Safer and more effective FIX therapeutics: impact of codon optimization

遗传密码是退化的。除了两种氨基酸（蛋氨酸和色氨酸）外，所有其他氨基酸 氨基酸残基各自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以及整个基因组中并不以相等的频率存在。这种非- 一致的密码子使用被称为密码子使用偏好。密码子使用偏好在生物体之间变化， 代表了生物体的独特特征。生物体特异性密码子选择与生物体特异性 同源tRNA群体的差异。在单细胞和多细胞生物中， 密码子使用和细胞tRNA含量之间存在强正相关，这意味着密码子偏好性 可能对翻译延伸率有直接影响。事实上，频繁使用/最佳密码子是，作为一个 规则，被发现翻译得比不经常使用的更快，因为更高的可用性（在 翻译）相应的常见同源tRNA。密码子偏好性的技术含义是， 用频繁使用的密码子替换罕见的同义密码子（密码子优化），可以增加蛋白质的 合成速率和由此的蛋白质产量。该平台技术已广泛应用于基础研究和 用于生产重组/治疗蛋白的生物技术工业。然而，最近的许多研究 鉴定了这种用于密码子优化的标准方法的一个重要缺点。虽然，同义词 假定这些变化是沉默的，但最近的数据表明，同义密码子替换可能 影响mRNA和蛋白质生物合成的许多方面。重要的是，它表明，同义词 密码子取代可能影响蛋白质折叠和翻译后修饰， 功能性后果。此外，同义密码子的变化被发现与50多个 这些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。但是，在这方面， 目前，对于为什么一些同义突变具有功能和临床意义， 后果，而其他人则没有。这些研究旨在阐明同义词的影响， 以F9基因编码的凝血因子IX（FIX）为模型，研究密码子替换对蛋白功能的影响 系统F9中的遗传缺陷是血友病B的原因;而几种疾病相关的同义词 已经在该基因中鉴定出突变。此外，FIX是一种易于进行密码子优化的药物产品 和密码子优化版本的F9用于基因治疗试验。我们的目标是在体外和体外 评估和理解密码子优化对FIX折叠和功能的影响的方法， F9 mRNA中的同义突变将是有害的区域。此外，免疫原性是另一个 在任何治疗性蛋白质的开发中的关键问题;然而，密码子的潜在影响- 尚未研究引发免疫应答的优化。通过评估呈现在细胞表面的肽， 抗原呈递细胞表面，通过MHC相关肽蛋白质组学（MAPPS）分析，我们 产生的初步数据表明野生型和密码子优化的FIX变体被加工， 呈现不同。我们将进一步研究所鉴定的FIX-肽诱导细胞凋亡的倾向。 通过研究T细胞反应的免疫反应。功能分析和免疫原性数据 研究将结合起来，以确定最佳的密码子优化策略，提供最高产量的完全 免疫原性不变的功能性蛋白，从而允许创建更安全和更有效的FIX 治疗学我们相信这种方法将适用于任何蛋白质治疗剂的设计。

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.