Tackling Emerging Threats to the Kentucky Racing Industry by Development of an Equine Gene Doping Program

RESEARCH PROPOSAL PREPARED FOR: Kentucky Horse Racing Commission – Equine Drug Research Council PREPARED BY: Scott D. Stanley, PhD and Cecily R. Wood, PhD University of Kentucky – College of Agriculture, Food and Environment M.H. Gluck Equine Research Center PROJECT TITLE: Tackling Emerging Threats to the Kentucky Horse Racing Industry: Development of Equine ‘Gene Doping’ Program Summary: Gene doping represents a threat to the integrity of the equine athlete and the horse racing industry. Expert groups have identified ‘gene doping’ as an activity which abuses and misuses gene therapy. Gene doping could be used to create a stronger, faster, or more resilient horse, all in the course of a single generation. To understand the molecular mechanisms of doping it is necessary identify the physiological essential mechanisms involved in the regulation of physical performance. These mechanisms include the regulation of skeletal muscle mass, oxygen delivery and modulation of energy metabolism. Consequently, gene doping has become a major concern in human sports and the horseracing industries. Many forms of drugs and gene manipulation are an acceptable and growing practice in human medicine for physical and mood modification. Recent advances in veterinary medical technologies have enabled gene therapy in horses as reported in clinical studies targeting tendon/ligament injuries and degenerative joint disease. In order to ensure safety and compliance with ethical standards for animal experimentation, the equine racing industry must establish methods to identify genetic alterations to effectively regulate professional misconduct. Genes for muscle mass, strength and healing: Myostatin is a cytokine known to influence the growth of skeletal muscle production and is expressed by the myostatin gene (MSTN). Thoroughbreds are physically characterized by an abundant mass of skeletal muscle with low body fat-to-muscle ratio with an extremely high percentage of fast twitch muscle fibers. These physical traits have been promulgated over centuries of selective breeding resulting in their natural athletic ability. Notwithstanding breeders selectivity, variation still exists within breeds that are contributing to horses’ unique performance capabilities. Splint horses need speed and power and are characterized by heavily muscled physique while distance horses, or those racing > 7 furlongs, have less skeletal muscle mass. Given these factors skeletal muscle mass production clearly plays a role in a horse’s athletic capability. Therefore, artificial enhancement through gene-doping targeting myostatin would be of particular interest to the horseracing industry. -1- Genes for oxygen consumption: Genes that influence oxygen consumption through their involvement in aerobic metabolism may also be targeted due to their performance enhancing potential. Physical performance is limited by the delivery of oxygenated blood to working muscles, and oxygen is the limiting factor to convert carbohydrates and fats into energy for muscle function. Horses hereditable traits evolved into structural modifications (e.g., heart size, cardiac output, stroke volume and splenic contraction) which produces naturally high oxygen consumption. Recombinant human erythropoietin (rhuEPO) and other synthetic analogs have been reported in horseracing as far back as the early 1990’s. In addition, horses produce antibodies to rhuEPO, making their abuse a major welfare concern as the antibodies have been reported to produce severe anemia in horses. Genes for angiogenesis: Genes that manipulate angiogenesis can increase blood flow to provide oxygen and nutrients for increased metabolic needs of contracting muscles, while simultaneously helping to maintain blood pressure. These processes are distinctively under the control of products formed in the angiogenesis pathway. For example, subsequent to aerobic exercise, creation of new capillaries and enlargement of new and existing vessels are activated by a combination of factors instigated by angiogenesis. The outcome of improved VO2Max is achieved by increasing blood flow and capillary population in smooth muscle delivering oxygen to enhance endurance. VEGF (vascular endothelial growth factor) are a family of polypeptide signaling proteins that work within the angiogenesis pathway. Throughout aerobic exercise skeletal muscle becomes hypoxic promoting HIF-1a transcription directing up-regulation of VEGF. Therefore, any substance or gene manipulation resulting in enhancement of the angiogenesis pathway would be an ideal doping agent. While detection methods are early in their development, innovative droplet digital polymerase chain reaction (ddPCR) approaches have been developed to provide high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for anti-doping. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions. The immense sample partitioning allowed through applications of ddPCR enable small differences in target DNA sequence between samples to be uniformly measured. -2-