Safer and more effective FIX therapeutics: impact of codon optimization

更安全、更有效的 FIX 疗法：密码子优化的影响

• 批准号: 10372111
• 负责人: Anton A. Komar
• 金额: $ 37.13万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372111
• 关键词: Academia; Affect; Amino Acid Sequence; Amino Acids; Antigen-Presenting Cells; Autoimmunity; Basic Science; Biogenesis; Biological Assay; Biological Models; Biotechnology; Blood coagulation; Brain hemorrhage; Calcium ion; Clinical; Code; Codon Nucleotides; Data; Development; Disease; F9 gene; Factor IX; Factor VIIIa; Factor X; Frequencies; Gene Proteins; Gene therapy trial; Genes; Genetic Code; Goals; Hemophilia B; Human; Immune response; In Vitro; Individual; Industry; Infusion procedures; Lead; Messenger RNA; Methionine; Modeling; Molecular Conformation; Mutation; Open Reading Frames; Organism; Patients; Pattern; Peptides; Pharmaceutical Preparations; Phospholipids; Population; Post-Translational Protein Processing; Preparation; Process; Production; Protein Biosynthesis; Protein Conformation; Proteins; Proteomics; Recombinants; Reporting; Ribosomes; Risk; Serine Protease; Site; Structure; Surface Antigens; T cell response; Technology; Testing; Therapeutic; Time; Transfer RNA; Translating; Translations; Trauma; Tryptophan; Variant; Vitamin K; Work; base; biopharmaceutical industry; design; immunogenicity; improved; protein folding; protein function; protein structure; protein structure function; ribosome profiling; therapeutic protein; whole genome

The genetic code is degenerate. With the exception of two amino acids (methionine and tryptophan), all other amino acid residues are each encoded by multiple, so-called synonymous codons. Synonymous codons are however not present at equal frequencies in individual mRNAs as well as entire genomes. This pattern of non- uniform codon use is known as codon usage bias. Codon usage bias varies between organisms and represents a unique feature of an organism. Organism-specific codon choice is related to organism-specific differences in populations of cognate tRNAs. In both unicellular and multicellular organisms there exists a strong positive correlation between codon usage and cellular tRNA content, meaning that codon bias would likely have a direct impact on translation elongation rates. Indeed, frequently used/optimal codons were, as a rule, are found to be translated more rapidly than infrequently used ones due to the higher availability (during translation) of corresponding frequent cognate tRNAs. A technological implication of codon bias is that substitution of rare synonymous codons with frequently used ones (codon optimization), can increase protein synthesis rates and thus protein yield. This platform technology has been widely used in basic research and in biotechnology industry for production of recombinant/therapeutic-proteins. However, numerous recent studies identified an important drawback of this standard approach for codon optimization. Although, synonymous changes were presumed to be silent, recent data have shown that synonymous codon substitutions may influence many aspects of mRNA and protein biogenesis. Importantly, it was demonstrated that synonymous codon substitutions may affect protein folding and post-translational modifications and thus may have functional consequences. Furthermore, synonymous codon changes were found to be associated with over 50 diseases unequivocally demonstrating the importance of codon usage for gene/protein function. However, currently, there is yet a limited understanding of why some synonymous mutations have functional and clinical consequences while others do not. The proposed studies are aimed at elucidating the effects of synonymous codon substitutions on protein function, using blood coagulation factor IX (FIX), coded by F9 gene as a model system. Genetic defects in F9 are responsible for hemophilia B; while several disease-associated synonymous mutations have been identified in this gene. Moreover, FIX is a drug-product amenable to codon-optimization and codon-optimized versions of F9 are used in gene therapy trials. Our goal is to use in vitro and ex vivo approaches to assess and understand the effects of codon optimization on FIX folding and function, and define regions in F9 mRNA in which synonymous mutations would be deleterious. Also, immunogenicity is another key concern in the development of any therapeutic protein; however, the potential influence of codon- optimization on eliciting immune responses has not been studied. By assessing the peptides presented on the surface of antigen presenting cells, through a MHC-associated peptide proteomics (MAPPS) assay, we generated preliminary data indicating that wild-type and codon-optimized FIX variants are processed and presented differently. We will further examine the propensity of the identified FIX-peptides to induce an immune response by investigating T cells responses. Data from functional analysis and immunogenicity studies will be combined to define the best codon optimization strategies that provide the highest yields of fully functional protein with unaltered immunogenicity, thus allowing the creation of safer and more effective FIX therapeutics. We believe this approach will be amenable to the design of any protein therapeutic.

遗传密码是退化的。除两个氨基酸（蛋氨酸和色氨酸）外，所有其他 氨基酸残基分别由多个所谓的同义密码子编码。同义密码子是 但是，在单个mRNA和整个基因组中不同等频率。这种非 - 均匀的密码子的使用被称为密码子使用偏差。密码子的使用偏见在生物和生物之间各不相同 代表有机体的独特特征。有机体特定的密码子选择与特定于生物的密码子有关 同源TRNA的种群差异。在单细胞和多细胞生物中都存在 密码子使用和细胞tRNA含量之间的强正相关，这意味着密码子偏差将会 可能会直接影响翻译伸长率。确实，经常使用/最佳密码子是 由于较高的可用性，发现规则比不经常使用的规则更快（在 相应频繁的同源trnas的翻译）。密码子偏见的技术含义是 用常用的密码子替换稀有的同义密码子（密码子优化）可以增加蛋白质 合成速率，因此蛋白质产量。该平台技术已被广泛用于基础研究和 生物技术行业用于生产重组/治疗性蛋白质。但是，最近的许多研究 确定了这种标准方法的重要缺点，以进行密码子优化。虽然，同义词 假定变化是沉默的，最近的数据表明，同义的密码子替代可能 影响mRNA和蛋白质生物发生的许多方面。重要的是，证明了同义词 密码子取代可能会影响蛋白质折叠和翻译后修饰，因此可能具有 功能后果。此外，发现同义密码子更改与50多个 疾病明确地证明了密码子使用对基因/蛋白质功能的重要性。然而， 目前，人们对某些同义突变具有功能性和临床的理解有限 后果而其他人则没有。拟议的研究旨在阐明同义词的影响 使用血液凝结因子IX（FIX）的蛋白质功能的密码子取代，由F9基因编码为模型 系统。 F9中的遗传缺陷负责血友病B；而几个与疾病相关的同义词 该基因已鉴定出突变。此外，FIX是一种可容纳密码子优化的药物产品 基因治疗试验中使用了F9的密码子优化版本。我们的目标是在体外和体内使用 评估和理解密码子优化对修复折叠和功能的影响的方法，并定义 F9 mRNA中的区域将是有害的。另外，免疫原性是另一种 开发任何治疗蛋白的关键问题；但是，密码子的潜在影响 尚未研究引起免疫反应的优化。通过评估在 通过MHC相关的肽蛋白质组学（MAPP）测定，抗原呈递细胞的表面，我们 生成的初步数据表明，处理了野生型和密码子优化的修复变体，并且已处理 呈现不同。我们将进一步研究已鉴定的固定肽的倾向，以诱导 通过研究T细胞反应的免疫反应。功能分析和免疫原性的数据 研究将合并以定义最佳的密码子优化策略，以提供最高的收益率 功能性蛋白质具有不变的免疫原性，从而允许产生更安全，更有效的固定 疗法。我们认为，这种方法适合于任何蛋白质治疗的设计。