Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma.,Drug Testing and Analysis

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Detection of intra-articular gene therapy in horses using quantitative real time PCR in synovial fluid and plasma.
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2020-03-16 , DOI: 10.1002/dta.2785
Joanne Haughan ₁ , Zibin Jiang ₁ , Darko Stefanovski ₁ , Kaitlyn L Moss ₁ , Kyla F Ortved ₁ , Mary A Robinson _{1,

2}

Affiliation

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.

中文翻译：

在滑液和血浆中使用定量实时PCR检测马的关节内基因治疗。

基因疗法可促进缺失或缺陷基因的表达，单剂给药后可治愈。监管机构禁止在马匹运动员中进行基因疗法，因为这种方法极有可能被滥用，并且需要采用新颖的分析方法进行检测。这项研究的目的是检测一种实验性基因疗法的实施：重组腺相关病毒载体（rAAV）带有抗炎细胞因子IL-10（rAAV-IL10）的转基因。随机分配12匹马接受rAAV-IL10或磷酸盐缓冲盐水（载体）的关节内注射至腕中关节。在第0、1、2、4、7、14、28、56和84天收集血浆和滑液。设计引物对以检测rAAV的两个独特区域。使用定量实时PCR，在所有六只接受治疗的马中，两组引物在关节中检测到rAAV持续14-28天，在血浆中检测到4天。在滑液中，两种引物组在第56天在4/6马中检测到rAAV，在1/6马中在第84天通过一种引物检测到rAAV。在血浆中，通过一套引物在5/6马中检测到rAAV 7天，在2/6马中检测14天，在1/6马中检测28天，在1/6马中检测到rAAV长达14天。其他引物组。这项研究是首次证实定量实时PCR可用于系统检测基因治疗产品对马的局部给药。一个引物组在5/6马中检测到rAAV 7天，在2/6马中检测14天，在1/6马中检测28天，另一引物在1/6马中检测到14天。组。这项研究是首次证实定量实时PCR可用于系统检测基因治疗产品对马的局部给药。一个引物组在5/6马中检测到rAAV 7天，在2/6马中检测14天，在1/6马中检测28天，另一引物在1/6马中检测到14天。组。这项研究首次证实了定量实时PCR可用于系统检测基因治疗产品对马的局部给药。

更新日期：2020-03-16

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