Development and Validation of a Novel Cas13a and Nanoparticle Guide-RNA Delivery System that Allows Precise Ablation of Host Macrophage Populations in a Humanized Mouse Model

项目摘要/摘要 总体目标是开发一种新的基因截取方法，并通过创建一种 巨噬细胞耗竭人源化小鼠模型。人源化小鼠的实验使用正在迅速发展 人类传染病和癌症研究进展。然而，尽管人类红细胞 (RBC)是在人源化小鼠植入人类造血细胞后在骨髓中产生的 干细胞(HSC)，成熟的人红细胞能被小鼠巨噬细胞迅速吞噬，并被清除 循环，阻碍了人红细胞的体内研究。在这里，我们建议开发和测试一种基因 该工具将专门摧毁老鼠体内的小鼠巨噬细胞。为此，我们将使用NOD.Cg- Prkdccerd IL2rgtm1Wji/SzJ(NSG)小鼠，支持更高水平的植入和重建 人肝干细胞，在内源性位点表达一种新的CRISPR相关蛋白13a(Cas13a)， 表达由单核/巨噬细胞特异性启动子CD68驱动。这将与以下内容相结合 CD44靶向透明质酸(HA)纳米颗粒的输送，这些纳米颗粒被巨噬细胞吞噬和 它将包含一个DNA载体，驱动针对以下基因的引导RNAs的表达 巨噬细胞存活，导致巨噬细胞自毁。CD68基因座和CD68基因座的联合应用 以CD44为靶点的HA纳米颗粒将为耗竭小鼠巨噬细胞提供高度的特异性。 此外，尽管移植到NSG小鼠体内的人巨噬细胞会吞噬纳米颗粒，但它们将 由于缺乏内源Cas13a而未受影响。具体目标是1)开发和描述一个 通过插入框内附件使用Bxb1位点特异性重组酶的新“基因截取”方法 由CRISPR/Cas9介导的CD68基因翻译起始ATG附近的(AttP)3‘位 NSG小鼠同源定向修复寡核苷酸后整合Cas13a-EGFP attB 基因整合到新整合的attP位点，使其表达受控于小鼠内源性 CD68启动子，导致Cas13a在小鼠巨噬细胞中特异性表达；2)验证我们的CD44- NSG-TG(Cas13a-EGFP)小鼠的靶向纳米颗粒递送系统，与Amiji博士合作，在 东北大学，通过提供针对巨噬细胞必需的两个基因的mRNAs的引导RNA 存活率(GAPDH(甘油醛-3-磷酸脱氢酶)和Tak1(TGFb激活的激酶)) 检测CD34+人造血干细胞移植后人红细胞在循环中的存活情况。 这些小鼠用于研究循环中的人红细胞，这将使研究1)遗传学 疾病，包括血红蛋白疾病，如镰状细胞性贫血和β-地中海贫血；二)传染病， 包括疟疾；和iii)患者来源的肿瘤异种移植。此外，我们的基因拦截和纳米颗粒 Delivery‘方法有可能应用于调节其他类型的细胞，提供了广泛的适用性 多个研究领域。

PROJECT SUMMARY/ABSTRACT The overall objective is to develop a novel gene-intercept methodology and to validate it by creating a macrophage-depleted humanized mouse model. The experimental use of humanized mice is driving rapid progress in the study of human infectious diseases and cancer. However, although human red blood cells (RBCs) are produced in the bone marrow following engraftment of humanized mice with human hematopoietic stem cells (HSC), mature human RBCs are rapidly phagocytized by mouse macrophages and are cleared from the circulation, preventing the in vivo study of human RBCs. Here, we propose to develop and test a genetic tool that will specifically destroy mouse macrophages in mice. To accomplish this, we will use NOD.Cg- Prkdcscid IL2rgtm1Wji/SzJ (NSG) mice, which support heightened levels of engraftment and reconstitution with human HSCs, to express a novel CRISPR associated protein 13a (Cas13a) at an endogenous locus, with expression being driven by a monocyte/macrophage-specific promoter, CD68. This will be combined with delivery of CD44-targeting hyaluronic acid (HA) nanoparticles, which are phagocytosed by macrophages and which will contain a DNA vector driving the expression of guide RNAs targeting genes essential for macrophage survival, leading to macrophage self-destruction. The combined use of the CD68 locus and CD44-targeting HA nanoparticles will provide a high level of specificity for depleting mouse macrophages. Further, although human macrophages engrafted in NSG mice will phagocytose nanoparticles, they will remain unaffected due to their lack of endogenous Cas13a. The specific aims are to 1) Develop and characterize a novel “gene intercept” methodology using Bxb1 site-specific recombinase by inserting an in-frame attachment (attP) site 3’ and adjacent to the translation initiation ATG of the CD68 gene, by CRISPR/Cas9-mediated homology-directed repair of an oligodeoxynucleotide in NSG mice followed by integrating a Cas13a-EGFP attB gene into the newly integrated attP site, such that its expression is under control of the mouse endogenous CD68 promoter, leading to Cas13a expression specifically in mouse macrophages, and 2) Validate our CD44- targeting nanoparticle delivery system in NSG-Tg(Cas13a-EGFP) mice, in collaboration with Dr. Amiji at Northeastern University, by delivering guide RNAs targeting mRNAs for two genes essential for macrophage survival (Gapdh (glyceraldehyde-3-phosphate dehydrogenase) and Tak1 (TGFb activated kinase)) followed by testing the survival of human RBCs in the circulation following engraftment with human CD34+ HSCs. Development of these mice for the study of circulating human RBCs will enable the study of i) genetic disorders, including hemoglobinopathies such as sickle cell anemia and β-thalassemia; ii) infectious diseases, including malaria; and iii) patient-derived tumor xenografts. Further, our ‘gene interception and nanoparticle delivery’ approach has the potential to be applied to modulating other cell types, providing broad applicability in a number of research fields.