Structural, functional, and immunogenicity implications of F9 gene recoding.
Structural, functional, and immunogenicity implications of F9 gene recoding.
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F9基因重编码的结构、功能和免疫原性意义。
DOI：
10.1182/bloodadvances.2022007094
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发表时间：
2022-07-12
期刊：
影响因子：
7.5
通讯作者：
Kimchi-Sarfaty, Chava
中科院分区：
文献类型：
--
作者：
Katneni, Upendra K.;Alexaki, Aikaterini;Hunt, Ryan C.;Hamasaki-Katagiri, Nobuko;Hettiarachchi, Gaya K.;Kames, Jacob M.;McGill, Joseph R.;Holcomb, David D.;Athey, John C.;Lin, Brian;Parunov, Leonid A.;Kafri, Tal;Lu, Qi;Peters, Robert;V. Ovanesov, Mikhail;Freedberg, Daron I.;Bar, Haim;Komar, Anton A.;Sauna, Zuben E.;Kimchi-Sarfaty, Chava
凝血FIX的结构变化可能由基因重新编码诱导，并可能影响免疫原性。血友病B是由凝血因子IX（FIX）活性缺陷引起的凝血障碍。目前已批准多种重组FIX蛋白治疗血友病B，目前正在开发几种基因治疗产品。密码子优化是制药工业中经常使用的技术，用于通过使用多个同义密码子取代重新编码编码序列来改善重组蛋白表达。这种基因重编码的基本假设是同义替换不会改变蛋白质的特征，因为蛋白质的一级序列保持不变。然而，大量关键证据表明，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蛋白的结构、功能和体内免疫原性。在这里，我们评估了F9的多种重新编码变体，旨在进一步探索密码子使用偏好对蛋白质性质的影响。这些构建体的详细评价显示改变的构象，并通过核糖体分析的翻译动力学评估显示局部翻译动力学的差异。使用主要组织相容性复合体（MHC）相关肽蛋白质组学分析评估野生型和重编码构建体，显示FIX衍生肽与MHC II类分子结合的不同表达，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，基因编码的过度表达导致次优的翻译后加工。总的来说，我们的结果突出了与基因编码的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其他基因编码的重组蛋白的影响。
Structural changes in coagulation FIX may be induced by recoding of the gene and could impact immunogenicity. 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.
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影响因子：
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