Laboratory of Molecular Immunology, The Rockefeller University;
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
J Vis Exp. 2023 Feb 10(192). doi: 10.3791/64858.
B cells and their progeny are the sources of highly expressed antibodies. Their high protein expression capabilities together with their abundance, easy accessibility via peripheral blood, and amenability to simple adoptive transfers have made them an attractive target for gene editing approaches to express recombinant antibodies or other therapeutic proteins. The gene editing of mouse and human primary B cells is efficient, and mouse models for in vivo studies have shown promise, but feasibility and scalability for larger animal models have so far not been demonstrated. We, therefore, developed a protocol to edit rhesus macaque primary B cells in vitro to enable such studies. We report conditions for in vitro culture and gene-editing of primary rhesus macaque B cells from peripheral blood mononuclear cells or splenocytes using CRISPR/Cas9. To achieve the targeted integration of large (<4.5 kb) cassettes, a fast and efficient protocol was included for preparing recombinant adeno-associated virus serotype 6 as a homology-directed repair template using a tetracycline-enabled self-silencing adenoviral helper vector. These protocols enable the study of prospective B cell therapeutics in rhesus macaques.
B 细胞及其后代是高表达抗体的来源。它们具有高蛋白表达能力，丰富，易于通过外周血获得，并且易于进行简单的过继转移，这使得它们成为表达重组抗体或其他治疗性蛋白的基因编辑方法的有吸引力的靶标。对小鼠和人原代 B 细胞的基因编辑效率很高，并且体内研究的小鼠模型显示出了前景，但迄今为止，尚未证明其在较大动物模型中的可行性和可扩展性。因此，我们开发了一种方案，以在体外编辑恒河猴原代 B 细胞，从而能够进行此类研究。我们报告了使用 CRISPR/Cas9 从外周血单核细胞或脾细胞体外培养和编辑恒河猴原代 B 细胞的条件。为了实现对大型（<4.5kb）盒的靶向整合，我们纳入了一种快速有效的方案，用于使用四环素激活的自我沉默腺病毒辅助载体制备重组腺相关病毒血清型 6，作为同源定向修复模板。这些方案使我们能够在恒河猴中研究潜在的 B 细胞治疗药物。
