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A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2021-08-19 , DOI: 10.1002/dta.3152
Zibin Jiang 1 , Joanne Haughan 1 , Kaitlyn L Moss 1 , Darko Stefanovski 1 , Kyla F Ortved 1 , Mary A Robinson 1, 2
Affiliation  

Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra-articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2–4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses.

中文翻译:

腺相关病毒载体2介导的基因掺杂定量PCR筛选方法

目前禁止人类和马运动员进行基因治疗,需要新的分析方法对其进行检测。大多数体内产品使用源自腺相关病毒 (AAV) 的非整合重组病毒载体将转基因递送到细胞中,在那里它们被转录并翻译成功能性蛋白质。尽管从重组 AAV (rAAV) 载体中去除了大部分野生型 AAV (WTAAV) DNA,但一些序列是保守的。本研究的目的是开发一种针对保守 AAV 序列的定量聚合酶链反应 (QPCR) 筛选测试,以实现对所有 rAAV 基因治疗产品的理论检测,无论编码的转基因如何,同时排除马体内 WTAAV DNA 的存在。开发并验证了引物组以靶向在 rAAV 病毒载体中高度保守的 AAV2 序列和仅在野生型 AAV2 (WTAAV2) 中发现的序列。对六匹马进行了关节内注射 rAAV。在第 0、1、2、4、7、14、28、56 和 84 天收集血浆和滑液。使用 QPCR,在所有马的血浆中检测到 rAAV 长达 2-4 天。rAAV DNA 在两匹马的滑液中检测了 28 天,这些马的滑液样本可用。在任何样品中均未检测到 WTAAV2 DNA。这是第一项开发 QPCR 测试的研究,该测试能够筛选可用于马基因掺杂的 rAAV 载体。2、4、7、14、28、56 和 84。使用 QPCR,在所有马的血浆中检测到 rAAV 长达 2-4 天。rAAV DNA 在两匹马的滑液中检测了 28 天,这些马的滑液样本可用。在任何样品中均未检测到 WTAAV2 DNA。这是第一项开发 QPCR 测试的研究,该测试能够筛选可用于马基因掺杂的 rAAV 载体。2、4、7、14、28、56 和 84。使用 QPCR,在所有马的血浆中检测到 rAAV 长达 2-4 天。rAAV DNA 在两匹马的滑液中检测了 28 天,这些马的滑液样本可用。在任何样品中均未检测到 WTAAV2 DNA。这是第一项开发 QPCR 测试的研究,该测试能够筛选可用于马基因掺杂的 rAAV 载体。
更新日期:2021-08-19
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