Division of Plasma Protein Therapeutics, Hemostasis Branch, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation & Research, US Food and Drug Administration (FDA), Silver Spring, MD.
Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.
Blood Adv. 2022 Jul 12;6(13):3932-3944. doi: 10.1182/bloodadvances.2022007094.
Hemophilia B is a blood clotting disorder caused by deficient activity of coagulation factor IX (FIX). Multiple recombinant FIX proteins are currently approved to treat hemophilia B, and several gene therapy products are currently being developed. Codon optimization is a frequently used technique in the pharmaceutical industry to improve recombinant protein expression by recoding a coding sequence using multiple synonymous codon substitutions. The underlying assumption of this gene recoding is that synonymous substitutions do not alter protein characteristics because the primary sequence of the protein remains unchanged. However, a critical body of evidence shows that synonymous variants can affect cotranslational folding and protein function. Gene recoding could potentially alter the structure, function, and in vivo immunogenicity of recoded therapeutic proteins. Here, we evaluated multiple recoded variants of F9 designed to further explore the effects of codon usage bias on protein properties. The detailed evaluation of these constructs showed altered conformations, and assessment of translation kinetics by ribosome profiling revealed differences in local translation kinetics. Assessment of wild-type and recoded constructs using a major histocompatibility complex (MHC)-associated peptide proteomics assay showed distinct presentation of FIX-derived peptides bound to MHC class II molecules, suggesting that despite identical amino acid sequence, recoded proteins could exhibit different immunogenicity risks. Posttranslational modification analysis indicated that overexpression from gene recoding results in suboptimal posttranslational processing. Overall, our results highlight potential functional and immunogenicity concerns associated with gene-recoded F9 products. These findings have general applicability and implications for other gene-recoded recombinant proteins.
血友病 B 是一种凝血障碍疾病，由凝血因子 IX（FIX）活性缺乏引起。目前有多种重组 FIX 蛋白被批准用于治疗血友病 B，并且目前正在开发几种基因治疗产品。密码子优化是制药行业中常用的一种技术，通过使用多个同义密码子替换重新编码编码序列来提高重组蛋白的表达。这种基因重编码的基本假设是，同义替换不会改变蛋白质特性，因为蛋白质的一级序列保持不变。然而，大量证据表明，同义变体可以影响共翻译折叠和蛋白质功能。基因重编码可能会改变重编码治疗性蛋白的结构、功能和体内免疫原性。在这里，我们评估了设计用于进一步探索密码子使用偏性对蛋白质特性影响的多个 F9 重编码变体。这些构建体的详细评估显示出构象的改变，并且通过核糖体分析评估翻译动力学揭示了局部翻译动力学的差异。使用主要组织相容性复合体（MHC）相关肽蛋白质组学测定法对野生型和重编码构建体进行评估显示，FIX 衍生肽与 MHC 类 II 分子结合的情况明显不同，这表明尽管氨基酸序列相同，但重编码蛋白可能表现出不同的免疫原性风险。翻译后修饰分析表明，基因重编码的过表达导致翻译后加工不理想。总体而言，我们的结果强调了与基因重编码 F9 产品相关的潜在功能和免疫原性问题。这些发现具有普遍适用性和对其他基因重编码重组蛋白的影响。
