Administration and detection of a multi-target rAAV gene doping vector in horses using multiple matrices and molecular techniques

IF 4.6 3区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Jillian Maniego, Caitlin Harding, Jocelyn Habershon-Butcher, Pamela Hincks, Edward Ryder
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引用次数: 0

Abstract

Gene doping, which includes the non-therapeutic use of genes or genetic elements that have the capacity to enhance athletic performance, is prohibited in horseracing and equestrian sports. To provide a comprehensive assessment of matrix and detection techniques, a custom adeno-associated virus serotype 8 vector was designed to include PCR binding sites for multiple target genes and assay types. The vector was injected via an intramuscular route into two Thoroughbred horses and matrices collected at defined timepoints. DNA was analysed using 3 detection methods: qPCR, digital PCR, and NGS. Overall, there was a strong correlation across the different detection methods employed, although digital PCR was less sensitive at lower concentrations. High concentrations of vector were detected at early timepoints in plasma and whole blood, which rapidly dropped after 0.5 d to trace levels by 4 d and 9 d post-administration respectively, following a similar pattern to previous studies. Vector was detected in dried blood spots at lower levels than whole blood, but with a similar detection time. Detection in hair root bulbs in one horse was observed at over a month post-administration, which opens new avenues for future gene doping testing in humans and animals.

Abstract Image

Abstract Image

利用多种基质和分子技术对马体内的多靶点 rAAV 基因兴奋剂载体进行管理和检测。
在赛马和马术运动中禁止使用基因兴奋剂,其中包括非治疗性使用具有提高运动成绩能力的基因或遗传元素。为了对基质和检测技术进行全面评估,我们设计了一种定制的腺相关病毒血清型 8 载体,其中包括多个目标基因和检测类型的 PCR 结合位点。该载体通过肌肉注射的方式注入两匹纯血马体内,并在规定的时间点收集基质。使用 3 种检测方法分析 DNA:qPCR、数字 PCR 和 NGS。总体而言,不同检测方法之间存在很强的相关性,但数字 PCR 对较低浓度的敏感性较低。在血浆和全血的早期时间点检测到高浓度的载体,0.5 天后迅速下降,分别在给药后 4 天和 9 天降至痕量水平,这与以往研究的模式相似。在干血斑中检测到的 Vector 含量低于全血,但检测时间相似。一匹马在用药后一个多月才在毛根球茎中检测到 Vector,这为今后在人类和动物中进行基因兴奋剂检测开辟了新的途径。
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来源期刊
Gene Therapy
Gene Therapy 医学-生化与分子生物学
CiteScore
9.70
自引率
2.00%
发文量
67
审稿时长
4-8 weeks
期刊介绍: Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.
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