Gene Doping - 101

from The Equine Veterinary Practitioner September 2021

A synopsis edited from: Tozaki T and Hamilton NA (2021). Control of gene doping in human and horse sports. Gene Ther 10.1038/s41434-021-00267-5

INTRODUCTION

Gene doping is prohibited in human and equine competition alike. The World Anti-Doping Agency (WADA), Fédération Equestre Internationale (FEI) and International Federation of Horseracing Authorities (IFHA) are the internationally recognised industry bodies that oversee governance of the Olympics, equestrian events and horseracing internationally. These organisations have introduced regulations to prohibit gene doping. The WADA (https://www.wada-ama.org/) and IFHA (https://www.ifhaonline.org/) have also published definitions of gene doping (FIGURE 1) while the FEI (https:// inside.fei.org/) is yet to do so. In humans, gene editing is most likely to be performed on adults, and as such will not affect the germline. This contrasts with the situation in horses which could be gene edited as adults or prenatally, as zygotes or embryos. In fact, editing horses as early as possible in development is appealing to reduce mosaicism and increase the efficiency of the edit. The editing of racehorse embryos to create genetically modified animals would be a direct contravention of Stud Book and horseracing rules. With the recent publication of a method to gene edit horse embryos, this is a particular concern for Horseracing Authorities. Further, in human sports, the recipient of gene doping can consent to the doping treatment, while clearly horses cannot, adding a further dimension to the ethical problem of gene doping in animals.

WHAT IS GENE DOPING?

WADA defines gene doping as the use of nucleic acids with the potential to enhance sport performance and can be broadly grouped into gene transfer, gene silencing and gene editing. Gene transfer involves the introduction of exogenous transgenes into living cells that normally is aimed at treating genetic disorders by facilitating expression of the transgene in the targeted cells. Erythropoietin gene therapy has been explored to treat renal failure, and insulin-like growth factor 1 to treat various causes of skeletal muscle loss. However, these two genes are very likely to be targeted for gene doping since the administration of their recombinant proteins is already prohibited by both equine and human anti-doping agencies. Gene silencing technology suppresses translation by hybridising the mRNA with artificially synthesised antisense oligonucleotides. Gene doping by gene suppression or silencing would target genes with products that can negatively affect performance, such as myostatin (MSTN), which contains a variant in Thoroughbred racehorses that is associated with racing performance. Gene editing is a technique used to perform substitutions, insertions, deletions, inversions, or translocations. The three best known gene-editing systems are zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR being the fastest and cheapest to use leading to easy and reliable editing of targeted genes.

CONCLUSION

Gene doping presents a progressing threat to the integrity of all forms of competitive sport. Although there is no evidence indicating that gene doping is being used currently in horses, the technology to apply many forms of gene doping already exists. Tests to detect inserted transgenes have been developed, but the detection of gene editing is still very difficult. While the most effective measure for anti-doping control is the introduction of doping tests, the broad range of possible targets means that a successful anti-gene doping programme will combine different types of detection testing, education, and surveillance, to send the message that gene doping will not be tolerated.

REGULATORY CONSIDERATIONS

Gene doping regulations published online for the International Federation of Horseracing Authorities, IFHA International Agreement on Breeding, Racing and Wagering (IABRW) are continually updated and are summarised in TABLE 1. The most up to date versions are found at IFHA International Agreement.

ARTICLE 6 B (RACING) - PROHIBITION OF GENETIC THERAPY, GENE EDITING AND GENOME EDITING

1. Prohibition on use or administration of Genetic

Therapy a. Definition of Genetic Therapy

Genetic Therapy is defined as including any therapy, method or process which involves the use or administration of: i. oligomers or polymers of nucleic acid ii. nucleic acid analogues iii. genetically modified cells iv. gene editing agents which are capable, at any time, of directly or indirectly causing an action or effect on, and/or manipulating, gene expression in any mammalian body, including but not limited to gene editing agents with the capacity to alter genome sequences and/or the transcriptional, post-transcriptional or epigenetic regulation of gene expression. For the avoidance of doubt, the use or administration of autologous conditioned serum or ‘platelet-rich plasma’ treatments which do not involve the transfer of whole cells / DNA are not defined as a Genetic Therapy for the purpose of the IABRW.

b. Except as provided by this Article, the use or administration of a Genetic Therapy on or to a horse is prohibited at all times. c. Exempted Genetic Therapy

Genetic Therapy may be used or administered to a specific horse with the express prior approval of a Racing Authority if that Genetic Therapy is used to treat an injury or disorder formally diagnosed by a veterinarian, and: i. is not capable of modifying a horse heritable genome ii. does not pose a threat to the welfare of horses iii. does not pose a threat to the integrity of racing, either by having the potential to enhance or harm the performance of a horse in a race

d. Control and recording

The owner or trainer has the responsibility to inform and obtain the approval of the relevant Racing Authority of any intended Genetic Therapy to a horse prior to such treatment, irrespective of whether it is to be administered before, in or out of training. The owner or trainer must maintain full and accurate records of all such therapies – which must be kept for a minimum of five years and be readily available for inspection by regulatory officials when requested.

2. Prohibition on Gene Editing and Genome Editing a. Definition of Gene Editing

Gene Editing is defined as any process or treatment in respect of a horse which involves the insertion, deletion and/or replacement of DNA at a specific site in the genome of the horse.

b. Definition of Genome Editing

Genome Editing is defined as any process or treatment in respect of a horse which involves the insertion, deletion and/or replacement of DNA in the genome of the horse.

c. The use on, or administration or application to, any horse of Gene Editing or Genome Editing is prohibited at all times.

Last update: JANUARY 2020

Gates S et al. A Case Series of Five Horses with Superficial Digital Flexor Tendon [SDFT] Lesions in the Carpal Canal. J Eq Vet Sci. 2021; 103: 103656. DOI 10.1016/j.jevs.2021.103656 The five horses were 11-28 years old and presented with an acute, unilateral forelimb lameness which worsened following proximal limb flexion and was accompanied by a mild swelling of the palmar carpal region in most cases. Ulnar nerve block abolished the lameness. Ultrasound evaluation revealed an increased cross-sectional area (CSA) of the SDFT tendon and a loss of tendon fibre architecture within the palmar aspect of the affected tendon, most readily identifiable upon longitudinal images. Three out of the five horses returned to their previous level of performance, and two were euthanized. A back-at-the-knee conformation was associated with a poorer prognosis in these cases. An intratendinous fibroma was identified histopathologically in one horse following euthanasia.