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

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。CD7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CD7单克隆抗体（α-hCD7） 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（Cas9P 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-hCD7 mAb结合，可以有效地递送到CD4 + 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CD4 + 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Cas9P LV被 用Sindbis/ZZ结构域包膜假型化，它可以通过抗体的Fc部分与α-hCD 7 mAb偶联 mAb并递送到CD4 + T细胞中。最后，我们发现，一旦CCR5单向导RNA（sgRNA）被激活， 共包装到与α-hCD7 mAb连接的Cas9P 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-hCD7 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-hCD7 mAb介导的 Cas9 RNP和Cas9P LV用于递送CRISPR-Cas9机制以靶向CCR5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-hCD7 mAb介导的Cas9 RNP和Cas9P LV递送靶向CCR5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）ccr5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。

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.