Integrating Chemistry and Evolution to Illuminate Biology and Enable Novel Therapeutics

整合化学和进化来照亮生物学并实现新的治疗方法 项目摘要 我们的实验室在化学和进化的界面上领导了三个研究项目。上 程序，我们开发了DNA模板合成作为一种新的方法来合成和发现生物活性 结合了生物进化和有机化学的小分子和聚合物。在第二个节目中， 我们开发了prime editing和base editing两种基因组编辑技术， 在哺乳动物基因组中的靶位点安装点突变、插入和缺失， 需要双链DNA断裂或供体DNA模板。在第三个程序中，我们应用了这些 拯救人类遗传疾病动物模型的技术。这MIRA更新旨在推进这些 三个针对新型小分子和基因组编辑疗法的研究项目。 在第一个程序中，我们开发了DNA模板合成，产生了DNA模板小分子文库， 包含> 250，000个独特大环的分子，并对这些文库进行体外选择， 发现新的激酶和蛋白酶抑制剂，包括第一个胰岛素降解的生理抑制剂 酶（IDE）。最近，我们发现了一类新的大环亲环蛋白抑制剂，包括CypD- 特定的变体，并解决了与CypD结合的几种抑制剂的X射线共晶结构。我们建议 了解这些大环化合物和CypD之间的相互作用，确定这些大环化合物的细胞活性， 化合物，并开发其他亲环素的特异性抑制剂。我们还将筛选一种新的DNA模板 约640，000个大环化合物文库，针对包括DNA修复蛋白在内的其他治疗目标， 肿瘤抑制因子、E3泛素连接酶、白细胞介素和白细胞介素受体、去泛素酶和ATP酶。 在第二个程序中，我们开发了基础编辑和主要编辑，这两种方法使 在哺乳动物细胞中进行精确的基因组编辑，而不需要双链DNA断裂或供体DNA 模板碱基编辑使用可编程的DNA结合来安装转换突变（C·G→T·A，或A·T→G·C 融合到天然或实验室进化的核碱基脱氨酶的蛋白质，而引物编辑使 安装所有可能类型的小取代、插入、缺失及其组合， Cas9-逆转录酶融合物的用途，所述融合物从工程化引物复制编辑的DNA信息， 将引导RNA直接编辑到哺乳动物基因组中的靶位点。我们建议通过以下方式推进主要编辑 揭示了有效的Prime编辑的细胞状态和细胞类型要求，工程改进了Prime编辑 系统，并开发用于将主要编辑器递送到人类遗传疾病的动物模型中的方法。 在第三个程序中，我们将碱基编辑与体内递送方法相结合，以直接校正DNA序列。 导致遗传疾病的突变，在酪氨酸血症动物模型中实现表型拯救75，遗传 肥胖症53，76，尼曼-皮克综合征55和早衰症。我们建议将基础编辑和主要编辑应用于 验证永久治疗慢性疼痛和脊髓性肌萎缩症（SMA）的新型体内方法。

Integrating Chemistry and Evolution to Illuminate Biology and Enable Novel Therapeutics Project Summary Our laboratory has led three research programs at the interface of chemistry and evolution. In the first program, we developed DNA-templated synthesis as a novel approach to synthesize and discover bioactive small molecules and polymers that combines biological evolution with organic chemistry. In the second program, we developed prime editing and base editing, two genome editing technologies that collectively enable precise installation of point mutations, insertions, and deletions at targeted sites in mammalian genomes without requiring double-stranded DNA breaks or donor DNA templates. In the third program, we have applied these technologies to rescue animal models of human genetic diseases. This MIRA renewal seeks to advance these three research programs towards novel small-molecule and genome-editing therapeutics. In the first program, we developed DNA-templated synthesis, generated libraries of DNA-templated small molecules containing >250,000 unique macrocycles, and performed in vitro selections on these libraries to discover novel kinase and protease inhibitors, including the first physiological inhibitor of insulin-degrading enzyme (IDE). Recently, we discovered a new class of macrocyclic cyclophilin inhibitors that include CypD- specific variants, and solved X-ray co-crystal structures of several inhibitors bound to CypD. We propose to understand the interactions between these macrocycles and CypD, determine the cellular activity of these compounds, and develop specific inhibitors of other cyclophilins. We will also screen a new DNA-templated library of ~640,000 macrocycles against additional targets of therapeutic interest including DNA repair proteins, tumor suppressors, E3 ubiquitin ligases, interleukin and interleukin receptors, deubiquitinases, and ATPases. In the second program, we developed base editing and prime editing, the two methods that enable precision genome editing in mammalian cells without requiring double-strand DNA breaks or donor DNA templates. Base editing installs transition mutations (C•G→T•A, or A•T→G•C) using programmable DNA-binding proteins fused to natural or laboratory-evolved nucleobase deaminase enzymes, while prime editing enables the installation of all possible types of small substitutions, insertions, deletions, and combinations thereof through the use of Cas9–reverse transcriptase fusions that copy edited DNA information from an engineered prime editing guide RNA directly into targeted sites in mammalian genomes. We propose to advance prime editing by revealing the cell-state and cell-type requirements for efficient prime editing, engineering improved prime editing systems, and developing methods for prime editor delivery into animal models of human genetic disease. In the third program, we have integrated base editing with in vivo delivery methods to directly correct the mutations that cause genetic diseases, achieving phenotypic rescue in animal models of tyrosinemia75, genetic deafnesses53,76, Niemann-Pick syndrome55, and progeria. We propose to apply base editing and prime editing to validate novel in vivo approaches to permanently treat chronic pain and spinal muscular atrophy (SMA).