Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection

Tozaki, Teruaki; Gamo, Shiori; Takasu, Masaki; Kikuchi, Mio; Kakoi, Hironaga; Hirota, Kei‐ichi; Kusano, Kanichi; Nagata, Shun‐ichi

doi:10.1186/s13104-018-3815-6

Cited by 28 publications

(28 citation statements)

References 10 publications

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“…This is slightly longer than the plasma detection time of 2 days for an equine erythropoietin (EPO) transgene using digital droplet PCR (ddPCR), after intramuscular administration to a single horse 7 . Interestingly, a hEPO transgene administered to a single pig could be detected in small amounts in plasma for up to 3 weeks using ddPCR 6 and even longer detection times are described for rEPO transgenes in NHP serum 8 using qPCR. In humans, rAAV gene therapy was detected for up to 14 weeks in serum 28,29 using PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, novel analytical methods are needed for the detection of this treatment modality. Previous work has focused on using polymerase chain reaction (PCR) to detect transgenes, which can be differentiated from naturally occurring genes via missing regions of non‐coding DNA 5–7 . However, to be successful, the exact sequence of the targeted transgene must be known in advance and some PCR assays for detection of transgene sequences are less efficient in the presence of host DNA than those targeting vector‐specific DNA sequences 8 …”

Section: Introductionmentioning

confidence: 99%

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Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Haughan

Jiang

Stefanovski

et al. 2020

Drug Testing and Analysis

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Gene therapy promotes the expression of missing or defective genes and can be curative following administration of a single dose. Gene therapy is prohibited in equine athletes by regulatory bodies due to the high potential for abuse and novel analytical methods are needed for detection. The goal of this study was to detect the administration of an experimental gene therapy: a recombinant adeno‐associated viral vector (rAAV) carrying a transgene for the anti‐inflammatory cytokine IL‐10 (rAAV‐IL10). Twelve horses were randomly assigned to receive an intra‐articular injection of rAAV‐IL10 or phosphate buffered saline (vehicle) into a middle carpal joint. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Primer pairs were designed to detect two unique regions of rAAV. Using quantitative real time PCR, both sets of primers detected rAAV for 14–28 days in joints and up to 4 days in plasma, in all six treated horses. In synovial fluid, rAAV was detected on day 56 in 4/6 horses by both primer sets, and on day 84 in 1/6 horses by one primer set. In plasma, rAAV was detected for 7 days in 5/6 horses, 14 days in 2/6 horses, and 28 days in 1/6 horses by one primer set, and was detected for up to 14 days in 1/6 horses by the other primer set. This study is the first to validate that quantitative real time PCR can be used to systemically detect the local administration of a gene therapy product to horses.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Haughan

Jiang

Stefanovski

et al. 2020

Drug Testing and Analysis

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“…This is broadly in line with the position in human sport, where the World Anti-Doping Agency (WADA) prohibits the use of gene editing for any purpose (Le Page 2017). The enforcement on such bans is, however, dependent upon the ability to detect genetic editing of an athlete, and such work is underway in equine doping (Teruaki et al 2019;Tozaki et al 2018).…”

Section: Gene Editingmentioning

confidence: 99%

“…2019). We note whilst the reasoning behind such regulation is based in the ethical principle of justice, the application of the principle-through enforcement of regulation-is necessarily predicated on the availability of reliable tests for gene editing that is nascent in scientific terms (Teruaki et al 2019;Tozaki et al 2018).…”

Section: The Principle Of Justice Applied To Editing the Equine Genomementioning

confidence: 99%

Ethics, Genetic Technologies and Equine Sports: The Prospect of Regulation of a Modified Therapeutic Use Exemption Policy

Campbell

McNamee

2020

Sport, Ethics and Philosophy

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This article critically reviews the current availability and selected use of genetic technologies for horses, before undertaking an ethical evaluation of current practice and regulatory positions in comparative relation to debates surrounding genetic testing, preimplantation genetic testing and gene editing in humans. We argue that genetic testing for hereditary disorders is not only justified but should be encouraged on welfare grounds and that genetic testing for performance traits is ethically permissible based on a restricted imperative to genetically edit horses and horse embryos to reduce genetic predisposition to disease and injury. Given the current state of the science, where the effects of gene editing on health and welfare are currently undetermined, space is created for an analytical distinction between equine gene editing for 'treatment' and for 'enhancement'. Gene editing is only justified for purposes of correcting/preventing disease and injury. Current regulation is challenged by apparently conflicting welfare-based ethical imperatives with respect to welfare-based gene editing. We propose modifications to the blanket bans on gene editing with a case-by-case assessment of applications to permit gene editing, based on best welfare interests underwritten by the aim of facilitating fair sport that adapt WADAs International Standard for Therapeutic Use Exemptions, adding an important reporting element. We reject the use of gene editing to obtain currently prohibited competitive advantages. In order to safeguard the welfare of human and equine athletes, we argue that regulatory institutions should urgently collaborate to develop cross-sport international regulations for the use of gene editing, including obligatory reporting of data about the health and welfare of genetically edited horses.

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“…Currently published methods for detection of gene doping use PCR-based methods or loop-mediated isothermal amplification (LAMP) that target unique sequences in a doping gene corresponding to exon-exon junctions in the intron-less transgene [3,[5][6][7][8][9][10][11][12][13]. However, because the exon-exon junctions of doping genes are known and the short PCR primers are even interrupted by the slightest change of the sequence, it is relatively simple to evade detection using current PCR-based methods by modifying the doping gene with for example synonymous mutations, which will then give a false-negative result.…”

Section: Introductionmentioning

confidence: 99%

A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

et al. 2019

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Gene doping confers health risks for athletes and is a threat to fair competition in sports. Therefore the anti-doping community has given attention on its detection. Previously published polymerase chain reaction-based methodologies for gene doping detection are targeting exon-exon junctions in the intron-less transgene. However, because these junctions are known, it would be relatively easy to evade detection by tampering with the copyDNA sequences. We have developed a targeted nextgeneration sequencing based assay for the detection of all exon-exon junctions of the potential doping genes, EPO, IGF1, IGF2, GH1, and GH2, which is resistant to tampering. Using this assay, all exon-exon junctions of copyDNA of doping genes could be detected with a sensitivity of 1296 copyDNA copies in 1000 ng of genomic DNA. In addition, promotor regions and plasmid-derived sequences are readily detectable in our sequence data. While we show the reliability of our method for a selection of genes, expanding the panel to detect other genes would be straightforward. As we were able to detect plasmidderived sequences, we expect that genes with manipulated junctions, promotor regions, and plasmid or virus-derived sequences will also be readily detected.

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Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection

Cited by 28 publications

References 10 publications

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Detection of intra‐articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma

Ethics, Genetic Technologies and Equine Sports: The Prospect of Regulation of a Modified Therapeutic Use Exemption Policy

A next-generation sequencing method for gene doping detection that distinguishes low levels of plasmid DNA against a background of genomic DNA

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