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（由适用提供）：绝大多数当前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，最近的发展使越来越现实地开发了不仅会结合疾病中实施的靶标，还可以改变基因和基因产物的共价结构的可能性，以更有效治疗或什至治愈多种疾病的方式改变了基因和基因产品的共价结构。尽管哺乳动物细胞中精确操纵基因和蛋白质的可能性，最终在人类中具有增强的潜力，但必须克服一些主要挑战才能充分实现这一愿景。这些挑战中最重要的可能是有效地创造了具有高度选择性和效力的基因组或蛋白质所需的大分子。为了实现一种视觉，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迅速产生具有精度，量身定制的特性的大分子。在最后一份授予期间，我们开发了一个使蛋白质能够在实验室中不断发展的系统，几乎不需要研究人员干预。所得系统的噬菌体辅助连续演化（PACE）使蛋白质可以以比常规方法快〜100倍的速度进行定向演化。在PACE的首次应用中，我们迅速进化了具有动态不同DNA启动子规范的RNA聚合酶。我们还通过使用PACE进化了对人类临床试验中目前使用的HCV蛋白抑制剂的耐药蛋白来确定候选药物对耐药性进化的脆弱性。此外，我们开发了重要的步伐功能，超出了基本的积极选择，包括对未耶稣活动的小分子调制和否定的选择。这些最初的研究将PACE确定为一个坚固且一般的平台，可以以前所未有的速度发展具有量身定制特性的蛋白质。 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 therapy: recombinase enzymes that insert DNA of interest into safe-harbor locali in the human genome, proteins that specifically clear disease-associated proteins,正交CAS9（CRISPR）核武器具有改变PAM特异性和增强活动的核，并且“智能” CAS9指导RNA仅在特定疾病相关的细胞状态的细胞中介导基因组工程。成功将建立这些蛋白质和RNA的新型治疗潜力，以解决包括许多人类遗传疾病在内的广泛的人类疾病。