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

项目总结/摘要 总体目标是开发一种新的基因拦截方法，并通过创建一个 巨噬细胞耗尽的人源化小鼠模型。人源化小鼠的实验性应用正在迅速发展 人类传染病和癌症的研究进展。然而，尽管人类的红细胞 在用人造血干细胞移植人源化小鼠后，在骨髓中产生红细胞（RBC）。 在造血干细胞（HSC）中，成熟的人RBC被小鼠巨噬细胞迅速吞噬，并从 循环，阻止人体红细胞的体内研究。在这里，我们建议开发和测试一种基因 一种专门破坏小鼠巨噬细胞的工具。为了实现这一点，我们将使用NOD. Cg- Prkdcscid IL2rgtm1Wji/SzJ（NSG）小鼠，其支持植入和重建的提高水平， 人HSC，在内源基因座表达新的CRISPR相关蛋白13a（Cas13a）， 表达由单核细胞/巨噬细胞特异性启动子CD68驱动。这将与 递送靶向CD44的透明质酸（HA）纳米颗粒，其被巨噬细胞吞噬， 其将包含驱动导向RNA表达的DNA载体，所述导向RNA靶向对于以下过程至关重要的基因： 巨噬细胞存活，导致巨噬细胞自我毁灭。联合使用CD68基因座和 靶向CD44的HA纳米颗粒将为消耗小鼠巨噬细胞提供高水平的特异性。 此外，尽管移植到NSG小鼠中的人巨噬细胞将吞噬纳米颗粒，但它们仍将保留。 因为它们缺乏内源性Cas13a而不受影响。具体目标是：（1）开发和表征 通过插入框内连接使用Bxb1位点特异性重组酶的新的"基因拦截"方法 通过CRISPR/Cas9介导的方法，在CD68基因的翻译起始ATG附近的3 '（attP）位点， NSG小鼠中寡脱氧核苷酸的同源性定向修复，随后整合Cas13a-EGFP attB 将该基因插入新整合的attP位点，使得其表达在小鼠内源性ATTP基因的控制下。 CD68启动子，导致Cas13a在小鼠巨噬细胞中特异性表达，和2）激活CD44- 与Amiji博士合作，在NSG-Tg（Cas13a-EGFP）小鼠中靶向纳米颗粒递送系统 东北大学，通过递送靶向巨噬细胞所必需的两个基因的mRNA的指导RNA， 存活（Gapdh（甘油醛-3-磷酸脱氢酶）和Tak1（TGF β激活的激酶）），然后 测试在植入人CD34 + HSC后人RBC在循环中的存活。 开发这些小鼠用于研究循环人RBC将使得能够研究i）遗传 病症，包括血红蛋白病如镰状细胞性贫血和β-地中海贫血; ii）感染性疾病， 包括疟疾;和iii）患者来源的肿瘤异种移植物。此外，我们的"基因拦截和纳米粒子" “递送”方法具有应用于调节其他细胞类型的潜力，在细胞内提供广泛的适用性。 一些研究领域。

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.