Improving gene expression via Massively Parallel Synonymous Codon Variant Screening

我们的总体目标是实践一种创新的新方法来经验地识别最优的 任何旨在最大化蛋白质积累的基因的密码子使用，无论是在异源还是 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的工作原理并进行演示 通过鉴定人类凝血因子IX基因的高表达密码子变体在植物和 哺乳动物的表达系统。我们的方法是基于观察到的高度相关性 我们植物瞬时表达系统中的mRNA和蛋白质的积累之间的关系 在哺乳动物和酵母系统中也支持这种相关性。我们最近发现，当四个人 一种免疫球蛋白重链的不同同义密码子变体在同一 植物中，这四个mrna的相对丰度与它们的相对丰度在表达时非常接近。 分开的。由此，我们设想了一种新的方法，我们称之为大规模平行同义密码子变体 筛查(MPSCVS)，这应该允许比较非常大量的不同基因变异在一个 单次实验。 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 目的：在腺相关病毒(AAV)基因治疗载体中获得凝血因子IX。我们将使用AAV文库来转换 活体小鼠的肝脏。将从小鼠的肝脏中分离出RNA。我们将把库的一份等分克隆到 一种植物表达载体及其转化用于烟草的根癌农杆菌 Benthamiana)变换。我们将在烟草叶片中瞬时表达该文库，收获叶片组织和 提取总RNA。来自植物和人类细胞的信使核糖核酸将被用来生产双链cdna， 下一代测序将对其进行“计数”，以确定两个系统中最丰富的mRNAs。 高表达密码子变体的克隆将分别在 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 并确定MPSCVS在确定最佳表达变异体方面的总体效率。

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.