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Improving gene expression via Massively Parallel Synonymous Codon Variant Screening

通过大规模并行同义密码子变体筛选提高基因表达

基本信息

批准号：
9908223
负责人：
KEITH WYCOFF
金额：
$ 29.86万
依托单位：
PLANET BIOTECHNOLOGY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2022-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9908223
关键词：
Algorithm Design Algorithms Aliquot Antibodies Biotechnology Blood Coagulation Factor Cells Code Codon Nucleotides Complementary DNA Consumption DNA DNA Library Data Dependovirus Dideoxy Chain Termination DNA Sequencing Enzyme-Linked Immunosorbent Assay Factor IX Gene Expression Gene Transduction Agent Genes Genetic Transcription Goals Harvest Heavy-Chain Immunoglobulins Human In Vitro Individual Libraries Literature Liver Measures Messenger RNA Methods Motivation Mus Nicotiana Organism Outcome Phase Planets Plant Extracts Plant Genes Plant Leaves Plants Play Production Proteins RNA Recombinant Proteins Recombinants Research Personnel Rhizobium radiobacter Ribosomal RNA Role Small RNA System Technology Testing Therapeutic Time Tissues Tobacco Transcript Vaccines Variant Work Yeasts base cDNA Library commercialization design experimental study expression vector gene therapy genetic variant improved in vivo innovation next generation sequencing particle protein expression screening single molecule real time sequencing therapeutic protein therapy design vector

项目摘要

Our overall goal is to reduce to practice an innovative new method for empirically identifying the optimal codon usage for any gene where the intent is to maximize protein accumulation, in either heterologous or homologous expression systems. In this Phase I project we explain how this method will work and demonstrate its usefulness by identifying highly expressing codon variants of a human Factor IX gene in both plant and mammalian expression systems. Our method is based on the observation that there is a high degree of correlation between accumulation of mRNA and protein in our plant transient expression system and there is literature support for such a correlation in mammalian and yeast systems as well. We recently found that when four divergent synonymous codon variants of one immunoglobulin heavy chain were expressed together in the same plant, the relative abundance of the four mRNAs was a close match for their relative abundance when expressed separately. From this we conceived of a new method that we call Massively Parallel Synonymous Codon Variant Screening (MPSCVS), which should allow the comparison of a very large number of different gene variants in a single experiment. Demonstration of our system will begin with a library of approximately 59,000 synonymous codon variants of Factor IX in an adeno-associated virus (AAV) gene therapy vector. We will use the AAV library to transduce the livers of mice in vivo. RNA will be isolated from the mouse livers. We will subclone an aliquot of the library into a plant expression vector and use that to transform Agrobacterium tumefaciens, which is used for plant (Nicotiana benthamiana) transformation. We will express the library transiently in tobacco leaves, harvest leaf tissue and isolate total RNA. The mRNA from both plants and human cells will be used to produce double stranded cDNA, which will be “counted” by next generation sequencing to identify the most abundant mRNAs in both systems. Clones of high-expressing codon variants will be tested for protein and RNA expression individually in the appropriate expression system. We will then analyze the degree of correlation between RNA and protein expression and determine the overall efficiency of MPSCVS in identifying the best expressing variants.

我们的总体目标是减少实践一个创新的新方法，以经验确定最佳的对于任何基因的密码子使用，其目的是使蛋白质积累最大化，在异源或同源表达系统。在这个第一阶段的项目中，我们将解释这种方法是如何工作的，并演示通过鉴定植物和植物中高度表达的人因子IX基因的密码子变体，哺乳动物表达系统。我们的方法是基于这样一种观察：在我们的植物瞬时表达系统中mRNA和蛋白质的积累之间的关系，也支持哺乳动物和酵母系统中的这种相关性。我们最近发现，一条免疫球蛋白重链的不同同义密码子变体在相同的细胞中一起表达。在植物中，四种mRNA的相对丰度与其表达时的相对丰度密切匹配分开由此，我们设想了一种新的方法，我们称之为大规模并行同义密码子变异筛选（MPSCVS），这应该允许在一个非常大的数量的不同基因变异的比较，单一实验我们的系统演示将从一个包含大约59，000个同义密码子变体的文库开始开始在腺相关病毒（AAV）基因治疗载体中的因子IX。我们将使用AAV库来检索小鼠体内的肝脏。将从小鼠肝脏中分离RNA。我们将文库的一部分亚克隆到一种植物表达载体，并用其转化农杆菌，用于植物（烟草 benthamiana）转化。我们将在烟草叶中瞬时表达文库，收获叶组织，分离总RNA。来自植物和人类细胞的mRNA将用于产生双链cDNA，其将通过下一代测序进行“计数”以鉴定两种系统中最丰富的mRNA。高表达密码子变体的克隆将分别测试蛋白质和RNA表达。适当的表达系统。然后我们将分析RNA和蛋白质之间的相关程度表达，并确定MPSCVS在鉴定最佳表达变体中的总体效率。