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.

项目概要/摘要在大多数生物体中，遗传密码由 64 个核苷酸三联体组成，编码 20 个氨基酸结构。 PI 研究计划的总体目标是开发用于蛋白质合成的模块。重新编程遗传密码以精确探测和操纵生物系统的跨学科工具。重新编程遗传密码的核心是我们向蛋白质中添加非规范氨基酸 (ncAA) 的能力该提案的总体目标是开发能够生物合成和利用 ncAA 的细胞并进行探索。这些非自然生物在蛋白质进化和治疗开发中的效用。为了实现这一目标，第一个研究方向将集中于生成具有各种 21 世纪的完全自主的有机体具有第21个氨基酸的原核和真核细胞将具有生物合成途径并且用于新氨基酸构建模块的生物正交氨酰基-tRNA 合成酶 (aaRS)/tRNA 对。 ncAA 的生物合成途径将从其他物种或通过代谢重新利用获得。专门将这些 ncAA 整合到蛋白质中，我们将进化生物正交 aaRS/tRNA 对并将其添加所产生的具有第 21 个氨基酸的生物体将允许具有新颖蛋白质的进化。活性以及新疗法的开发，而不是发展新的或增强的酶活性。通过 20 种规范氨基酸可访问，将生成包含 ncAA 的酶突变体的文库非自然生物体并进行基于荧光激活细胞分选（FACS）的或生存进化的 ncAA 依赖性酶可用于防止非预期的增殖。接下来，我们将探讨这些的用途。具有用于治疗开发的额外蛋白质构建块的非天然生物体。将使用能够通过生物正交手柄生物合成和利用氨基酸的真核细胞来生产。具有优化治疗功效的抗体变体，具有额外的构建模块。将允许重新定向嵌合抗原受体（CAR）免疫细胞的特异性，从而提供我们在这个项目中的努力将产生一系列可切换 CAR 免疫细胞的新设计策略。具有额外氨基酸构建模块的生物体，并将产生基于 ncAA 的多功能平台可能彻底改变现代医学的蛋白质进化或治疗性蛋白质。