Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina.
Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina.
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DOI：
10.1016/j.omtn.2016.12.006
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发表时间：
2017-03-17
期刊：
影响因子：
--
通讯作者：
MacLaren RE
中科院分区：
文献类型：
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作者：
Patrício MI;Barnard AR;Orlans HO;McClements ME;MacLaren RE
土拨鼠肝炎病毒转录后调节元件(WPRE)已被包括在腺相关病毒(AAV)的转基因盒中，在包括遗传性视网膜疾病在内的几个基因治疗临床试验中。然而，WPRE在多大程度上增加了视网膜中的转基因表达仍不清楚。为了解决这个问题，我们首先对含有WPRE报告基因的AAV2载体进行了体外比较，然后通过小鼠视网膜下注射的方法进行了体内比较。在这两种情况下，WPRE的存在导致了眼底荧光、蛋白质印迹和免疫组织化学检测的转基因表达水平显著升高。这两个载体进一步在来自临床指征视网膜切除患者的人视网膜外植体中进行比较，其中WPRE的存在再次导致报告基因表达的增强。最后，一种使用目前用于脉络膜病临床试验的转基因的类似方法在体外和体内都产生了类似的结果，证实了WPRE效应是独立于转基因的。我们的数据完全支持将WPRE纳入正在进行的和未来的AAV视网膜基因治疗试验，在这些试验中，它可能允许在总体较低剂量的载体下实现治疗效果。
The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.