A novel form of light chain gene replacement

轻链基因替换的新形式

• 批准号: 10330601
• 负责人: Patrick C. Swanson
• 金额: $ 18.19万
• 依托单位: CREIGHTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-19 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10330601
• 关键词: Adaptive Immune System; Alleles; Antibodies; Antibody Formation; Antigen Receptors; B cell repertoire; B-Lymphocytes; Base Sequence; Binding; Categories; Cells; Chimera organism; Code; DNA; Data; Dependence; Development; Ephrin-A5; Epitopes; Event; Exons; Exploratory/Developmental Grant; Foundations; Framework Regions; Frequencies; Gene Conversion; Gene Rearrangement; Generations; Genes; Genetic Recombination; Genomic Instability; Heavy-Chain Immunoglobulins; Hybridomas; Hybrids; Immune; Immunity; Immunoglobulin Somatic Hypermutation; Immunoglobulins; Impairment; In Vitro; Individual; Knock-in; Lead; Lecithin; Light; Liposomes; Lymphocyte; Malignant Neoplasms; Mediating; Modeling; Mus; Mutation; Nonhomologous DNA End Joining; Organism; Pathogenicity; Peptide Signal Sequences; Population Heterogeneity; Process; Proteins; Receptor Gene; Recombinant Antibody; Recurrence; Reporting; Research; Sequence Analysis; Site; Structure; Surface Antigens; T-Lymphocyte; Testing; Therapeutic antibodies; Transgenic Mice; Work; activation-induced cytidine deaminase; adaptive immunity; base; chimeric gene; design; evidence base; gene replacement; genomic locus; high reward; high risk; immunoreactivity; insight; kappa-Chain Immunoglobulins; microbial; microorganism; novel; programs; rational design; receptor; repaired; single cell sequencing

PROJECT SUMMARY B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins cleave antigen receptor gene segments at recombination signal sequences (RSS) through a nick-hairpin mechanism, and is completed when the DNA breaks are sensed and repaired by non-homologous end-joining. Classically, B cell repertoire diversity is considered restricted by the number of functional V, D, and J gene segments in the immunoglobulin (Ig) heavy and light chain gene loci. However, forms of secondary V(D)J rearrangement have been reported in which an unrearranged Ig heavy chain V (VH) gene segment replaces a rearranged VH gene via recombination with a cryptic RSS embedded in the 3’ end of the VH gene (called VH gene replacement). Mechanistic insight into this process has been hampered by the low frequency of these events and, typically, the use of germline pre-rearranged VH alleles. Whether similar events occur in the light chain loci remains unclear. We recently performed bulk light chain repertoire sequencing of B cells from transgenic mice expressing the Ig VH12 heavy chain, sorted based on immunoreactivity to phosphatidylcholine. Interestingly, our analysis uncovered infrequent, but recurrent, endogenous hybrid rearranged Ig kappa V (KV) gene sequences, in which the 3’ end of the highly selected KV4-91 gene was replaced by another KV gene. Based on this preliminary data, our working hypothesis is that the RAG proteins mediate KV gene replacement in VH12 mice via cleavage of a cryptic RSS identified in framework region 3 of the KV gene (KV FR3 cRSS). The proposed project will extend our preliminary findings to confirm KV gene replacement leads to productive antibody generation in single B cells, test whether the KV FR3 cRSS supports RAG-mediated cleavage and rearrangement in vitro and in cells, and also exclude activation-induced cytidine deaminase (AID) as an alternative mechanism for KV gene replacement in VH12 mice. This project will challenge our current understanding of the theoretical constraints on antibody structural diversity, and lead to considering KV gene chimeras for rational design of therapeutic antibodies. This work will also highlight an important potential caveat of automated KV sequence analysis, because such events (regardless of origin) may be missed in KV gene calls or be mistaken for somatic hypermutation. The potential of this project to shift existing paradigms places the project in the “high risk-high reward” category for which the R21 was designed.

项目摘要 B和T淋巴细胞构成了我们自适应免疫系统的基础，该系统基于特定 通过结构潜水表面抗原受体对外国分子的识别。这些结构多样性 受体通过淋巴细胞期间抗原受体基因的位点特异性重排起源 发展。当RAG1/2蛋白质时，启动了称为V（d）J重组的重排过程 通过尼克哈普蛋白在重组信号序列（RSS）处的清除抗原受体基因段（RSS） 机理，当DNA断裂被非理论终端结合感知和修复时，机制就可以完成。 从经典上讲，B细胞库的多样性被功能V，D和J基因的数量限制。 免疫球蛋白（IG）重链和轻链基因基因座的段。但是，次级V（d）j的形式 已经报道了重排，其中透明的IG重链V（VH）基因段替代了 通过重组与嵌入在VH基因3'末端的加密rss通过重组重组（称为VH）（称为VH） 基因替代）。这些过程的机械洞察力受到这些过程的低频的阻碍 事件，通常是使用种系预先固定的VH等位基因的事件。是否在光线下发生类似事件 连锁基因座仍然不清楚。我们最近从 表达IG VH12重链的转基因小鼠，基于免疫反应性排序 磷脂酰胆碱。有趣的是，我们的分析发现了不经常但经常性的内生杂交 重新排列的IG Kappa V（KV）基因序列，其中高度选择的KV4-91基因的3'端为 取而代之的是另一个KV基因。基于此初步数据，我们的工作假设是抹布蛋白 通过在框架区域3中鉴定的加密rss的裂解3 KV基因（KV FR3 CRS）。拟议的项目将扩展我们的初步发现以确认KV基因 替换导致单个B细胞中产物抗体的产生，测试KV FR3 CRS是否支持 抹布介导的裂解和体外和细胞重排，也排除了激活诱导的胞苷 脱氨酶（AID）是VH12小鼠中KV基因替代的替代机制。这个项目将 挑战我们当前对抗体结构多样性的理论约束的理解，并导致 考虑KV基因嵌合体进行治疗抗体的合理设计。这项工作还将突出 自动化KV序列分析的重要潜在警告，因为此类事件（无论起源如何） 可能会错过KV基因呼叫，也可能被误认为是躯体超臭。这个项目的转变潜力 现有的范式将该项目属于R21设计的“高风险高奖励”类别。