Department of Clinical Studies, University of Pennsylvania School of Veterinary Medicine, New Bolton Center, Kennett Square, Pennsylvania, USA.
Pennsylvania Equine Toxicology and Research Laboratory, West Chester University, West Chester, Pennsylvania, USA.
Drug Test Anal. 2022 May;14(5):963-972. doi: 10.1002/dta.3152. Epub 2021 Sep 3.
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.
基因治疗目前在人类和赛马运动员中被禁止，因此需要新的分析方法来检测它。大多数体内产品使用非整合、源自腺相关病毒（AAV）的重组病毒载体将转基因导入细胞，在细胞中转录并翻译成功能性蛋白质。虽然重组 AAV（rAAV）载体中已去除了大多数野生型 AAV（WTAAV）DNA，但仍保留了一些保守序列。本研究旨在开发一种针对保守 AAV 序列的定量聚合酶链反应（QPCR）筛选测试，以便能够理论上检测所有 rAAV 基因治疗产品，而无需考虑编码的转基因，同时排除马体内 WTAAV DNA 的存在。开发并验证了引物组，以靶向 rAAV 病毒载体中高度保守的 AAV2 序列和仅在野生型 AAV2 中发现的序列（WTAAV2）。六匹马接受了关节内注射 rAAV。在第 0、1、2、4、7、14、28、56 和 84 天收集血浆和滑液。使用 QPCR，所有马的血浆中 rAAV 最多可在 2-4 天内检测到。有 2 匹马的滑液样本可用于检测，rAAV DNA 在滑液中可检测到 28 天。在任何样本中均未检测到 WTAAV2 DNA。这是第一项开发能够筛选可能用于赛马基因兴奋剂的 rAAV 载体的 QPCR 测试的研究。
