Advanced Delivery Platforms for Base Editing In Vivo

项目摘要 Cas9平台使基因组编辑、碱基编辑（BE）和引物编辑能够诱导基因表达。 敲除以及定制的序列改变。CRISPR-Cas9系统具有类似的潜力， 通过精确编辑疾病位点来彻底改变临床实践。我们已经开发了Nme 2Cas 9作为一种 编辑平台，其具有（1）紧凑的尺寸，促进单个腺相关病毒（AAV）递送;（2） 二核苷酸（N4 CC）原型间隔区邻近基序（PAM），其提供高靶位点密度;和（3）特殊的 精度最近，我们开发了Nme 2Cas 9腺嘌呤碱基编辑器（ABE）系统，作为第一批 可以在体内验证单AAV递送。AAV是一种有效的体内编辑递送方式，特别是在哺乳动物中。 肝外组织如中枢神经系统（CNS）。尽管如此，碱的治疗前景 编辑系统将取决于提高编辑效率，限制旁观者编辑（或其后果）， 最大化PAM依赖性靶向范围，并最小化免疫原性、毒性和延长 脱氨酶表达（其可损害编辑效率并导致安全性风险，例如肝毒性 以及不需要的编辑的积累）。在这里，我们建议利用我们建立的单矢量 Nme 2-ABE系统可开发新型碱基编辑功能，提高有效性、瞄准范围， 实用性和安全性，并验证这些系统在治疗小鼠CNS疾病模型中的作用。的目标 该建议是（1）开发下一代、单AAV、脱氨酶镶嵌的Nme 2-ABE和具有以下特征的指南： 提高效率，单核苷酸PAM，以及对旁观者编辑的更大控制;（2）使用下一个- 用于小鼠CNS中疾病基因的治疗性编辑的单AAV Nme 2-ABE系统 肌萎缩性侧索硬化症和Batten病模型;以及（3）开发使用抑制的系统， 内源性microRNA和药物依赖性剪接系统，以增强AAV递送的Nme 2- 体内ABE。这些安全性增强将减少抗Nme 2-ABE免疫应答，改善潜在的 对肝脏和特定CNS细胞类型的毒性作用，并限制可能由 编辑机器的持续表达。这些目标的成功实现将提供宝贵的 增强体内基因组编辑的递送、功效和特异性。

Project Summary The Cas9 platform has enabled genome editing, base editing (BE), and prime editing to induce gene knockouts as well as tailor-made sequence alterations. CRISPR-Cas9 systems have the potential to similarly revolutionize clinical practice through precise editing of disease loci. We have developed Nme2Cas9 as an editing platform with (1) compact size, facilitating single adeno-associated virus (AAV) delivery; (2) a dinucleotide (N4CC) protospacer-adjacent motif (PAM) that affords high target site density; and (3) exceptional accuracy. More recently, we developed Nme2Cas9 adenine base editor (ABE) systems as among the first to be validated in vivo for single-AAV delivery. AAV is a potent editing delivery modality in vivo, especially in extrahepatic tissues such as the central nervous system (CNS). Nonetheless, the therapeutic promise of base editing systems will hinge upon improving editing efficiency, limiting bystander edits (or their consequences), maximizing PAM-dependent targeting scope, and minimizing immunogenicity, toxicity, and prolonged deaminase expression (which can compromise editing efficiency and lead to safety risks such as hepatotoxicity and the accumulation of unwanted edits). Here we propose to capitalize on our establishment of single-vector Nme2-ABE systems to develop novel base editing capabilities with increased effectiveness, targeting scope, utility, and safety, and to validate these systems in the treatment of CNS disease models in mice. The goals of this proposal are (1) to develop next-generation, single-AAV, deaminase-inlaid Nme2-ABEs and guides with increased efficiency, a single-nucleotide PAM, and greater control over bystander editing; (2) to use next- generation, single-AAV Nme2-ABE systems for therapeutic editing of disease genes in the CNS of mouse models of amyotrophic lateral sclerosis and Batten disease; and (3) to develop systems that use repression by endogenous microRNAs and drug-dependent splicing systems to enhance the safety of AAV-delivered Nme2- ABE in vivo. These safety enhancements will reduce anti-Nme2-ABE immune responses, ameliorate potential toxic effects on the liver and on specific CNS cell types, and limit the off-target mutagenesis that can arise from sustained expression of the editing machinery. Successful completion of these aims will provide invaluable enhancements to the delivery, efficacy, and specificity of in vivo genome editing.