Development and Applications of Unnatural Organisms with a 21 Amino Acid Genetic Code

具有21个氨基酸遗传密码的非自然生物的开发与应用

基本信息

批准号：
10640232
负责人：
Han Xiao
金额：
$ 37.84万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2024-06-30
项目状态：
已结题

项目摘要

PROJECT SUMMARY/ABSTRACT In most organisms, the genetic code, consisting of 64 triplets of nucleotides, encodes 20 amino acid building blocks used in the synthesis of proteins. The overall goal of the PI’s research program is to develop interdisciplinary tools to reprogram the genetic code to precisely probe and manipulate biological systems. Central to reprogramming the genetic code is our ability to add noncanonical amino acids (ncAAs) to proteins of interest. The overall goal of this proposal is to develop cells able to biosynthesize and utilize ncAAs and explore the utility or these unnatural organisms in protein evolution and therapy development. To achieve this goal, the first research direction will focus on the generation of completely autonomous organisms with a variety of 21st amino acids. The prokaryotic and eukaryotic cells with the 21st amino acid will harbor a biosynthetic pathway and a bioorthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair for the new amino acid building block. The biosynthesis pathway of ncAAs will be obtained from other species or via metabolic repurposing. To site- specifically incorporate these ncAAs into proteins, we will evolve bioorthogonal aaRS/tRNA pairs and add them to the cells. The resulting organisms with a 21st amino acids will allow for the evolution of proteins with novel activities as well as the development of new therapies. To evolve novel or enhanced enzyme activity not accessible by the 20 canonical amino acids, a library of ncAA-containing enzyme mutants will be generated in the unnatural organisms and subjected to a fluorescence-activated cell sorting (FACS)-based or survival selection. The evolved ncAA-dependent enzymes can be used to prevent the unintended proliferation of genetically modified organisms or to prepare autotrophic vaccines. Next, we will explore the utility of these unnatural organisms with additional protein building blocks for therapeutic development. The prokaryotic and eukaryotic cells able to biosynthesize and utilize amino acids with bioorthogonal handles will be used to produce antibody variants with optimized therapeutic efficacy. Engineered immune cells with additional building blocks will allow for the redirection of the specificity of chimeric antigen receptor (CAR)-immune cells, thus providing a new design strategy for switchable CAR-immune cells. Our efforts in this project will yield a collection of organisms with additional amino acids building blocks, and will result in versatile platforms for ncAA-based protein evolution or therapeutic proteins that could revolutionize modern medicine.

项目摘要/摘要在大多数生物体中，遗传密码由64个核苷酸组成，编码20个氨基酸建筑物用于合成蛋白质的块。 PI研究计划的总体目标是开发跨学科工具将通用代码重新编程为精确探测和操纵生物系统。重编程通用代码的中心是我们将非规范氨基酸（NCAA）添加到蛋白质的能力兴趣。该提案的总体目标是开发能够生物合成和利用NCAA并探索的细胞蛋白质进化和治疗开发中的效用或这些不自然的生物。为了实现这一目标，第一个研究方向将集中于以各种第21的全部自主生物的产生氨基酸。 21氨基酸的原核和真核细胞将带有生物合成途径，并且新型氨基酸构建块的生物正交氨基酰基-TRNA合成酶（AARS）/TRNA对。这 NCAA的生物合成途径将从其他物种或通过代谢重新定位获得。到现场 - 特别将这些NCAA纳入蛋白质中，我们将进化生物正交的AARS/tRNA对并添加它们到细胞。带有21氨基酸的生物生物可以使蛋白质与新颖活动以及新疗法的发展。进化新颖或增强的酶活性而不是可以通过20个规范氨基酸来访问，将在含NCAA的酶突变体的库中产生不自然的组织，并受到荧光激活的细胞分选（FACS）的基础或存活选择。进化的NCAA依赖性酶可用于防止意外的增殖转基因的生物或制备自养疫苗。接下来，我们将探索这些效用不自然的生物具有其他蛋白质构建块，用于治疗性发育。原核生物和真核细胞可以和使用生物正交手柄的氨基酸来生产具有优化治疗有效性的抗体变体。具有其他构件的工程免疫细胞将允许重定向嵌合抗原受体（CAR） - 免疫细胞的特异性，从而提供了可开关的CAR-MMUNE细胞的新设计策略。我们在这个项目中的努力将产生带有其他氨基酸的生物体，并将为基于NCAA的多功能平台提供多功能平台蛋白质进化或可以彻底改变现代医学的蛋白质。