50 August / September 2019
A Marker-based Method to Detect Phosphorothioated Oligonucleotides in Equine Plasma Using Spectroscopic Analysis
1. Teruaki Tozaki* Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan Corresponding author:
ttozaki@lrc.or.jp
2. Kaoru Karasawa* AB Sciex, 4-7-35 Kitashinagawa, Shinagawa-ku, Tokyo 140-0001, Japan Corresponding author:
Kaoru.Karasawa@sciex.com 3. Yohei Minamijima Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan 4. Hideaki Ishii Drug Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan 5. Mio Kikuchi Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan 6. Hironaga Kakoi Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan 7. Kei-ichi Hirota Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan 8. Kanichi Kusano Racehorse Hospital Ritto Training Center, Japan Racing Association, 1028 Misono, Ritto, Shiga 520-3085, Japan 9. Shun-ichi Nagata Genetic Analysis Department, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan * Teruaki Tozaki and Kaoru Karasawa contributed equally to this study
Although there have been no known reports of gene doping in the horseracing industry to date, the relatively rapid advances and increasing accessibility of technologies for genetic modification may make this a reality. It is therefore prudent to investigate and develop methods to detect such practices. Therapeutic oligonucleotides technologies may be repurposed for gene doping. A common way of improving the structural integrity and stability of oligonucleotides to retard degradation when used for therapeutic applications is the addition of a phosphorothioated modification. This chemical moiety was employed as a viable marker for the detection of phosphorothioated oligonucleotides, using a method based on targeted and non-targeted spectroscopic analysis with liquid chromatography tandem mass spectrometry, including information- dependent data acquisition with dynamic background subtraction. It is proposed that this method be further investigated for use in the detection of equine gene doping.
Introduction
Maintaining the integrity and fairness of competitive sports has become an increasingly demanding effort. Since the establishment of the International Federation of Horseracing Authorities (IFHA) Gene Doping Control Subcommittee (GDCS) in March 2016 [1], several horseracing nations have decided to work together and invest in research to better understand this relatively new form of doping [2]. Although it is believed that gene doping has not been or is currently being misused to corrupt the sport, this investment demonstrates the commitment of these nations to stay ahead of potential cheating [2]. In Japan, the Laboratory of Racing Chemistry has been researching gene doping detection methods since 2017 and is currently developing nucleic acid medicine and transgene detection methods [3].
The doping could consist of the administration of oligomers or polymers of nucleic acid, nucleic acid analogues,
or genetically unmodifi ed or genetically modifi ed cells, resulting in the genetic manipulation of racehorses to enhance their performance [4,5]. Although this has not been demonstrated in equine species, studies in other mammalian species have shown the feasibility of gene doping to enhance athletic performance [6,7]. Through its potential ability to not only affect the genetics of an animal long-term but also the genomes of its offspring, gene doping threatens both horse racing and breeding [2,4]. This was recognised by David Sykes, the director of equine health and welfare at the British Horseracing Authority (BHA), who stated in April 2019 [2], “This is new technology that is unravelling all the time; none of us here think that there has probably been a previous incidence of it, but that doesn’t mean that we shouldn’t be looking forward into the next fi ve or 10 years and at least being able to identify if it is going to occur.”
This technology came about originally in efforts to develop new medicines to
treat previously undruggable, usually rare, human diseases, such as spinal muscle atrophy - a disease that is the most common hereditary cause of infant mortality [4,8,9]. The development of oligonucleotide medicines occurred in fi ts and starts with much hope and many setbacks over the last 30 years or so [10-12]. However, as the basic biology became better understood and chemistries improved, along with more sophisticated delivery systems, we are now seeing a renaissance in oligonucleotide therapies [10-12]. Several such rationally designed precision medicines have now
Figure 1: Chemical structure of phosphorothioated nucleic acids.
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
