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: 10600842
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10600842
• 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 therapy trial; Genes; Genetic Code; Goals; Hemophilia B; Hemorrhage; 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; Transfer RNA; Translating; Translations; Trauma; Tryptophan; Variant; Vitamin K; Work; biopharmaceutical industry; design; immunogenicity; improved; protein folding; protein function; protein structure; ribosome profiling; technology platform; 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.

遗传密码是简并的。除两种氨基酸（蛋氨酸和色氨酸）外，所有其他氨基酸 每个氨基酸残基都由多个所谓的同义密码子编码。同义密码子是 然而，在单个 mRNA 和整个基因组中出现的频率并不相同。这种模式非 统一的密码子使用被称为密码子使用偏差。密码子使用偏差因生物体而异 代表了有机体的独特特征。生物体特异性密码子的选择与生物体特异性有关 同源 tRNA 群体的差异。在单细胞和多细胞生物中都存在 密码子使用与细胞 tRNA 含量之间呈强正相关，这意味着密码子偏倚会 可能对平移伸长率产生直接影响。事实上，经常使用/最佳的密码子是 规则，由于可用性更高（在 相应的频繁同源 tRNA 的翻译）。密码子偏差的技术含义是 用常用密码子替换稀有同义密码子（密码子优化），可以增加蛋白质 合成率和蛋白质产量。该平台技术已广泛应用于基础研究和 用于生产重组/治疗性蛋白质的生物技术工业。然而，最近的大量研究 确定了这种密码子优化标准方法的一个重要缺点。虽然，同义 变化被认为是沉默的，最近的数据表明同义密码子替换可能 影响 mRNA 和蛋白质生物发生的许多方面。重要的是，事实证明同义 密码子替换可能会影响蛋白质折叠和翻译后修饰，因此可能具有 功能性后果。此外，同义密码子的变化被发现与超过 50 疾病明确证明了密码子使用对于基因/蛋白质功能的重要性。然而， 目前，对于为什么一些同义突变具有功能和临床作用的理解还很有限。 后果，而其他人却没有。拟议的研究旨在阐明同义的影响 使用 F9 基因编码的凝血因子 IX (FIX) 作为模型，进行密码子替换对蛋白质功能的影响 系统。 F9 的基因缺陷导致血友病 B；而一些与疾病相关的同义词 已在该基因中发现突变。此外，FIX 是一种适合密码子优化的药物产品 F9 的密码子优化版本用于基因治疗试验。我们的目标是在体外和离体使用 评估和理解密码子优化对 FIX 折叠和功能的影响的方法，并定义 F9 mRNA 中的同义突变是有害的区域。另外，免疫原性也是一个因素 任何治疗性蛋白质开发中的关键问题；然而，密码子的潜在影响 尚未研究引发免疫反应的优化。通过评估呈现在 抗原呈递细胞表面，通过 MHC 相关肽蛋白质组学 (MAPPS) 测定，我们 生成的初步数据表明野生型和密码子优化的 FIX 变体已被处理并 呈现方式不同。我们将进一步检查已识别的 FIX 肽诱导 通过研究 T 细胞反应来进行免疫反应。来自功能分析和免疫原性的数据 研究将结合起来以确定最佳密码子优化策略，以提供最高的完全产量 具有未改变免疫原性的功能蛋白，从而可以创建更安全、更有效的 FIX 疗法。我们相信这种方法将适用于任何蛋白质治疗的设计。

项目成果

In Silico Evaluation of Cyclophilin Inhibitors as Potential Treatment for SARS-CoV-2.

亲环蛋白抑制剂作为 SARS-CoV-2 潜在治疗方法的计算机评估。

• 发表时间: 2021-06
• 影响因子: 4.2
• 作者: Laurie, Kyle;Holcomb, David;Kames, Jacob;Komar, Anton A;DiCuccio, Michael;Ibla, Juan C;Kimchi
• 通讯作者: Kimchi

Gene variants of coagulation related proteins that interact with SARS-CoV-2.

与 SARS-CoV-2 相互作用的凝血相关蛋白的基因变体。

• 发表时间: 2021
• 影响因子: 4.3
• 作者: Holcomb, David;Alexaki, Aikaterini;Hernandez, Nancy;Hunt, Ryan;Laurie, Kyle;Kames, Jacob;Hamasaki;Komar, Anton A;DiCuccio, Michael;Kimchi
• 通讯作者: Kimchi

Potential impact on coagulopathy of gene variants of coagulation related proteins that interact with SARS-CoV-2.

与 SARS-CoV-2 相互作用的凝血相关蛋白的基因变异对凝血病的潜在影响。

• 发表时间: 2020-09-18
• 作者: Holcomb, David;Alexaki, Aikaterini;Hernandez, Nancy;Laurie, Kyle;Kames, Jacob;Hamasaki;Komar, Anton A;DiCuccio, Michael;Kimchi
• 通讯作者: Kimchi

Advances in methods for tRNA sequencing and quantification.

• DOI: 10.1016/j.tig.2023.11.001
• 发表时间: 2023-12-01
• 期刊: Trends in genetics : TIG
• 作者: Nigam H Padhiar;Upendra K. Katneni;Anton A Komar;Yuri Motorin;C. Kimchi
• 通讯作者: C. Kimchi

Sequence analysis of SARS-CoV-2 genome reveals features important for vaccine design.

SARS-CoV-2 基因组的序列分析揭示了对疫苗设计重要的特征。

• 发表时间: 2020
• 影响因子: 4.6
• 作者: Kames, Jacob;Holcomb, David D;Kimchi, Ofer;DiCuccio, Michael;Hamasaki;Wang, Tony;Komar, Anton A;Alexaki, Aikaterini;Kimchi
• 通讯作者: Kimchi