Continuous Evolution of Proteins with Novel Therapeutic Potential

具有新治疗潜力的蛋白质的不断进化

• 批准号: 8962813
• 负责人: DAVID R LIU
• 金额: $ 45.15万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8962813
• 关键词: Address; Bacteriophages; Binding; CRISPR/Cas technology; Cells; Cleaved cell; Clinical; Clinical Trials; DNA; DNA-Directed RNA Polymerase; Development; Directed Molecular Evolution; Disease; Drug resistance; Enzymes; Evolution; Gene Proteins; Gene Structure; Genes; Genome; Genome engineering; Genomics; Grant; Guide RNA; Health; Hepatitis C virus; Hereditary Disease; Human; Human Genetics; Human Genome; Intervention; Laboratories; Mammalian Cell; Mediating; Messenger RNA; Methods; Nature; Peptide Hydrolases; Property; Protease Inhibitor; Proteins; Proteome; Research Personnel; Resistance; Site; Specificity; Speed; System; TNF gene; Therapeutic; Therapeutic Agents; Time; Variant; Vision; drug candidate; gene product; human disease; interest; macromolecule; next generation; novel strategies; novel therapeutics; nuclease; promoter; recombinase; small molecule; success; therapeutic protein

 DESCRIPTION (provided by applicant): The vast majority of current therapeutic agents function by binding to disease-associated macromolecules and modulating their activity. Recent developments, however, have made increasingly realistic the possibility of developing next-generation therapeutics that do not simply bind targets implicated in disease, but instead alter the covalent structure of genes and gene products in ways that can more effectively treat-or even cure-many diseases. While the possibility of precisely manipulating genes and proteins in mammalian cells and, eventually, in humans, has enormous potential, several major challenges must be overcome to fully realize this vision. Perhaps the most significant of these challenges is the efficient creation of the macromolecules that are needed to alter genomes or proteomes with a high degree of selectivity and potency. To realize a vision in which arbitrary genes or proteins can be manipulated in mammalian cells to treat disease thus requires new approaches to rapidly generating macromolecules with precise, tailor-made properties. During the last granting period, we developed a system that enables proteins to evolve continuously in the laboratory, requiring virtually no researcher intervention. The resulting system, phage-assisted continuous evolution (PACE), allows proteins to undergo directed evolution at a rate ~100-fold faster than conventional methods. In the first applications of PACE, we rapidly evolved RNA polymerases with dramatically different DNA promoter specificities. We also identified the vulnerabilities of drug candidates to the evolution of drug resistance by using PACE to evolve proteases that are resistant to HCV protease inhibitors currently used in human clinical trials. In addition, we developed important PACE capabilities beyond basic positive selection, including small- molecule modulation of selection stringency and negative selection against undesired activities. These initial studies established PACE as a robust and general platform to evolve proteins with tailor-made properties at an unprecedented speed. In the next granting period, we propose to apply these developments to continuously evolve four classes of proteins or RNAs, each with the ability to manipulate the covalent structure of genes or gene products, and each with potential relevance to the development of next-generation human therapeutics: recombinase enzymes that insert DNA of interest into safe-harbor loci in the human genome, proteases that specifically cleave disease- associated proteins, orthogonal Cas9 (CRISPR) nucleases with altered PAM specificities and enhanced activities, and "smart" Cas9 guide RNAs that mediate genome engineering only in those cells that are in specific disease-associated cell states. Success would establish the novel therapeutic potential of these proteins and RNAs to address a wide range of human diseases, including many human genetic disorders.

 描述（由申请人提供）：目前绝大多数治疗剂通过与疾病相关的大分子结合并调节其活性来发挥作用，然而，最近的发展使得开发不仅仅结合靶点的下一代疗法的可能性变得越来越现实。与疾病有关，而是以更有效地治疗甚至治愈许多疾病的方式改变基因和基因产物的共价结构，而精确操纵哺乳动物细胞以及最终人类中的基因和蛋白质的可能性已经存在。巨大的为了充分实现这一愿景，必须克服几个主要挑战，也许这些挑战中最重要的是创建具有高度选择性和效力的改变基因组或蛋白质组所需的大分子。可以在哺乳动物细胞中操纵任意基因或蛋白质来治疗疾病，因此需要新的方法来快速生成具有精确、定制特性的大分子。几乎没有研究人员由此产生的系统，即噬菌体辅助连续进化（PACE），使蛋白质能够以比传统方法快约 100 倍的速度进行定向进化。在 PACE 的首次应用中，我们快速进化出具有显着不同的 DNA 启动子的 RNA 聚合酶。我们还通过使用 PACE 进化出对目前用于人体临床试验的 HCV 蛋白酶抑制剂具有抗药性的蛋白酶，确定了候选药物对耐药性进化的脆弱性。此外，我们还开发了超越基本的 PACE 功能。正选择，包括选择严格性的小分子调节和针对不良活动的负选择，这些初步研究将 PACE 建立为一个强大且通用的平台，以前所未有的速度进化具有定制特性的蛋白质。应用这些发展来不断进化四类蛋白质或RNA，每一种都具有操纵基因或基因产物的共价结构的能力，并且每一种都与下一代人类疗法的开发具有潜在的相关性：插入DNA的重组酶感兴趣人类基因组中的安全港位点、特异性切割疾病相关蛋白的蛋白酶、具有改变的 PAM 特异性和增强活性的正交 Cas9 (CRISPR) 核酸酶，以及仅在特定细胞中介导基因组工程的“智能”Cas9 引导 RNA特定疾病相关的细胞状态的成功将确立这些蛋白质和 RNA 的新治疗潜力，以解决广泛的人类疾病，包括许多人类遗传性疾病。