Chemically regulating AAV transgene expression with endogenous gene activators

摘要 AAV基因治疗已应用于多种遗传性疾病的治疗，并已被应用 到目前为止在1000多个人类中。使用AAV基因治疗疾病的乐观临床数据已被观察到 肌肉、血液、大脑和那些影响视力的东西。在所有这些应用中，管理的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。跟随 转导，AAV载体基因组形成环状串联，有限的研究证明了这些 异构体与组蛋白、转录激活因子和抑制因子有关。这些观察结果 暗示一种形式上的可能性，即AAV表观小体部分受到转基因表达的限制，暗指 调节其表观遗传成分以增强和/或抑制转录活性的能力。我们 S最近开发了一项名为化学表观遗传修饰剂的技术，该技术将蛋白质靶向偶联 用特定的小分子激活剂控制基因表达的启动子系统。这令人兴奋 技术已经证明了特定的基因在染色体水平上的激活和抑制。在预赛中 研究表明，我们的技术被用于招募特定的转录和表观遗传调节因子。 转导的AAV载体上的异构体。由此产生的数据表明特定的表观遗传学参与其中。 在固定载体剂量下将转基因表达提高10倍以上的修饰剂。此外，独特的 CEM设计允许结合激活分子的竞争抑制物，从而提供 转录抑制的机制。受到这些发现的鼓舞，我们提出了一个详细的机制 利用染色质体外检测AAV转基因表达控制平台 细胞转导后的免疫沉淀(目标1)。此外，此CEM方法的功能还包括 将在小鼠模型(AIM)中评估全身性AAV给药后的表型转基因调控 2)。总的来说，这里产生的数据将表征对AAV转基因表达的自然限制 在研究体内控制和特异性激活和抑制AAV的新方法时 转基因一般用于更安全的基因治疗应用。

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.