Adeno-associated virus (AAV) is a powerful gene therapy vector that has been used in several FDA-approved therapies as well as in multiple clinical trials. This vector has high therapeutic versatility with the ability to deliver genetic payloads to a variety of human tissue types, yet there is currently a lack of transgene expression control once the virus is administered. There are also times when transgene expression is too low for the desired therapeutic outcome, necessitating high viral dose administration resulting in possible immunological complications. Herein, we validate a chemically controllable AAV transgene expression technology in vitro that utilizes bifunctional molecules known as chemical epigenetic modifiers (CEMs). These compounds employ endogenous epigenetic machinery to specifically enhance transgene expression of episomal DNA. A recombinant AAV (rAAV) was designed to both deliver the reporter transgene as well as deliver a synthetic zinc finger (ZFs) protein fused to FK506 binding protein (FKBP). These synthetic ZFs target a DNA-binding array sequence upstream of the promoter expressing the AAV transgene to specifically enhance AAV transgene expression in the presence of a CEM. The transcriptional activating compound CEM87 functions by recruiting the epigenetic transcription activator bromodomain-containing protein 4 (BRD4), increasing AAV transgene activity up to fivefold in a dose-dependent manner in HEK293T cells. The highest levels of transgene product activity are seen 24 h following CEM87 treatment. Additionally, the CEM87-mediated enhancement of different transgene products with either Luciferase or green fluorescent protein (GFP) was observed in multiple cell lines and enhancement of transgene expression was capsid serotype independent. The impact of CEM87 activity can be disrupted through drug removal or chemical recruitment site competition with FK506, thus demonstrating the reversibility of the impact of CEM87 on transgene expression. Collectively, this chemically controllable rAAV transgene technology provides temporal gene expression control that could increase the safety and efficiency of AAV-based research and therapies.

中文翻译：

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腺相关病毒传递转基因的化学表观遗传调控。

腺相关病毒 (AAV) 是一种强大的基因治疗载体，已用于 FDA 批准的多种疗法以及多项临床试验。该载体具有高度的治疗多功能性，能够将遗传有效负载传递到各种人体组织类型，但目前一旦施用病毒，就缺乏转基因表达控制。有时转基因表达对于所需的治疗结果来说太低，需要高病毒剂量施用，从而导致可能的免疫并发症。在此，我们在体外验证了化学可控 AAV 转基因表达技术，该技术利用称为化学表观遗传修饰剂 (CEM) 的双功能分子。这些化合物利用内源性表观遗传机制来特异性增强附加型 DNA 的转基因表达。重组 AAV (rAAV) 设计用于递送报告基因转基因以及递送与 FK506 结合蛋白 (FKBP) 融合的合成锌指 (ZFs) 蛋白。这些合成的 ZF 靶向表达 AAV 转基因的启动子上游的 DNA 结合阵列序列，以在 CEM 存在的情况下特异性增强 AAV 转基因表达。转录激活化合物 CEM87 通过招募表观遗传转录激活剂含溴结构域蛋白 4 (BRD4) 发挥作用，在 HEK293T 细胞中以剂量依赖性方式将 AAV 转基因活性提高至五倍。CEM87 处理后 24 小时，转基因产物活性达到最高水平。此外，在多种细胞系中观察到 CEM87 介导的荧光素酶或绿色荧光蛋白 (GFP) 不同转基因产物的增强，并且转基因表达的增强与衣壳血清型无关。CEM87 活性的影响可以通过药物去除或与 FK506 的化学招募位点竞争来破坏，从而证明 CEM87 对转基因表达的影响是可逆的。总的来说，这种化学可控的 rAAV 转基因技术提供了时间基因表达控制，可以提高基于 AAV 的研究和治疗的安全性和效率。