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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

基本信息

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
项目类别：
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

项目摘要

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.

摘要这项拟议研究的目标是优化CRISPR-Cas9递送系统，以实现有效的体内基因转移。编辑T细胞。CRISPR-Cas9已经成为在不同生物体中进行基因组工程的强大工具，以及用于开发遗传和传染病的疗法。众所周知，T细胞在人类免疫系统中在协调细胞介导的免疫和体液免疫;因此，T细胞基因组工程提供了希望治疗艾滋病、癌症以及自身免疫性疾病。然而，在体内靶向递送所述化合物是不可能的。 CRISPR-Cas9机器到T细胞以有效地修饰其基因组仍然是一个主要困难。CD 7是一个泛T T细胞上特异性表达的细胞分子。该受体在抗体（Ab）结合，并已用于Ab介导的体内毒素递送，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CD 7单克隆抗体（α-hCD 7）通过Cas9核糖核蛋白（RNP）和Cas9蛋白（mAb）介导的Cas9蛋白向T细胞的递送预包装的慢病毒样颗粒（Cas9 P LV）将允许有效的体内基因编辑。在初步研究中，我们发现蛋白A ZZ结构域融合的荧光蛋白，一旦缀合，与α-hCD 7 mAb结合，可以有效地递送到CD 4 + T细胞;我们还发现， Cas9-ZZ融合蛋白可以有效地递送并内化到CD 4 + T细胞中。此外，为了克服为了解决Cas9蛋白直接递送的潜在免疫原性问题，我们开发了一种新的慢病毒（LV） - 基于颗粒的Cas9蛋白递送策略，以屏蔽LV颗粒中的Cas9蛋白。一旦这个Cas9 P LV被用Sindbis/ZZ结构域包膜假型化，它可以通过Fc区的Fc部分与α-hCD 7 mAb偶联。 mAb并递送到CD 4 + T细胞中。最后，我们发现，一旦CCR 5单向导RNA（sgRNA）被激活，共包装到与α-hCD 7 mAb连接的Cas9 P LV中，我们可以有效地在T细胞中进行基因编辑。这些概念验证结果证明了α-hCD 7 mAb介导的CRISPR-Cas9递送的潜力。植入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-hCD 7 mAb介导的 Cas9 RNP和Cas9 P LV用于递送CRISPR-Cas9机制以靶向CCR 5和PD-1基因。这些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-hCD 7 mAb介导的Cas9 RNP和Cas9 P LV递送靶向CCR 5和PD-1的编辑效率基因.我们还试图验证在人源化小鼠中有效破坏这些基因后的功能变化模型最后，我们将验证由体内基因编辑引起的恒河猴（RM）ccr 5的破坏，猴-人免疫缺陷病毒（SHIV）感染的RM模型中的功能变化。我们的研究旨在为临床应用开辟一条有效的T细胞体内基因编辑的新途径。