Gene and Cell Doping: The New Frontier - Beyond Myth or Reality

2 Jun 2017

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) 

  • PMID: 28578328
  • DOI: 10.1159/000465456

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

7 Jun 2016

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)

  • PMID: 27439362
  • DOI: 10.1038/gt.2016.47


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

11 Jan 2016

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)

  • PMID: 26752352
  • DOI: 10.1038/gt.2016.2


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

19 Dec 2013

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)

  • PMID: 24407183
  • PMCID: PMC3955335
  • DOI: 10.4103/1008-682X.122339


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.

Functional hypoxia drives neuroplasticity and neurogenesis via brain erythropoietin

9 Mar 2020

Functional hypoxia drives neuroplasticity and neurogenesis via brain erythropoietin / Debia Wakhloo, Franziska Scharkowski, Yasmina Curto, Umer Javed Butt, Vikas Bansal, Agnes A. Steixner-Kumar, Liane Wüstefeld, Ashish Rajput, Sahab Arinrad, Matthias R. Zillmann, Anna Seelbach, Imam Hassouna, Katharina Schneider, Abdul Qadir Ibrahim, Hauke B. Werner, Henrik Martens, Kamilla Miskowiak, Sonja M. Wojcik, Stefan Bonn, Juan Nacher, Klaus-Armin Nave, Hannelore Ehrenreich. - (Nature Communications 11 (2020) 1313 (9 March); p. 1-12)

  • PMID: 32152318
  • PMCID: PMC7062779
  • DOI: 10.1038/s41467-020-15041-1


Abstract

Erythropoietin (EPO), named after its role in hematopoiesis, is also expressed in mammalian brain. In clinical settings, recombinant EPO treatment has revealed a remarkable improvement of cognition, but underlying mechanisms have remained obscure. Here, we show with a novel line of reporter mice that cognitive challenge induces local/endogenous hypoxia in hippocampal pyramidal neurons, hence enhancing expression of EPO and EPO receptor (EPOR). High-dose EPO administration, amplifying auto/paracrine EPO/EPOR signaling, prompts the emergence of new CA1 neurons and enhanced dendritic spine densities. Single-cell sequencing reveals rapid increase in newly differentiating neurons. Importantly, improved performance on complex running wheels after EPO is imitated by exposure to mild exogenous/inspiratory hypoxia. All these effects depend on neuronal expression of the Epor gene. This suggests a model of neuroplasticity in form of a fundamental regulatory circle, in which neuronal networks-challenged by cognitive tasks-drift into transient hypoxia, thereby triggering neuronal EPO/EPOR expression.

Drug insight: Testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging

1 Mar 2006

Drug insight : Testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging Shalender Bhasin, Olga M. Calof, Thomas W. Storer, Martin L. Lee, Norman A. Mazer, Ravi Jasuja, Victor M. Montori, Wenqing Gao, James T. Dalton. - (Nature Clinical Practice Endocrinology & Metabolism 2 (2006) 3 (March); p. 146-159)

  • PMID: 16932274
  • PMCID: PMC2072878
  • DOI: 10.1038/ncpendmet0120


Abstract

Several regulatory concerns have hindered development of androgens as anabolic therapies, despite unequivocal evidence that testosterone supplementation increases muscle mass and strength in men; it induces hypertrophy of type I and II muscle fibers, and increases myonuclear and satellite cell number. Androgens promote differentiation of mesenchymal multipotent cells into the myogenic lineage and inhibit their adipogenic differentiation, by facilitating association of androgen receptors with beta-catenin and activating T-cell factor 4. Meta-analyses indicate that testosterone supplementation increases fat-free mass and muscle strength in HIV-positive men with weight loss, glucocorticoid-treated men, and older men with low or low-normal testosterone levels. The effects of testosterone on physical function and outcomes important to patients have not, however, been studied. In older men, increased hematocrit and increased risk of prostate biopsy and detection of prostate events are the most frequent, testosterone-related adverse events. Concerns about long-term risks have restrained enthusiasm for testosterone use as anabolic therapy. Selective androgen-receptor modulators that are preferentially anabolic and that spare the prostate hold promise as anabolic therapies. We need more studies to determine whether testosterone or selective androgen-receptor modulators can induce meaningful improvements in physical function and patient-important outcomes in patients with physical dysfunction associated with chronic illness or aging.

Characteristics and Attitudes of Men Using Anabolic Androgenic Steroids (AAS): A Survey of 2385 Men

14 Dec 2020

Characteristics and Attitudes of Men Using Anabolic Androgenic Steroids (AAS) : A Survey of 2385 Men / Alex K. Bonnecaze, Thomas O'Connor, Joseph A. Aloi. - (American Journal of Men's Health 14 (2020) 6 (November-December); p. 1-12)

  • PMID: 33307930
  • PMCID: PMC7739101
  • DOI: 10.1177/1557988320966536


Abstract

Additional characterization of patients using anabolic androgenic steroids (AAS) is needed to improve harm reduction and cessation resources for patients. Our group sought to expand upon the currently limited data regarding AAS use by performing a web-based survey assessing experiences of males using AAS. Participants included men over the age of 18 with history of AAS use within the past 5 years. Data were collected between August 2019 and April 2020. Primary outcome measures included age when starting AAS, dose of AAS, motivations for use, experiences with health-care professionals, and rate of successful cessation. The survey was accessed 3640 times, resulting in 2385 completed surveys meeting the inclusion criteria (68.93% participation rate).

Average participant age was 31.69 ± 10.09 years. Over half of respondents were from the United States (n = 1271, 53.3%). Motives to use AAS included improving appearance (n = 1959, 82.2%), strength gain (n = 1192, 50%), and self-esteem/body image issues (n = 712, 29.87%). Participants rated physicians poorly, regarding knowledge of AAS (4.08 ± 2.23). Most participants did not reveal AAS use to their health-care providers (n = 1338, 56.1%); of those that did, 55.30% (n = 579) reported feeling discriminated against for their use. Of 46.16% (n = 1101) attempting AAS cessation, 60.22% (n = 663) were unsuccessful. Challenges in the management of AAS use include early onset of use, supraphysiologic doses used, and frequently present body image disorders stress. Distrust of health-care providers, poor cessation rates, and lack of physician training further exacerbate this. These findings should serve to reinforce previous calls to action for further research on the treatment of AAS use disorder.

 

Long-term testosterone gel (AndroGel) treatment maintains beneficial effects on sexual function and mood, lean and fat mass, and bone mineral density in hypogonadal men

1 May 2004

Long-term testosterone gel (AndroGel) treatment maintains beneficial effects on sexual function and mood, lean and fat mass, and bone mineral density in hypogonadal men / Christina Wang, Glenn Cunningham, Adrian Dobs, Ali Iranmanesh, Alvin M. Matsumoto, Peter J. Snyder, Thomas Weber, Nancy Berman, Laura Hull, Ronald S. Swerdloff. - (The Journal of Clinical Endocrinology & Metabolism 89 (2004) 5 (1 May); p. 2085-2098)

  • PMID: 15126525
  • DOI: 10.1210/jc.2003-032006


Abstract

Transdermal testosterone (T) delivery represents an effective alternative to injectable androgens. We studied 163 hypogonadal men who applied 5, 7.5, or 10 g AndroGel (T gel) 1% CIII per day for up to 42 months. Efficacy data were presented in 123 subjects considered evaluable. Continuous AndroGel treatment normalized mean serum T and free T levels. Mean serum 5alpha-dihydrotestosterone concentrations and 5alpha-dihydrotestosterone/T ratio slightly increased, mean serum estradiol/T ratio doubled, and mean serum FSH and LH levels were suppressed by T replacement. Sexual function and mood parameters improved rapidly and were maintained throughout T treatment. Lean body mass increased (P = 0.0001) and fat mass decreased (P = 0.0001), and these changes were maintained with treatment but were not accompanied by significant increases in muscle strength. Increases in serum bone markers suggestive of increased bone formation were followed by gradual and progressive increases in bone mineral density more in the spine (P = 0.0001) than the hip (P = 0.0004). Mild local skin irritation occurred in 12 subjects, resulting in discontinuation in only one subject. Except for the anticipated increase in hematocrit and hemoglobin, there were no clinically significant changes in blood counts or biochemistry. In three subjects with elevated serum prostate-specific antigen, prostate biopsies showed cancer. We conclude that continued application of AndroGel resulted in beneficial effects similar to those with injectables and other transdermal preparations. This study was neither placebo controlled nor powered to determine the effects of T treatment on prostate cancer risk. Thus, monitoring for prostatic disease and assessment for erythrocytosis are strongly advised to reduce the risk of adverse events with T treatment of hypogonadal men.

SAIDS 2019 SAIDS vs Mamorallo Tjoka

14 Jan 2021

In June 2019 the South African Institute for Drugfree Sport (SAIDS) had reported anti-doping rule violations against the Lesotho Athlete Mamorallo Tjoka for Tampering and Evasion of sample collection. The Athlete filed a statement in her defence and failed to attend the hearing of the SAIDS Disciplinary Panel. 

Three Doping Control Officers (DCOs) testified that on 7 October 2018 they established that the Athlete was present in the house of her aunt in Lesotho and after introduction they notified her about the sample collection. Before the sample collection could commence the Athlete went to another room and hereafter the DCOs discovered that the Athlete had left the house and disappeared.

In her submissions the Athlete contradicted the statements of the DCOs and denied that she had been in the house of her aunt in Lesotho on 7 October 2018. She stated that she was in Johannesburg that day and involved in a car accident. After delays she produced an incident report and relevant passport information. 

SAIDS opened several investigations into the Athlete which revealed that the Athlete’s car accident on 7 October in Johannesburg was a fabrication. Cell phone data and other evidence confirmed that at the time of the alleged incident on 7 October 2018 she clearly was at her aunt’s house in Lesotho and not in Johannesburg. 

Furthermore it appeared that all the people involved in this alleged car accident had provided false statements. Cell phone data showed that they knew each other and there had been communications between them. In fact the cell phone data established de specific locations of these people and none of them were even close to the point of the alleged accident on that day. 

Considering the evidence in this case the Disciplinary Panel finds that the Athlete had committed the reported anti-doping rule violations and it rejected the false evidence she had produced in support of her assertions.

Because this is the Athlete’s second anti-doping rule violation the SAIDS Disciplinary Panel decides on 16 August 2019 to impose an 8 year period of ineligibility on the Athlete starting on the date she evaded the sample collection, i.e. on 7 October 2018. 

Due to the Athlete dishonest conduct the Panel deemed that the SAIDS’ costs for the results management, the conducted investigations and the legal costs shall be borne by the Athlete.

FEI 2020 FEI vs Tsutomu Inoue

4 Jan 2021

In March the International Equestrian Federation (FEI) has reported an anti-doping rule violation against the Japanese Parathlete Tsutomu Inoue after his sample tested positive for the prohibited substance Prednisone / Prednisolone. After notification the Athlete filed a statement in his defence.

The Parathlete admitted the violation, denied the intentional use of the substance. He had mentioned al his medications on the Doping Control Form and demonstred with medical evidence that suffered from several conditions. He had used the substance as prescribed medication since 1987 with no alternative medication available.

Since he had not received anti-doping education he was unaware he had to apply for TUE. Hereafter his application for a TUE which was granted by the TUE Committee in April 2020. His application for a retroactive TUE was denied.

The parties in this case reached an agreement and requested the FEI Tribunal to issue a Decision incorporating the terms of this agreement. The parties agreed that the violation was not intentional and that the Athlete bears No Significant Fault or Negligence with a light degree of Fault or Negligence. The Athlete accepted the sanction and agreed to fulfil the Education Requirement within 1 year.

Therefore the FEI Tribunal decides on 4 January 2021 in accordance with the mutual consent of the Parties to impose a fine and a 2 month period of ineligibility on the Athlete starting on the date of the decision.

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