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 氨基酸残基分别由多个所谓的同义密码子编码。同义密码子是 然而，在单个mRNAs和整个基因组中并不存在相同的频率。这种非-的模式 密码子的统一使用被称为密码子使用偏向。密码子的使用偏向因生物体和 代表了有机体的一种独特特征。特定生物体密码子的选择与特定生物体相关 同源tRNA的群体差异。在单细胞和多细胞生物体中都存在一个 密码子使用与细胞tRNA含量呈强正相关，这意味着密码子偏好性将 可能对翻译延伸率有直接影响。事实上，频繁使用的/最佳密码子是 由于更高的可用性(期间)，发现翻译规则比不常用的规则更快 对应的频繁同源tRNA的翻译)。密码子偏向的一个技术含义是 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 合成速度和蛋白质产量。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产业。然而，最近的许多研究 确定了这种用于密码子优化的标准方法的一个重要缺陷。虽然，同义词 改变被认为是沉默的，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多个方面。重要的是，它证明了同义词 密码子替换可能会影响蛋白质折叠和翻译后修饰，因此可能会 功能后果。此外，同义密码子的改变被发现与超过50个相关 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，对于为什么某些同义突变具有功能性和临床意义，人们的理解还很有限。 后果，而其他人则不会。拟议的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)为模型的密码子替换对蛋白质功能的影响 系统。F9的基因缺陷是血友病B的原因；而几种与疾病相关的同义词 已经在这个基因中发现了突变。此外，FIX是一种可以进行密码子优化的药物产品 密码子优化版本的F9被用于基因治疗试验。我们的目标是在体外和体外使用 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基因中同义突变有害的区域。此外，免疫原性也是另一个原因。 在任何治疗性蛋白质的开发中都是关键问题；然而，密码子的潜在影响- 在诱导免疫反应方面的优化还没有研究。通过评估呈现在 通过MHC相关多肽蛋白质组学(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变体被处理和 以不同的方式呈现。我们将进一步研究已识别的固定多肽诱导 通过研究T细胞的反应来进行免疫反应。来自功能分析和免疫原性的数据 研究将结合定义最佳密码子优化策略，以提供最高的产量完全 具有不变免疫原性的功能蛋白，从而允许创建更安全和更有效的修复 治疗学。我们相信，这种方法将服从于任何蛋白质疗法的设计。

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.