A polymerase chain reaction-based methodology to detect gene doping

Abstract

The non-therapeutic use of genes to enhance athletic performance (gene doping) is a novel threat to the World of Sports. Skeletal muscle is a prime target of gene therapy and we asked whether we can develop a test system to produce and detect gene doping. Towards this end, we introduced a plasmid (pCMV-FAK, 3.8 kb, 50 g) for constitutive expression of the chicken homologue for the regulator of muscle growth, focal adhesion kinase (FAK), via gene electro transfer in the anti-gravitational muscle, m. soleus, or gastrocnemius medialis of rats. Activation of hypertrophy signalling was monitored by assessing the ribosomal kinase p70S6K and muscle Wbre cross section. Detectability of the introduced plasmid was monitored with polymerase chain reaction in deoxyribonucleic acids (DNA) from transfected muscle and serum. Muscle transfection with pCMV-FAK elevated FAK expression 7-and 73-fold, respectively, and increased mean cross section by 52 and 16% in targeted muscle Wbres of soleus and gastrocnemius muscle 7 days after gene electro transfer. Concomitantly p70S6K content was increased in transfected soleus muscle (+110%). Detection of the exogenous plasmid sequence was possible in DNA and cDNA of muscle until 7 days after transfection, but not in serum except close to the site of plasmid deposition, 1 h after injection and surgery. The Wndings suggest that the reliable detection of gene doping in the immoral athlete is not possible unless a change in the current practice of tissue sampling is applied involving the collection of muscle biopsy close to the site of gene injection.

Gene doping: an overview and current implications for athletes

“…Gene transfer has been shown to be detectable in a biopsy for up to a decade 48. Since knowledge about the injection site is required, and biopsies are generally considered to be too invasive, methods using only blood, urine, serum, hair, saliva or a combination are needed 6 23 36 75 128 130…”

“…In mice injected intramuscularly with AAV-mediated gene therapy, PCR allowed detection in the blood for several weeks,37 though in another test it was undetectable in blood after half an hour 130. However, PCR is less useful for detecting doping using genes with introns because it presents problems with alternative splicing and efficacy 12 127.…”

“…This might make gene doping more attractive for athletes considering cheating 23 44 127 128. Currently, no specific test to detect gene doping has been approved by the WADA or used by a WADA-accredited laboratory 5 10 23 129 130…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…The approach was applied to a non‐human primate EPO gene doping model providing proof‐of‐concept data, which should be corroborated with further analyses demonstrating that the principle can be applied to other DNA targets as well. In a different study, an attempt was conducted to determine the intramuscularly administered plasmid (cytomegalovirus‐focal adhesion kinase) in rats using PCR . While tissue sampling of the transfected muscle allowed for the detection of the exogenous DNA sequence for up to seven days, essentially all serum samples returned negative test results, demonstrating the challenging aspect of sports drug testing since tissue sampling will not be an option in doping controls.…”

Myostatin: genetic variants, therapy and gene doping

“…Another issue in the scientific community and sports organizations is the concern to control this type of high-tech doping in competitions. As yet there are no antidoping methods available to screen and identify transgenic athletes, but some studies are underway to develop these Scarano et al, 2011;Carter, Flueck, 2012).…”