene doping is a concern for biodiversity and ethics as the modified genome introduced into an egg would be heritable.

As the Guest Editor of IJMS for this Special Issue, “Gene Doping in Human and Animal Sports”, I expect submissions from many researchers working across the wide spectrum of gene doping control in human and animal sports. This special issue deals with the latest advances in gene therapy and genome editing therapy, detection methods for monitoring these techniques, problems in sports and horseracing caused by gene doping, and the corresponding welfare and ethical issues of gene doping.

Dr. Teruaki Tozaki
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • cell therapy
  • cell doping
  • gene doping
  • gene editing
  • gene silencing
  • gene therapy
  • transgene
  • CRISPR, therapeutic oligonucleotide

Published Papers (5 papers)

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Research

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16 pages, 3797 KiB  
Article
Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography–High-Resolution Mass Spectrometry
by Emily Helmes, Jacob Montgomery, Gwendolyne Alarcio, Herra G. Mendoza, Jeffrey A. Blea, Peter A. Beal and Benjamin C. Moeller
Int. J. Mol. Sci. 2024, 25(11), 5752; https://doi.org/10.3390/ijms25115752 - 25 May 2024
Viewed by 322
Abstract
There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression [...] Read more.
There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts. Full article
(This article belongs to the Special Issue Gene Doping Control in Human and Animal Sports)
21 pages, 778 KiB  
Article
PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry
by Tessa Wilkin, Natasha A. Hamilton, Adam T. Cawley, Somanath Bhat and Anna Baoutina
Int. J. Mol. Sci. 2024, 25(5), 2570; https://doi.org/10.3390/ijms25052570 - 22 Feb 2024
Viewed by 879
Abstract
The term ‘gene doping’ is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based [...] Read more.
The term ‘gene doping’ is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia. Full article
(This article belongs to the Special Issue Gene Doping Control in Human and Animal Sports)
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13 pages, 1573 KiB  
Article
Identification of Potential miRNA Biomarkers to Detect Hydrocortisone Administration in Horses
by Mio Kikuchi, Taichiro Ishige, Yohei Minamijima, Kei-ichi Hirota, Shun-ichi Nagata, Teruaki Tozaki, Hironaga Kakoi, Toshina Ishiguro-Oonuma and Keiichiro Kizaki
Int. J. Mol. Sci. 2023, 24(19), 14515; https://doi.org/10.3390/ijms241914515 - 25 Sep 2023
Cited by 1 | Viewed by 967
Abstract
Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this [...] Read more.
Circulating microRNAs (miRNAs) are stable in bodily fluids and are potential biomarkers of various diseases and physiological states. Although several studies have been conducted on humans to detect drug doping by miRNAs, research on drugs and miRNAs in horses is limited. In this study, circulating miRNAs in horses after hydrocortisone administration were profiled and variations in miRNAs affected by hydrocortisone administration during endogenous hydrocortisone elevation were examined. The miRNAs were extracted from thoroughbred horse plasma before and after hydrocortisone administration and subjected to small RNA sequencing and reverse transcription quantitative PCR (RT-qPCR). RT-qPCR validation was performed for the 20 miRNAs that were most affected by hydrocortisone administration. The effects of elevated endogenous hydrocortisone levels due to exercise and adrenocorticotropic hormone administration were also confirmed. The validation results showed that approximately half of the miRNAs showed the same significant differences as those obtained using small RNA sequencing. Among the twenty miRNAs, two novel miRNAs and miR-133a were found to vary differently between exogenous hydrocortisone administration and endogenous hydrocortisone elevation. This study provides basic knowledge regarding the circulating miRNA profile of horses after hydrocortisone administration and identifies three miRNAs that could potentially be used as biomarkers to detect hydrocortisone administration. Full article
(This article belongs to the Special Issue Gene Doping Control in Human and Animal Sports)
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Review

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13 pages, 655 KiB  
Review
Whispers in the Blood: Leveraging MicroRNAs for Unveiling Autologous Blood Doping in Athletes
by Mehdi Hassanpour and Amankeldi A. Salybekov
Int. J. Mol. Sci. 2024, 25(1), 249; https://doi.org/10.3390/ijms25010249 - 23 Dec 2023
Cited by 1 | Viewed by 859
Abstract
The prevalence of autologous blood transfusions (ABTs) presents a formidable challenge in maintaining fair competition in sports, as it significantly enhances hemoglobin mass and oxygen capacity. In recognizing ABT as a prohibited form of doping, the World Anti-Doping Agency (WADA) mandates stringent detection [...] Read more.
The prevalence of autologous blood transfusions (ABTs) presents a formidable challenge in maintaining fair competition in sports, as it significantly enhances hemoglobin mass and oxygen capacity. In recognizing ABT as a prohibited form of doping, the World Anti-Doping Agency (WADA) mandates stringent detection methodologies. While current methods effectively identify homologous erythrocyte transfusions, a critical gap persists in detecting autologous transfusions. The gold standard practice of longitudinally monitoring hematological markers exhibits promise but is encumbered by limitations. Despite its potential, instances of blood doping often go undetected due to the absence of definitive verification processes. Moreover, some cases remain unpenalized due to conservative athlete-sanctioning approaches. This gap underscores the imperative need for a more reliable and comprehensive detection method capable of unequivocally differentiating autologous transfusions, addressing the challenges faced in accurately identifying such prohibited practices. The development of an advanced detection methodology is crucial to uphold the integrity of anti-doping measures, effectively identifying and penalizing instances of autologous blood transfusion. This, in turn, safeguards the fairness and equality essential to competitive sports. Our review tackles this critical gap by harnessing the potential of microRNAs in ABT doping detection. We aim to summarize alterations in the total microRNA profiles of erythrocyte concentrates during storage and explore the viability of observing these changes post-transfusion. This innovative approach opens avenues for anti-doping technologies and commercialization, positioning it as a cornerstone in the ongoing fight against doping in sports and beyond. The significance of developing a robust detection method cannot be overstated, as it ensures the credibility of anti-doping efforts and promotes a level playing field for all athletes. Full article
(This article belongs to the Special Issue Gene Doping Control in Human and Animal Sports)
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Other

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10 pages, 2981 KiB  
Case Report
Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs
by Nana Naumann, Alina Paßreiter, Andreas Thomas, Oliver Krug, Katja Walpurgis and Mario Thevis
Int. J. Mol. Sci. 2023, 24(21), 15835; https://doi.org/10.3390/ijms242115835 - 31 Oct 2023
Cited by 3 | Viewed by 1322
Abstract
Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use [...] Read more.
Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time. Full article
(This article belongs to the Special Issue Gene Doping Control in Human and Animal Sports)
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