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利用基因组编辑技术重编程 B 细胞的抗原特异性。

Reprogramming the antigen specificity of B cells using genome-editing technologies.

机构信息

Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, United States.

International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, United States.

出版信息

Elife. 2019 Jan 17;8:e42995. doi: 10.7554/eLife.42995.

DOI:10.7554/eLife.42995
PMID:30648968
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6355199/
Abstract

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.

摘要

我们开发了一种方法，通过使用基因组编辑技术直接修饰成熟 B 细胞的免疫球蛋白 (Ig) 基因，将新的抗原结合部位引入人类抗体库。我们使用 CRISPR-Cas9 进行同源定向修复策略，用来自 HIV 广谱中和抗体 (bnAb) PG9 的重链 (HC) 可变区替换 B 细胞系中的重链 (HC) 可变区。我们的策略旨在针对经历 VDJ 重组的细胞起作用，这些细胞可以使用任何组合的可变 (V)、多样性 (D) 和连接 (J) 基因。修饰后的基因座表达 PG9 HC，与天然轻链 (LC) 配对，导致 HIV 特异性 B 细胞受体 (BCR) 的细胞表面表达。工程细胞中的内源性激活诱导胞嘧啶脱氨酶 (AID) 允许 Ig 类别转换，并产生具有改善的 HIV 中和活性的 BCR 变体。因此，以这种方式工程化的 BCR 保留了在适应性免疫反应期间通常需要的亲和力成熟的遗传灵活性。使用这种策略编辑了来自三个不同供体的外周血源性原代 B 细胞。经工程化的细胞能够结合 PG9 表位，测序 mRNA 显示，在与 CD40 配体和 IL-4 共培养后，PG9 HC 转录为几种不同的同型。

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