Gene doping detection in the era of genomics.

Publisher: John Wiley & Sons Country of Publication: England NLM ID: 101483449 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1942-7611 (Electronic) Linking ISSN: 19427603 NLM ISO Abbreviation: Drug Test Anal Subsets: MEDLINE

Original Publication: Chichester, UK : John Wiley & Sons

Doping in Sports*/prevention & control ; Doping in Sports*/methods ; Genomics*/methods ; Substance Abuse Detection*/methods ; High-Throughput Nucleotide Sequencing*/methods; Humans ; Real-Time Polymerase Chain Reaction/methods

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.
(© 2024 John Wiley & Sons Ltd.)

Bizzotto S, Dou Y, Ganz J, et al. Landmarks of human embryonic development inscribed in somatic mutations. Science. 2021;371(6535):1249‐1253. doi:10.1126/science.abe1544.
Rockweiler NB, Ramu A, Nagirnaja L, et al. The origins and functional effects of postzygotic mutations throughout the human life span. Science. 2023;380(6641):eabn7113. doi:10.1126/science.abn7113.
Tomasetti C, Li L, Vogelstein B. Stem cell divisions, somatic mutations, cancer etiology, and cancer prevention. Science. 2017;355(6331):1330‐1334. doi:10.1126/science.aaf9011.
Cortés‐Ciriano I, Gulhan DC, Lee JJ‐K, Melloni GEM, Park PJ. Computational analysis of cancer genome sequencing data. Nat Rev Genet. 2022;23(5):298‐314. doi:10.1038/s41576‐021‐00431‐y.
Saha K, Sontheimer EJ, Brooks PJ, et al. The NIH somatic cell genome editing program. Nature. 2021;592(7853):195‐204. doi:10.1038/s41586‐021‐03191‐1.
Collins FS, Doudna JA, Lander ES, Rotimi CN. Human molecular genetics and genomics—important advances and exciting possibilities. New Engl J Med. 2021;384(1):1‐4. doi:10.1056/NEJMp2030694.
Prince HM. Gene transfer: a review of methods and applications. Pathology. 1998;30(4):335‐347. doi:10.1080/00313029800169606.
Sun W, Wang H. Recent advances of genome editing and related technologies in China. Gene Ther. 2020;27(7‐8):312‐320. doi:10.1038/s41434‐020‐0181‐5.
Li G, Li X, Zhuang S, et al. Gene editing and its applications in biomedicine. Sci China Life Sci. 2022;65(4):660‐700. doi:10.1007/s11427‐021‐2057‐0.
Wang JY, Doudna JA. CRISPR technology: a decade of genome editing is only the beginning. Science. 2023;379(6629):251. doi:10.1126/science.add8643.
Shivram H, Cress BF, Knott GJ, Doudna JA. Controlling and enhancing CRISPR systems. Nat Chem Biol. 2021;17(1):10‐19. doi:10.1038/s41589‐020‐00700‐7.
Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 2021;6(1):53. doi:10.1038/s41392‐021‐00487‐6.
Maeder ML, Gersbach CA. Genome‐editing technologies for gene and cell therapy. Mol Ther. 2016;24(3):430‐446. doi:10.1038/mt.2016.10.
Doudna JA. The promise and challenge of therapeutic genome editing. Nature. 2020;578(7794):229‐236. doi:10.1038/s41586‐020‐1978‐5.
Kan MJ, Doudna JA. Treatment of genetic diseases with CRISPR genome editing. Jama. 2022;328(10):980‐981. doi:10.1001/jama.2022.13468.
Arnold C. Record number of gene‐therapy trials, despite setbacks. Nat Med. 2021;27(8):1312‐1315. doi:10.1038/s41591‐021‐01467‐7.
Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359(6372):175. doi:10.1126/science.aan4672.
Reardon S. 'It's a vote for hope': first gene therapy for muscular dystrophy nears approval, but will it work? Nature. 2023;618(7965):451‐453. doi:10.1038/d41586‐023‐01799‐z.
Wong C. UK first to approve CRISPR treatment for diseases: what you need to know. Nature. 2023;623(7988):676‐677. doi:10.1038/d41586‐023‐03590‐6.
Fischetto G, Bermon S. From gene engineering to gene modulation and manipulation: can we prevent or detect gene doping in sports? Sports Med. 2013;43(10):965‐977. doi:10.1007/s40279‐013‐0075‐4.
Unal M, Ozer Unal D. Gene doping in sports. Sports Med. 2004;34(6):357‐362. doi:10.2165/00007256‐200434060‐00002.
Azzazy HME. Gene doping. In: Thieme D, Hemmersbach P, eds. Doping in Sports: Biochemical Principles, Effects and Analysis. Springer Berlin Heidelberg; 2010:485‐512.
Gould D. Gene doping: gene delivery for Olympic victory. Br J Clin Pharmacol. 2013;76(2):292‐298. doi:10.1111/bcp.12010.
Battery L, Solomon A, Gould D. Gene doping: Olympic genes for Olympic dreams. J R Soc Med. 2011;104(12):494‐500. doi:10.1258/jrsm.2011.110240.
Filipp F. Is science killing sport? Gene therapy and its possible abuse in doping. EMBO Rep. 2007;8(5):433‐435. doi:10.1038/sj.embor.7400968.
Bird SR, Goebel C, Burke LM, Greaves RF. Doping in sport and exercise: anabolic, ergogenic, health and clinical issues. Ann Clin Biochem. 2016;53(2):196‐221. doi:10.1177/0004563215609952.
Schneider AJ, Friedmann T. The problem of doping in sports. In: Advances in Genetics. Vol.51. Academic Press; 2006:1‐9. doi:10.1016/S0065‐2660(06)51001‐6.
Saugy M, Lundby C, Robinson N. Monitoring of biological markers indicative of doping: the athlete biological passport. Br J Sports Med. 2014;48(10):827‐832. doi:10.1136/bjsports‐2014‐093512.
WADA. 2021. Athlete biological passport operating guidelines. https://www.wada-ama.org/sites/default/files/resources/files/guidelines_abp_v8_final.pdf.
Pincock S. Gene doping. Lancet. 2005;366:S18‐S19. doi:10.1016/S0140‐6736(05)67829‐4.
Friedmann T, Rabin O, Frankel MS. Gene doping and sport. Science. 2010;327(5966):647‐648. doi:10.1126/science.1177801.
Gineviciene V, Utkus A, Pranckeviciene E, et al. Perspectives in sports genomics. Biomedicine. 2022;10(2):298. doi:10.3390/biomedicines10020298.
Lopez S, Meirelles J, Rayol V, et al. Gene doping and genomic science in sports: where are we? Bioanalysis. 2020;12(11):801‐811. doi:10.4155/bio‐2020‐0093.
Diamanti‐Kandarakis E, Konstantinopoulos PA, Papailiou J, Kandarakis SA, Andreopoulos A, Sykiotis GP. Erythropoietin abuse and erythropoietin gene doping—detection strategies in the genomic era. Sports Med. 2005;35(10):831‐840. doi:10.2165/00007256‐200535100‐00001.
Baoutina A, Coldham T, Bains GS, Emslie KR. Gene doping detection: evaluation of approach for direct detection of gene transfer using erythropoietin as a model system. Gene Ther. 2010;17(8):1022‐1032. doi:10.1038/gt.2010.49.
Baoutina A, Bhat S, Li DK, Emslie KR. Towards a robust test to detect gene doping for anabolic enhancement in human athletes. Drug Test Anal. 2023;15(3):314‐323. doi:10.1002/dta.3411.
Yanazawa K, Sugasawa T, Aoki K, Nakano T, Kawakami Y, Takekoshi K. Development of a gene doping detection method to detect overexpressed human follistatin using an adenovirus vector in mice. PeerJ. 2021;9:e12285. doi:10.7717/peerj.12285.
van der Gronde T, de Hon O, Haisma HJ, Pieters T. Gene doping: an overview and current implications for athletes. Br J Sports Med. 2013;47(11):670‐678. doi:10.1136/bjsports‐2012‐091288.
Kovac M, Litvin YA, Aliev RO, et al. Gene therapy using plasmid DNA encoding vascular endothelial growth factor 164 and fibroblast growth factor 2 genes for the treatment of horse tendinitis and desmitis: case reports. Front Vet Sci. 2017;4:168. doi:10.3389/fvets.2017.00168.
Kovac M, Litvin YA, Aliev RO, et al. Gene therapy using plasmid DNA encoding VEGF164 and FGF2 genes: a novel treatment of naturally occurring tendinitis and desmitis in horses. Front Pharmacol. 2018;9:978. doi:10.3389/fphar.2018.00978.
Bara S, Barczuk P, Gałajda E, et al. A review of the possibilities of gene doping in sports focused on the advantages and disadvantages. J Educ Health Sport. 2023;42(1):35‐45. doi:10.12775/JEHS.2023.42.01.003.
Fallahi A, Ravasi A, Farhud D. Genetic doping and health damages. Iran J Public Health. 2011;40(1):1‐14.
Friedmann T. How close are we to gene doping? Hastings Cent Rep. 2010;40(2):20‐22. doi:10.1353/hcr.0.0246.
Li CW, Samulski RJ. Engineering adeno‐associated virus vectors for gene therapy. Nat Rev Genet. 2020;21(4):255‐272. doi:10.1038/s41576‐019‐0205‐4.
Smalley E. FDA warns public of dangers of DIY gene therapy. Nat Biotechnol. 2018;36(2):119‐120. doi:10.1038/nbt0218‐119.
Raguram A, Banskota S, Liu DR. Therapeutic in vivo delivery of gene editing agents. Cell. 2022;185(15):2806‐2827. doi:10.1016/j.cell.2022.03.045.
Picanco‐Castro V, Pereira CG, Covas DT, et al. Emerging patent landscape for non‐viral vectors used for gene therapy. Nat Biotechnol. 2020;38(2):151‐158. doi:10.1038/s41587‐019‐0402‐x.
Kaiser J. How safe is a popular gene therapy vector? Science. 2020;367(6474):131. doi:10.1126/science.367.6474.131.
Brown J. Genetic doping: WADA we do about the future of ‘cheating’ in sport? The Int Sports Law J. 2019;19(3‐4):258‐280. doi:10.1007/s40318‐019‐00141‐y.
Ehrbar JT. Ethical considerations of genetic manipulation in sport. Sport J. 2015. doi:10.17682/sportjournal/2015.018.
Triviño JLP. Gene doping and the ethics of sport: between enhancement and posthumanism. Int J Sports Sci. 2011;1:1‐8.
Oliveira RS, Collares TF, Smith KR, Collares TV, Seixas FK. The use of genes for performance enhancement: doping or therapy? Braz J Med Biol Res. 2011;44(12):1194‐1201. doi:10.1590/S0100‐879X2011007500145.
Koerner S. Spill‐over effect and functional illegality—towards a sociology of gene doping. Adv Phys Educ. 2017;7(01):60‐69. doi:10.4236/ape.2017.71006.
Thompson H. Performance enhancement: superhuman athletes. Nature. 2012;487(7407):287‐289. doi:10.1038/487287a.
Foddy B, Savulescu J. Ethics of performance enhancement in sport: drugs and gene doping. In: Principles of Health Care Ethics; 2006:511‐519. doi:10.1002/9780470510544.ch70.
Cantelmo RA, da Silva AP, Mendes‐Junior CT, Dorta DJ. Gene doping: present and future. Eur J Sport Sci. 2020;20(8):1093‐1101. doi:10.1080/17461391.2019.1695952.
Wells DJ. Gene doping: possibilities and practicalities. Med Sport Sci. 2009;54:166‐175. doi:10.1159/000235703.
WADA. 2019. Prohibited list. https://www.wada-ama.org/sites/default/files/wada_2019_english_prohibited_list.pdf.
WADA. 2021. Laboratory guidelines‐gene doping detection based on polymerase chain reaction (PCR). https://www.wada-ama.org/en/resources/laboratory-guidelines-gene-doping-detection-based-polymerase-chain-reaction-pcr.
Parzeller M. The punishment of gene doping—the relation between WADA prohibited lists, German Medicinal Products Act, German Doping Agents Amounts Ordinance, and Basic Law of the Federal Republic of Germany. Drug Test Anal. 2011;3(10):688‐694. doi:10.1002/dta.326.
Lu Y, Yan J, Ou G, Fu L. A review of recent Progress in drug doping and gene doping control analysis. Molecules. 2023;28(14):5483. doi:10.3390/molecules28145483.
Baoutina A. A brief history of the development of a gene doping test. Bioanalysis. 2020;12(11):723‐727. doi:10.4155/bio‐2020‐0056.
Baoutina A, Bhat S, Zheng M, et al. Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy. Gene Ther. 2016;23(10):708‐717. doi:10.1038/gt.2016.47.
Aoki K, Sugasawa T, Yanazawa K, et al. The detection of trans gene fragments of hEPO in gene doping model mice by Taqman qPCR assay. PeerJ. 2020;8:e8595. doi:10.7717/peerj.8595.
Sugasawa T, Aoki K, Watanabe K, et al. Detection of transgenes in gene delivery model mice by adenoviral vector using ddPCR. Gene. 2019;10(6):436. doi:10.3390/genes10060436.
Scarano S, Ermini ML, Spirit MM, et al. Simultaneous detection of transgenic DNA by surface plasmon resonance imaging with potential application to gene doping detection. Anal Chem. 2011;83(16):6245‐6253. doi:10.1021/ac200877m.
Paßreiter A, Thomas A, Grogna N, Delahaut P, Thevis M. First steps toward uncovering gene doping with CRISPR/Cas by identifying SpCas9 in plasma via HPLC–HRMS/MS. Anal Chem. 2020;92(24):16322‐16328. doi:10.1021/acs.analchem.0c04445.
Baoutina A, Alexander IE, Rasko JE, et al. Developing strategies for detection of gene doping. J Gene Med. 2008;10(1):3‐20. doi:10.1002/jgm.1114.
Naumann N, Paßreiter A, Thomas A, Krug O, Walpurgis K, Thevis M. Analysis of potential gene doping preparations for transgenic DNA in the context of sports drug testing programs. Int J Mol Sci. 2023;24(21):15835. doi:10.3390/ijms242115835.
Minunni M, Scarano S, Mascini M. Affinity‐based biosensors as promising tools for gene doping detection. Trends Biotechnol. 2008;26(5):236‐243. doi:10.1016/j.tibtech.2008.02.005.
Salamin O, Kuuranne T, Saugy M, Leuenberger N. Loop‐mediated isothermal amplification (LAMP) as an alternative to PCR: a rapid on‐site detection of gene doping. Drug Test Anal. 2017;9(11‐12):1731‐1737. doi:10.1002/dta.2324.
Beiter T, Zimmermann M, Fragasso A, et al. Establishing a novel single‐copy primer‐internal intron‐spanning PCR (spiPCR) procedure for the direct detection of gene doping. Exerc Immunol Rev. 2008;14:73‐85.
Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real‐time RT‐PCR. Nat Protoc. 2006;1(3):1559‐1582. doi:10.1038/nprot.2006.236.
Baoutina A, Coldham T, Fuller B, Emslie KR. Improved detection of transgene and nonviral vectors in blood. Hum Gene Ther Methods. 2013;24(6):345‐354. doi:10.1089/hgtb.2013.128.
Moser DA, Braga L, Raso A, Zacchigna S, Giacca M, Simon P. Transgene detection by digital droplet PCR. PLoS ONE. 2014;9(11):e111781. doi:10.1371/journal.pone.0111781.
Wang J, Bi X, Chen W, et al. Identification of the insertion site of transgenic DNA based on cyclization of the target gene with the flanking sequence and nested inverse PCR. Talanta Open. 2021;3:100033. doi:10.1016/j.talo.2021.100033.
Neuberger EWI, Perez I, Le Guiner C, et al. Establishment of two quantitative nested qPCR assays targeting the human EPO transgene. Gene Ther. 2016;23(4):330‐339. doi:10.1038/gt.2016.2.
Yi JY, Kim M, Min H, et al. New application of the CRISPR‐Cas9 system for site‐specific exogenous gene doping analysis. Drug Test Anal. 2021;13(4):871‐875. doi:10.1002/dta.2980.
Passreiter A, Naumann N, Thomas A, et al. How to detect CRISPR with CRISPR—employing SHERLOCK for doping control purposes. Analyst. 2022;147(23):5528‐5536. doi:10.1039/D2AN01318E.
Yan J, Xu Z, Zhou H, et al. Integration of CRISPR/Cas12a and multiplexed RPA for fast detection of gene doping. Anal Chem. 2022;94(47):16481‐16490. doi:10.1021/acs.analchem.2c04079.
Yi J‐Y, Kim M, Ahn JH, Kim BG, Son J, Sung C. CRISPR/deadCas9‐based high‐throughput gene doping analysis (HiGDA): a proof of concept for exogenous human erythropoietin gene doping detection. Talanta. 2023;258:124455. doi:10.1016/j.talanta.2023.124455.
de Boer EN, van der Wouden PE, Johansson LF, van Diemen CC, Haisma HJ. A next‐generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA. Gene Ther. 2019;26(7‐8):338‐346. doi:10.1038/s41434‐019‐0091‐6.
Wonkam A. Sequence three million genomes across Africa. Nature. 2021;590(7845):209‐211. doi:10.1038/d41586‐021‐00313‐7.
Editorials. The next 20 years of human genomics must be more equitable and more open. Nature. 2021;590(7845):183‐184. doi:10.1038/d41586‐021‐00328‐0.
Azzazy HME, Mansour MMH, Christenson RH. Gene doping: of mice and men. Clin Biochem. 2009;42(6):435‐441. doi:10.1016/j.clinbiochem.2009.01.001.
Neuhaus CP, Parent B. Gene doping‐in animals? Ethical issues at the intersection of animal use, gene editing, and sports ethics. Camb Q Healthc Ethics. 2019;28(1):26‐39. doi:10.1017/S096318011800035X.
Cyranoski D. The CRISPR‐baby scandal: what's next for human gene‐editing. Nature. 2019;566(7745):440‐442. doi:10.1038/d41586‐019‐00673‐1.
Wilkin T, Baoutina A, Hamilton N. Equine performance genes and the future of doping in horseracing. Drug Test Anal. 2017;9(9):1456‐1471. doi:10.1002/dta.2198.
Tozaki T, Hamilton NA. Control of gene doping in human and horse sports. Gene Ther. 2022;29(3‐4):107‐112. doi:10.1038/s41434‐021‐00267‐5.
Tozaki T, Kwak HG, Nakamura K, et al. Sequence determination of phosphorothioated oligonucleotides using MALDI–TOF mass spectrometry for controlling gene doping in equestrian sports. Drug Test Anal. 2022;14(1):175‐180. doi:10.1002/dta.3154.
Ohnuma A, Tozaki T, Kikuchi M, et al. Multiplex detection of transgenes using πCode technology for gene doping control. Anal Chem. 2023;95(27):10149‐10154. doi:10.1021/acs.analchem.3c00988.
Wong KS, Cheung HW, Szeto CWL, Tsang CYN, Wan TSM, Ho ENM. A multiplex qPCR assay for transgenes detection: a novel approach for gene doping control in horseracing using conventional laboratory setup. Drug Test Anal. 2023;15(8):879‐888. doi:10.1002/dta.3483.
Tozaki T, Ohnuma A, Kikuchi M, et al. Design and storage stability of reference materials for microfluidic quantitative PCR‐based equine gene doping tests. J Equine Sci. 2021;32(4):2125. doi:10.1294/jes.32.125.
Haughan J, Jiang Z, Stefanovski D, Moss KL, Ortved KF, Robinson MA. Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma. Drug Test Anal. 2020;12(6):743‐751. doi:10.1002/dta.2785.
Cheung HW, Wong KS, Lin VYC, Wan TSM, Ho ENM. A duplex qPCR assay for human erythropoietin (EPO) transgene to control gene doping in horses. Drug Test Anal. 2021;13(1):113‐121. doi:10.1002/dta.2907.
Cheung HW, Wong KS, Lin VYC, et al. Optimization and implementation of four duplex quantitative polymerase chain reaction assays for gene doping control in horseracing. Drug Test Anal. 2022;14(9):1587‐1598. doi:10.1002/dta.3328.
Tozaki T, Ohnuma A, Kikuchi M, et al. Microfluidic quantitative PCR detection of 12 transgenes from horse plasma for gene doping control. Gene. 2020;11(4):457. doi:10.3390/genes11040457.
Jiang Z, Haughan J, Moss KL, Stefanovski D, Ortved KF, Robinson MA. A quantitative PCR screening method for adeno‐associated viral vector 2‐mediated gene doping. Drug Test Anal. 2022;14(5):963‐972. doi:10.1002/dta.3152.
Tozaki T, Ohnuma A, Iwai S, et al. Robustness of digital PCR and real‐time PCR in transgene detection for gene‐doping control. Anal Chem. 2021;93(18):7133‐7139. doi:10.1021/acs.analchem.1c01173.
Tozaki T, Gamo S, Takasu M, et al. Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection. BMC Res Notes. 2018;11(1):708. doi:10.1186/s13104‐018‐3815‐6.
Tozaki T, Ohnuma A, Takasu M, et al. Droplet digital PCR detection of the erythropoietin transgene from horse plasma and urine for gene‐doping control. Gene. 2019;10(3):243. doi:10.3390/genes10030243.
Tozaki T, Ohnuma A, Kikuchi M, et al. Robustness of digital PCR and real‐time PCR against inhibitors in transgene detection for gene doping control in equestrian sports. Drug Test Anal. 2021;13(10):1768‐1775. doi:10.1002/dta.3131.
Tozaki T, Ohnuma A, Hamilton NA, et al. Low‐copy transgene detection using nested digital polymerase chain reaction for gene‐doping control. Drug Test Anal. 2022;14(2):382‐387. doi:10.1002/dta.3173.
Tozaki T, Ohnuma A, Kikuchi M, et al. Rare and common variant discovery by whole‐genome sequencing of 101 thoroughbred racehorses. Sci Rep. 2021;11(1):16057. doi:10.1038/s41598‐021‐95669‐1.
Maniego J, Pesko B, Hincks P, et al. Direct sequence confirmation of qPCR products for gene doping assay validation in horses. Drug Test Anal. 2022;14(6):1017‐1025. doi:10.1002/dta.3219.
Maniego J, Pesko B, Habershon‐Butcher J, et al. Screening for gene doping transgenes in horses via the use of massively parallel sequencing. Gene Ther. 2022;29(5):236‐246. doi:10.1038/s41434‐021‐00279‐1.
Tozaki T, Ohnuma A, Nakamura K, et al. Detection of indiscriminate genetic manipulation in thoroughbred racehorses by targeted resequencing for gene‐doping control. Gene. 2022;13(9):1589. doi:10.3390/genes13091589.
Maniego J, Pesko B, Habershon‐Butcher J, et al. Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer. Drug Test Anal. 2022;14(8):1429‐1437. doi:10.1002/dta.3267.
Tozaki T, Ohnuma A, Kikuchi M, et al. Identification of processed pseudogenes in the genome of thoroughbred horses: possibility of gene‐doping detection considering the presence of pseudogenes. Anim Genet. 2022;53(2):183‐192. doi:10.1111/age.13174.
Tozaki T, Ohnuma A, Takasu M, et al. Detection of non‐targeted transgenes by whole‐genome resequencing for gene‐doping control. Gene Ther. 2021;28(3‐4):199‐205. doi:10.1038/s41434‐020‐00185‐y.
Tozaki T, Ohnuma A, Kikuchi M, et al. Whole‐genome resequencing using genomic DNA extracted from horsehair roots for gene‐doping control in horse sports. J Equine Sci. 2020;31(4):75‐83. doi:10.1294/jes.31.75.
Wells DJ. Gene doping: the hype and the reality. Br J Pharmacol. 2008;154(3):623‐631. doi:10.1038/bjp.2008.144.
Gaffney GR, Parisotto R. Gene doping: a review of performance‐enhancing genetics. Pediatr Clin North am. 2007;54(4):807‐822. doi:10.1016/j.pcl.2007.04.004.
Ostrander EA, Huson HJ, Ostrander GK. Genetics of athletic performance. Annu Rev Genomics Hum Genet. 2009;10(1):407‐429. doi:10.1146/annurev‐genom‐082908‐150058.
Varillas‐Delgado D, Del Coso J, Gutierrez‐Hellin J, et al. Genetics and sports performance: the present and future in the identification of talent for sports based on DNA testing. Eur J Appl Physiol. 2022;122(8):1811‐1830. doi:10.1007/s00421‐022‐04945‐z.
Brzeziańska E, Domańska D, Jegier A. Gene doping in sport—perspectives and risks. Biol Sport. 2014;31(4):251‐259. doi:10.5604/20831862.1120931.
Beiter T, Zimmermann M, Fragasso A, et al. Direct and long‐term detection of gene doping in conventional blood samples. Gene Ther. 2011;18(3):225‐231. doi:10.1038/gt.2010.122.
Marchand A, Roulland I, Semence F, Ericsson M. EPO transgene detection in dried blood spots for antidoping application. Drug Test Anal. 2021;13(11‐12):1888‐1896. doi:10.1002/dta.3059.
Tozaki T, Ohnuma A, Kikuchi M, et al. Investigation of optimal procedures for storage and use of plasma samples suitable for gene doping tests. J Equine Sci. 2023;34(2):2232. doi:10.1294/jes.34.21.
Carter A, Flueck M. A polymerase chain reaction‐based methodology to detect gene doping. Eur J Appl Physiol. 2012;112(4):1527‐1536. doi:10.1007/s00421‐011‐2113‐y.
Ni W, Le Guiner C, Gernoux G, et al. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping. Gene Ther. 2011;18(7):709‐718. doi:10.1038/gt.2011.19.
Sugasawa T, Nakano T, Fujita S‐I, et al. Proof of gene doping in a mouse model with a human erythropoietin gene transferred using an adenoviral vector. Gene. 2021;12(8):1249. doi:10.3390/genes12081249.
Sugasawa T, Aoki K, Yanazawa K, Takekoshi K. Detection of multiple transgene fragments in a mouse model of gene doping based on plasmid vector using TaqMan‐qPCR assay. Gene. 2020;11(7):750. doi:10.3390/genes11070750.
Mauro VP, Chappell SA. A critical analysis of codon optimization in human therapeutics. Trends Mol Med. 2014;20(11):604‐613. doi:10.1016/j.molmed.2014.09.003.
Burgess‐Brown NA, Sharma S, Sobott F, Loenarz C, Oppermann U, Gileadi O. Codon optimization can improve expression of human genes in Escherichia coli: a multi‐gene study. Protein Expr Purif. 2008;59(1):94‐102. doi:10.1016/j.pep.2008.01.008.
Devi N, Adivitiya, Khasa YP. A combinatorial approach of N‐terminus blocking and codon optimization strategies to enhance the soluble expression of recombinant hIL‐7 in E‐coli fed‐batch culture. Appl Microbiol Biotechnol. 2016;100(23):9979‐9994. doi:10.1007/s00253‐016‐7683‐5.
Chin JX, Chung BKS, Lee DY. Codon optimization online (COOL): a web‐based multi‐objective optimization platform for synthetic gene design. Bioinformatics. 2014;30(15):2210‐2212. doi:10.1093/bioinformatics/btu192.
Ranaghan MJ, Li JJ, Laprise DM, Garvie CW. Assessing optimal: inequalities in codon optimization algorithms. BMC Biol. 2021;19(1):36. doi:10.1186/s12915‐021‐00968‐8.
Hernandez‐Alias X, Benisty H, Radusky LG, Serrano L, Schaefer MH. Using protein‐per‐mRNA differences among human tissues in codon optimization. Genome Biol. 2023;24(1):34. doi:10.1186/s13059‐023‐02868‐2.
Mauro VP, Chappell SA. Considerations in the use of codon optimization for recombinant protein expression. Methods Mol Biol. 2018;1850:275‐288. doi:10.1007/978‐1‐4939‐8730‐6_18.
Byrd JB, Greene AC, Prasad DV, Jiang X, Greene CS. Responsible, practical genomic data sharing that accelerates research. Nat Rev Genet. 2020;21(10):615‐629. doi:10.1038/s41576‐020‐0257‐5.
Lewis D. China's souped‐up data privacy laws deter researchers. Nature. 2023. doi:10.1038/d41586‐41023‐01638‐41581.
Lim B, Lin YY, Navin N. Advancing cancer research and medicine with single‐cell genomics. Cancer Cell. 2020;37(4):456‐470. doi:10.1016/j.ccell.2020.03.008.
Evrony GD, Hinch AG, Luo C. Applications of single‐cell DNA sequencing. Annu Rev Genomics Hum Genet. 2021;22(1):171‐197. doi:10.1146/annurev‐genom‐111320‐090436.
Gawad C, Koh W, Quake SR. Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single‐cell genomics. Proc Natl Acad Sci U S a. 2014;111(50):17947‐17952. doi:10.1073/pnas.1420822111.
Wang Y, Waters J, Leung ML, et al. Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature. 2014;512(7513):155‐160. doi:10.1038/nature13600.
Su X, Zhao L, Shi Y, et al. Clonal evolution in liver cancer at single‐cell and single‐variant resolution. J Hematol Oncol. 2021;14(1):22. doi:10.1186/s13045‐021‐01036‐y.
Su X, Bai S, Xie G, et al. Accurate tumor clonal structures require single‐cell analysis. Ann N Y Acad Sci. 2022;1517(1):213‐224. doi:10.1111/nyas.14897.
Gonzalez‐Pena V, Natarajan S, Xia Y, et al. Accurate genomic variant detection in single cells with primary template‐directed amplification. Proc Natl Acad Sci U S a. 2021;118(24):e2024176118. doi:10.1073/pnas.2024176118.
Zong C, Lu S, Chapman AR, Xie XS. Genome‐wide detection of single‐nucleotide and copy‐number variations of a single human cell. Science. 2012;338(6114):1622‐1626. doi:10.1126/science.1229164.
Chen C, Xing D, Tan L, et al. Single‐cell whole‐genome analyses by linear amplification via transposon insertion (LIANTI). Science. 2017;356(6334):189‐194. doi:10.1126/science.aak9787.
Logsdon GA, Vollger MR, Eichler EE. Long‐read human genome sequencing and its applications. Nat Rev Genet. 2020;21(10):597‐614. doi:10.1038/s41576‐020‐0236‐x.
Eid J, Fehr A, Gray J, et al. Real‐time DNA sequencing from single polymerase molecules. Science. 2009;323(5910):133‐138. doi:10.1126/science.1162986.
Clarke J, Wu HC, Jayasinghe L, Patel A, Reid S, Bayley H. Continuous base identification for single‐molecule nanopore DNA sequencing. Nat Nanotechnol. 2009;4(4):265‐270. doi:10.1038/nnano.2009.12.
Wang YH, Zhao Y, Bollas A, et al. Nanopore sequencing technology, bioinformatics and applications. Nat Biotechnol. 2021;39(11):1348‐1365. doi:10.1038/s41587‐021‐01108‐x.
Maniego J, Giles O, Hincks P, Stewart G, Proudman C, Ryder E. Long‐read sequencing assays designed to detect potential gene editing events in the myostatin gene revealed distinct haplotype signatures in the thoroughbred horse population. Anim Genet. 2023;54(4):470‐482. doi:10.1111/age.13332.

YG2023QNB06 SJTU Interdisciplinary Program; YG2021QN80 SJTU Interdisciplinary Program; World Anti-Doping Agency; Shanghai Gaofeng and Gaoyuan Project

Keywords: detection method; gene doping; gene editing; high‐throughput sequencing; qPCR

Date Created: 20240226 Date Completed: 20241212 Latest Revision: 20241212

20241212

10.1002/dta.3664

38403949