Finding the golden genes : Advances in gene therapy could tempt some athletes to enhance their genetic makeup, leading some researchers to work on detection methods just in case / Patrick Barry. - (Science News 174 (2008) 3 (2 August); p. 16-21)
New application of the CRISPR-Cas9 system for site-specific exogenous gene doping analysis / Joon-Yeop Yi, Minyoung Kim, Hophil Min, Byung-Gee Kim, Junghyun Son, Oh-Seung Kwon, Changmin Sung. - (Drug Testing and Analysis (2020) 17 November)
Abstract
The increased potential for gene doping since the introduction of gene therapy presents the need to develop anti-doping assays. We therefore aimed to develop a quick and simple method for the detection of specifically targeted exogenous doping genes utilizing an in vitro clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. A human erythropoietin (hEPO) is a drug frequently used for doping in athletes, and gene doping using gene transfer techniques may be attempted. Therefore, we selected hEPO gene as a model of exogenous doping gene, and complemental single guide RNA (sgRNA) was designed to specifically bind to the four exon-exon junctions in the hEPO cDNA. For the rapid reaction of CRISPR-Cas9, further optimization was performed using an open-source program (CRISPOR) that avoids TT and GCC motifs before the protospacer adjacent motif (PAM) domain and predicts the efficiency of the sgRNA. We optimized the in vitro Cas9 assay and dual use of sgRNA for double cleavage and identified the limit of detection (LOD) of the 1.25 nM of the double cleavage method. We expect that the improved CRISPR-Cas9 method can be used for anti-doping analyses of gene doping.
Detection of non-targeted transgenes by whole-genome resequencing for gene-doping control / Teruaki Tozaki, Aoi Ohnuma, Masaki Takasu, Kotono Nakamura, Mio Kikuchi, Taichiro Ishige, Hironaga Kakoi, Kei-Ichi Hirora, Norihisa Tamura, Kanichi Kusano, Shun-Ichi Nagata. - (Gene Therapy (2020) 7 August)
Abstract
Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.
Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig : a model case study for gene doping detection / Teruaki Tozaki, Shiori Gamo, Masaki Takasu, Mio Kikuchi, Hironaga Kakoi, Kei-Ichi Hirota, Kanichi Kusano, Shun-Ichi Nagata. - (BMC Res Notes 11 (2018) 10 October)
Abstract
Objective: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 μg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction.
Results: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.
Gene and Cell Doping : The New Frontier - Beyond Myth or Reality / Elmo W.I. Neuberger, Perikles Simon. - (Medicine and sport science 62 (2017) 2 June; p. 91-106)
Abstract
The advent of gene transfer technologies in clinical studies aroused concerns that these technologies will be misused for performance-enhancing purposes in sports. However, during the last 2 decades, the field of gene therapy has taken a long and winding road with just a few gene therapeutic drugs demonstrating clinical benefits in humans. The current state of gene therapy is that viral vector-mediated gene transfer shows the now long-awaited initial success for safe, and in some cases efficient, gene transfer in clinical trials. Additionally, the use of small interfering RNA promises an efficient therapy through gene silencing, even though a number of safety concerns remain. More recently, the development of the molecular biological CRISPR/Cas9 system opened new possibilities for efficient and highly targeted genome editing. This chapter aims to define and consequently demystify the term "gene doping" and discuss the current reality concerning gene- and cell-based physical enhancement strategies. The technological progress in the field of gene therapy will be illustrated, and the recent clinical progress as well as technological difficulties will be highlighted. Comparing the attractiveness of these technologies with conventional doping practices reveals that current gene therapy technologies remain unattractive for doping purposes and unlikely to outperform conventional doping. However, future technological advances may raise the attractiveness of gene doping, thus making it easier to develop detection strategies. Currently available detection strategies are introduced in this chapter showing that many forms of genetic manipulation can already be detected in principle.
Synthetic certified DNA reference material for analysis of human erythropoietin transgene and transcript in gene doping and gene therapy / A. Baoutina, S. Bhat, M. Zheng, L. Partis, M. Dobeson, I.E. Alexander, K.R. Emslie. - (Gene Therapy 23 (2016) 10 (October; p. 708-717)
Abstract
There is a recognised need for standardisation of protocols for vector genome analysis used in vector manufacturing, to establish dosage, in biodistribution studies and to detect gene doping in sport. Analysis of vector genomes and transgene expression is typically performed by qPCR using plasmid-based calibrants incorporating transgenic sequences. These often undergo limited characterisation and differ between manufacturers, potentially leading to inaccurate quantification, inconsistent inter-laboratory results and affecting clinical outcomes. Contamination of negative samples with such calibrants could cause false positive results. We developed a design strategy for synthetic reference materials (RMs) with modified transgenic sequences to prevent false positives due to cross-contamination. When such RM is amplified in transgene-specific assays, the amplicons are distinguishable from transgene's amplicons based on size and sequence. Using human erythropoietin as a model, we produced certified RM according to this strategy and following ISO Guide 35. Using non-viral and viral vectors, we validated the effectiveness of this RM in vector genome analysis in blood in vitro. The developed design strategy could be applied to production of RMs for other transgenes, genes or transcripts. Together with validated PCR assays, such RMs form a measurement tool that facilitates standardised, accurate and reliable genetic analysis in various applications.
Establishment of two quantitative nested qPCR assays targeting the human EPO transgene / E.W.I. Neuberger, I. Perez, C. Le Guiner, D. Moser, T. Ehlert, M. Allais, P. Moullier, P. Simon, R.O. Snyder. - (Gene Therapy 23 (2016) 4 (April); p. 330-339)
Abstract
For ethical and safety reasons it is critical to develop easily implemented assays with high sensitivity and specificity for gene doping surveillance. Two nested quantitative real-time PCR (qPCR) assays were developed that target the human EPO (hEPO) cDNA sequence in a circular form, representative of recombinant adeno-associated viral (rAAV) vector genomes found in vivo. Through an interlaboratory evaluation, the assays were validated and utilized in an in vitro blinded study. These assays are specific and extremely sensitive with a limit of detection (LOD) of 1 copy of circular plasmid DNA and a limit of quantification (LOQ) of 10 to 20 copies in the presence of 500 ng of human genomic DNA (hgDNA) extracted from WBCs. Additionally, using the two nested qPCR assays in a non-human primate study, where macaques were injected intramuscularly with a rAAV8 vector harboring a promoterless hEPO cDNA sequence, the viral vector was detected 8 to 14 weeks post-injection in macaque WBCs. The high sensitivity of the nested qPCR approach along with the capability of quantifying target DNA, make this approach a reliable tool for gene doping surveillance and the monitoring of exogenous DNA sequences.
Selective androgen receptor modulators for the treatment of late onset male hypogonadism / Christopher C. Coss, Amanda Jones, Michael L. Hancock, Mitchell S. Steiner, James T. Dalton. - (Asian Journal of Andrology 16 (2014) 2 (March-April); p. 256-261)
Abstract
Several testosterone preparations are used in the treatment of hypogonadism in the ageing male. These therapies differ in their convenience, flexibility, regional availability and expense but share their pharmacokinetic basis of approval and dearth of long-term safety data. The brevity and relatively reduced cost of pharmacokinetic based registration trials provides little commercial incentive to develop improved novel therapies for the treatment of late onset male hypogonadism. Selective androgen receptor modulators (SARMs) have been shown to provide anabolic benefit in the absence of androgenic effects on prostate, hair and skin. Current clinical development for SARMs is focused on acute muscle wasting conditions with defined clinical endpoints of physical function and lean body mass. Similar regulatory clarity concerning clinical deficits in men with hypogonadism is required before the beneficial pharmacology and desirable pharmacokinetics of SARMs can be employed in the treatment of late onset male hypogonadism.
Excretion of 19-norandrosterone after consumption of boar meat / Frank Hülsemann, Gregor Fußhöller, Christine Lehn, Mario Thevis. - (Drug Testing and Analysis (2020) Special Issue (30 October); p. 1-7)
Abstract
The consumption of the offal of noncastrated pigs can lead to the excretion of 19-norandrosterone (NorA) in urine of humans. In doping control, GC/C/IRMS is the method of choice to differentiate between an endogenous or exogenous origin of urinary NorA. In some cases, after the consumption of wild boar offal, the δ13 C values of urinary NorA fulfill the criteria of an adverse analytical finding due to differing food sources of boar and consumer. However, consumption of wild boar's offal is not very common in Germany, and thus, the occurrence of such an analytical finding is unlikely. In contrast, the commerce with wild boar meat has increased in Germany within the last years. Up to 20,000 tons of wild boar meat are annually consumed. In order to probe for the probability of the occurrence of urinary NorA after consumption of wild boar meat, human urine samples were tested following the ingestion of commercially available game. In approximately half of the urine samples, traces of NorA were detected postadministration of 200 to 400 g boar meat. The highest urinary concentration was 2.9 ng/ml, and significant amounts were detected up to 9 h after the meal. δ13 C values ranged from -18.5‰ to -23.5‰, which would have led to at least two adverse analytical findings if the samples were collected in an antidoping context. IRMS analysis on German boar tissue samples showed that δ13 C values for wild boar's steroids are unpredictable and may vary seasonally.
Tissue selectivity and potential clinical applications of trenbolone (17beta-hydroxyestra-4,9,11-trien-3-one) : A potent anabolic steroid with reduced androgenic and estrogenic activity / Joshua F. Yarrow, Sean C. McCoy, Stephen E. Borst. - (Steroids 75 (2010) 6 (June); p. 377-389)
Abstract
Recently, the development of selective androgen receptor modulators (SARMs) has been suggested as a means of combating the deleterious catabolic effects of hypogonadism, especially in skeletal muscle and bone, without inducing the undesirable androgenic effects (e.g., prostate enlargement and polycythemia) associated with testosterone administration. 17beta-Hydroxyestra-4,9,11-trien-3-one (trenbolone; 17beta-TBOH), a synthetic analog of testosterone, may be capable of inducing SARM-like effects as it binds to androgen receptors (ARs) with approximately three times the affinity of testosterone and has been shown to augment skeletal muscle mass and bone growth and reduce adiposity in a variety of mammalian species. In addition to its direct actions through ARs, 17beta-TBOH may also exert anabolic effects by altering the action of endogenous growth factors or inhibiting the action of glucocorticoids. Compared to testosterone, 17beta-TBOH appears to induce less growth in androgen-sensitive organs which highly express the 5alpha reductase enzyme (e.g., prostate tissue and accessory sex organs). The reduced androgenic effects result from the fact that 17beta-TBOH is metabolized to less potent androgens in vivo; while testosterone undergoes tissue-specific biotransformation to more potent steroids, dihydrotestosterone and 17beta-estradiol, via the 5alpha-reductase and aromatase enzymes, respectively. Thus the metabolism of 17beta-TBOH provides a basis for future research evaluating its safety and efficacy as a means of combating muscle and bone wasting conditions, obesity, and/or androgen insensitivity syndromes in humans, similar to that of other SARMs which are currently in development.
