Chemically regulating AAV transgene expression with endogenous gene activators

摘要 AAV基因疗法可用于治疗多种遗传性疾病，并且已经应用于 已经有超过1,000人死亡已经观察到使用AAV基因疗法治疗疾病的乐观临床数据。 肌肉、血液、大脑以及影响视力的物质。在所有这些应用中，施用的AAV载体 对人类来说有一个共同点：它们在转基因表达水平上是不可控的。以下 转导后，AAV载体基因组形成环状多联体，有限的研究已经证明了这些功能。 附加体与组蛋白以及转录激活因子和抑制因子相关。这些观察结果 这表明了AAV附加体部分地限制转基因表达的形式可能性，暗示了 调节其表观遗传组成以增强和/或抑制转录活性的能力。我们 最近开发了一种称为化学表观遗传修饰剂（CEM）的技术， 在启动子处的系统，以用特定的小分子激活剂控制基因表达。这个令人兴奋 技术已经证明了在染色体水平上的特异性基因激活和抑制。初步 研究中，我们的技术进行了评估，招聘特定的转录和表观遗传调节 转导的AAV载体附加体。由此产生的数据表明，参与特定的表观遗传 修饰剂以在固定载体剂量下使转基因表达增强10倍以上。此外，独特的 CEM设计允许活化分子的竞争性抑制剂的结合，从而提供了一种新的免疫抑制剂。 转录抑制机制。这些发现令人兴奋，我们提出了一个详细的机制 使用染色质在体外检查用于受控AAV转基因表达的该平台 细胞转导后的免疫沉淀（目的1）。此外，此CEM方法的功能 将在小鼠模型中全身施用AAV后评价附加型转基因调节（Aim 2）。总的来说，本文产生的数据将表征对AAV转基因表达的天然限制 在研究用于AAV的受控和特异性激活和抑制的体内新方法的同时， 转基因技术在更安全的基因治疗中的应用。

Abstract AAV gene therapy has applications for the treatment of diverse genetic diseases and has already been applied in over 1,000 humans to date. Optimistic clinical data using AAV gene therapy has been observed for disorders of the muscle, blood, brain, and those affecting vision. In all these applications, the AAV vectors administered to humans have one thing in common: they are uncontrollable at the level of transgene expression. Following transduction, AAV vector genomes form circular concatemers and limited studies have demonstrated these episomes associate with histones as well as transcriptional activators and repressors. These observations suggest a formal possibility that AAV episomes are, in part, restricted for transgene expression, alluding to the ability to modulate their epigenetic composition to enhance and/or repress the transcriptional activity. We recently developed a technology termed chemical epigenetic modifiers (CEM)s that couple a protein targeting system at a promoter to control gene expression with a specific small molecule activator. This exciting technology has demonstrated specific gene activation and repression at the chromosomal level. In preliminary studies, our technology was evaluated for the recruitment of specific transcriptional and epigenetic regulators to transduced AAV vector episomes. The resultant data demonstrate the involvement of specific epigenetic modifiers to enhance transgene expression greater than 10-fold at a fixed vector dose. Additionally, the unique CEM design allows binding of competitive inhibitors of the activation molecules, thereby providing a mechanism for transcriptional repression. Excited by these findings, we propose a detailed mechanistic examination of this platform for controlled AAV transgene expression in vitro using chromatin immunoprecipitation following cell transduction (Aim 1). Additionally, the functionality of this CEM approach for episomal transgene regulation will be evaluated following systemic AAV administration in a mouse model (Aim 2). Collectively, data generated herein will characterize the natural restriction on AAV transgene expression while investigating a novel approach in vivo for the controlled and specific activation and repression of AAV transgenes towards safer gene therapy applications in general.