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: 10246267
• 负责人: Guohua Yi
• 金额: $ 66.97万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246267
• 关键词: Ablation; Adaptive Immune System; Antibodies; Antibody Response; Autoimmune Diseases; Binding; Biodistribution; C57BL/6 Mouse; CCR5 gene; CD4 Positive T Lymphocytes; CD7 gene; CRISPR/Cas technology; Cells; Cellular Immunity; Chimeric Proteins; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communicable Diseases; DNA; Deoxyribonuclease I; Drug Kinetics; Electroporation; Engineering; Fluorescence; Gene Delivery; Genes; Genetic Diseases; Genome; Genome engineering; Goals; Guide RNA; HIV Infections; HIV-1; Human; Humoral Immunities; Immunity; Infection; Integrase; Length; Lentivirus; Link; Macaca mulatta; Malignant Neoplasms; Mediating; Methodology; Methods; Modeling; Monoclonal Antibodies; Mus; Organism; Phase; Plasmids; Play; Production; Proteins; RNA Sequences; Reagent; Reporting; Research; Ribonucleoproteins; Role; Safety; Self Tolerance; Site; Stimulus; Surface; System; T-Cell Lymphoma; T-Lymphocyte; Testing; Therapeutic; Toxicity Tests; Toxin; Viral; Viral Vector; Virus Diseases; base; cancer therapy; chemokine; clinical application; cytokine; design; efficacy testing; engineered T cells; humanized mouse; immunogenicity; in vivo; leukemia/lymphoma; mouse model; novel; particle; preclinical study; programmed cell death protein 1; receptor; recruit; scale up; simian human immunodeficiency virus; targeted delivery; tool

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 这项拟议的研究的目的是优化CRISPR-CAS9递送系统，以有效地体内基因 T细胞的编辑。 CRISPR-CAS9已成为潜水生物体基因组工程的强大工具， 以及开发遗传和传染病的治疗。已知T细胞起着关键作用 在策划细胞介导的免疫学和体液免疫学方面；因此，T细胞基因组工程提供了承诺 治疗HIV感染，癌症以及自身免疫性疾病。但是，在体内靶向交付 CRISPR-CAS9机械可有效地修改其基因组，仍然是一个主要的困难。 CD7是PAN-T 特异性在T细胞上表达的细胞分子。该接收器在抗体（AB）后迅速内化 结合，它已用于AB介导的毒素体内递送以治疗T细胞淋巴瘤和白血病 在临床前研究和临床试验中。我们假设抗人CD7单克隆抗体（α-HCD7） mAb）通过Cas9核糖核蛋白（RNP）和Cas9蛋白介导的Cas9蛋白递送至T细胞 预包装的慢病毒样颗粒（CAS9P LV）将允许体内有效的基因编辑。 在初步研究中，我们发现蛋白A Zz结构域融合的荧光蛋白曾经结合过 使用α-HCD7 mAb，可以有效地输送到CD4+ T细胞中。我们还发现α-HCD7 mAb偶联 Cas9-ZZ融合蛋白可以有效地传递并内化为CD4+ T细胞。此外，要克服 潜在的免疫原性问题通过直接递送Cas9蛋白，我们开发了一种新型的慢病毒（LV） 基于粒子的CAS9蛋白输送策略，以屏蔽LV颗粒中的Cas9蛋白。一旦这个cas9p lv是 用sindbis/zz结构域伪造，可以通过FC部分连接到α-HCD7 mAb。 mab并输送到CD4+ T细胞中。最后，我们发现一旦CCR5单引导RNA（SGRNA）已 共包装到与α-HCD7 mAb相关的CAS9P LV中，我们可以在T细胞中有效地进行基因编辑。 这些概念验证结果表明了α-HCD7 mAb介导的CRISPR-CAS9的递送的潜力 进入T细胞进行体内基因编辑。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可提供CRISPR-CAS9机械，以瞄准CCR5和PD-1基因。这些 基因在HIV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递送来靶向CCR5和PD-1的编辑效率 基因。我们还寻求在人源化小鼠中有效破坏这些基因后验证功能变化 型号。最后，我们将验证由体内基因编辑和 猿猴免疫缺陷病毒（SHIV）感染的RM模型的功能变化。我们的研究有 我们旨在为T细胞的临床应用开辟新的途径，以有效地体内基因编辑。