A novel form of light chain gene replacement

轻链基因替换的新形式

• 批准号: 10191435
• 负责人: Patrick C. Swanson
• 金额: $ 21.83万
• 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 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191435
• 关键词: Adaptive Immune System; Alleles; Antibodies; Antibody Formation; Antigen Receptors; B cell repertoire; B-Lymphocytes; Base Sequence; Binding; Categories; Cells; Chimera organism; Cleaved cell; 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; 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 在这些结构抗原受体上识别外国分子。 受体来自淋巴细胞期间特定的抗原重排 开发。 在重组信号序列（RSS）上切割抗原受体基因片段通过尼克·希尔平 当DNA断裂通过非同源末端连接来感知和修复时，机理被构成。 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）中的片段重链基因基因座。 已经报道了重排，其中透明的Ig重链V（VH）基因段取代了 通过重组与嵌入在VH基因的3'端中的隐性RSS重组（称为VH）通过重新组合（称为VH） 基因替代）对此过程的洞察力受到了低频 事件，通常是使用种系预先恢复的VH Allels的事件。 链基因座尚不清楚。 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。 重排IG Kappa V（KV）基因序列，其中高度选择的KV4-91基因的3'端为 取代了另一个KV基因。 通过在框架区域3中鉴定的密码RSS的裂解3 KV基因（KV FR3 CRS）。 替换会导致单个B细胞的生产抗体产生，测试KV FR3 CRSS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）作为VH12小鼠的KV基因替代的替代机制。 挑战我们当前对理论约束Ontibopy结构多样性的理解，并领导 考虑KV基因嵌合体进行治疗抗体的合理设计。 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，也可以误认为该项目的可能性。 现有的范式将该项目列入了R21设计的“高风险高奖励”类别。