Novel CRISPR-Cas9 protein delivery to T cells in vivo by targeting CD7

通过靶向 CD7 将新型 CRISPR-Cas9 蛋白递送至体内 T 细胞

• 批准号: 10045353
• 负责人: Guohua Yi
• 金额: $ 70.77万
• 依托单位: UNIVERSITY OF TEXAS HLTH CTR AT TYLER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-20 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10045353
• 关键词: Novel; CRISPR; Cas9; protein; delivery

Abstract The goal of this proposed research is to optimize the CRISPR-Cas9 delivery system for efficient in vivo gene editing of T cells. CRISPR-Cas9 has emerged as a powerful tool for genome engineering in diverse organisms, as well as for developing therapeutics for genetic and infectious diseases. T cells are known to play critical role in orchestrating cell-mediated immunity and humoral immunity; thus, T cell genome engineering offers promise to treat HIV infection, cancer, as well as autoimmune diseases. However, in vivo targeting delivery of the CRISPR-Cas9 machinery to T cells to efficiently modify their genome remains a major difficulty. CD7 is a pan-T cell molecule that is specifically expressed on T cells. This receptor is rapidly internalized after antibody (Ab) binding, and it has been used for Ab-mediated in vivo delivery of toxins to treat T cell lymphomas and leukemias in preclinical studies and clinical trials. We hypothesize that anti-human CD7 monoclonal antibody (α-hCD7 mAb)-mediated delivery of Cas9 protein to T cells via Cas9 ribonucleoprotein (RNP) and Cas9 protein prepackaged lentivirus-like particles (Cas9P LV) will allow efficient gene editing in vivo. In preliminary studies, we found that protein A ZZ domain-fused fluorescence protein, once conjugated with α-hCD7 mAb, could be effectively delivered into CD4+ T cells; we also found that α-hCD7 mAb-conjugated Cas9-ZZ fusion protein could be efficiently delivered and internalized into CD4+ T cells. Additionally, to overcome the potential immunogenicity problem by direct delivery of Cas9 protein, we developed a novel lentiviral (LV) particle-based Cas9 protein delivery strategy to shield Cas9 protein in the LV particles. Once this Cas9P LV was pseudotyped with a Sindbis/ZZ domain envelope, it could be conjugated to α-hCD7 mAb via Fc portion of the mAb and delivered into CD4+ T cells. Finally, we found that once a CCR5 single guide RNA (sgRNA) had been co-packaged into a Cas9P LV linked to an α-hCD7 mAb, we could efficiently perform gene editing in T cells. These proof-of-concept results demonstrate the potential of α-hCD7 mAb-mediated delivery of CRISPR-Cas9 into T cells for in vivo gene editing. Therefore, in this proposed research, we will use α-hCD7 mAb-mediated Cas9 RNP and Cas9P LV to deliver the CRISPR-Cas9 machinery to target the CCR5 and PD-1 genes. These genes have been shown to be important in HIV infection and cancer. We will test and compare the in vivo gene editing efficiency using α-hCD7 mAb-mediated Cas9 RNP and Cas9P LV delivery to target the CCR5 and PD-1 genes. We also seek to validate functional changes after efficient disruption of these genes in humanized mouse models. Finally, we will validate the disruption of rhesus monkey (RM) ccr5 caused by in vivo gene editing and functional changes in a RM model of simian-human immunodeficiency virus (SHIV) infection. Our research has been designed to open a new avenue toward efficient in vivo gene editing of T cells for clinical applications.

抽象的 这项研究的目标是优化 CRISPR-Cas9 递送系统，以实现高效的体内基因 CRISPR-Cas9 已成为多种生物体基因组工程的强大工具， 众所周知，T 细胞在开发遗传性疾病和传染病疗法方面发挥着关键作用。 因此，T 细胞基因组工程在协调细胞介导的免疫和体液免疫方面提供了希望。 治疗艾滋病毒感染、癌症以及自身免疫性疾病。然而，体内靶向递送。 CRISPR-Cas9机制对T细胞有效地修改其基因组仍然是泛T细胞的一个主要困难。 T 细胞上特异表达的细胞分子，该受体在抗体 (Ab) 后迅速内化。 结合，它已被用于抗体介导的体内毒素递送，以治疗 T 细胞淋巴瘤和白血病 我们在临床前研究和临床试验中追求抗人CD7单克隆抗体（α-hCD7）。 mAb）介导的 Cas9 蛋白通过 Cas9 核糖核蛋白 (RNP) 和 Cas9 蛋白递送至 T 细胞 预包装的慢病毒样颗粒（Cas9P LV）将允许在体内进行有效的基因编辑。 在初步研究中，我们发现蛋白质 A ZZ 结构域融合荧光蛋白，一旦缀合 与 α-hCD7 mAb 一起，可以有效地递送至 CD4+ T 细胞；我们还发现 α-hCD7 mAb 缀合 Cas9-ZZ融合蛋白可以有效地递送并内化到CD4+T细胞中。 针对直接传递Cas9蛋白潜在的免疫原性问题，我们开发了一种新型慢病毒（LV） 基于颗粒的 Cas9 蛋白递送策略，一旦 Cas9P LV 被屏蔽，即可将 Cas9 蛋白屏蔽在 LV 颗粒中。 带有 Sindbis/ZZ 包膜结构域的假型化，它可以通过 Fc 部分与 α-hCD7 mAb 缀合 mAb 并递送至 CD4+ T 细胞中，最后，我们发现 CCR5 单引导 RNA (sgRNA) 已被导入。 将其共同包装到与 α-hCD7 mAb 连接的 Cas9P LV 中，我们可以在 T 细胞中有效地进行基因编辑。 这些概念验证结果证明了 α-hCD7 mAb 介导的 CRISPR-Cas9 递送的潜力 因此，在这项拟议的研究中，我们将使用 α-hCD7 mAb 介导的。 Cas9 RNP 和 Cas9P LV 用于传递 CRISPR-Cas9 机制来靶向 CCR5 和 PD-1 基因。 基因已被证明在艾滋病毒感染和癌症中很重要，我们将测试和比较体内基因。 使用 α-hCD7 mAb 介导的 Cas9 RNP 和 Cas9P LV 递送靶向 CCR5 和 PD-1 的编辑效率 我们还试图验证人源化小鼠中这些基因被有效破坏后的功能变化。 最后，我们将验证体内基因编辑对恒河猴 (RM) ccr5 的破坏。 我们的研究发现了猿猴人类免疫缺陷病毒（SHIV）感染的 RM 模型的功能变化。 旨在为临床应用中 T 细胞的有效体内基因编辑开辟一条新途径。