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 ?-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.

Public Health Relevance

The use of ?humanized? mice?mice that can be injected with human cells and tissues, enabling study of human diseases in a living experimental organism?is advancing progress in the study of human infectious diseases and cancer. However, in current strains of humanized mice, the mouse immune system removes human red blood cells (RBCs) from the circulation following injection, preventing the use of these valuable strains for the study of diseases involving RBCs, such as malaria and similar blood-borne diseases, or sickle cell anemia. In the proposed project we will develop and test, in mice, a novel technique to deplete the specific mouse immune-system cells that eliminate RBCs from circulation, yielding a novel strain of mice that can be used to study RBC-related diseases, and yielding a technique that other researchers can use to remove cell types of their choice from mice, potentially advancing research in a broad range of human diseases.

National Institute of Health (NIH)
Office of The Director, National Institutes of Health (OD)
Exploratory/Developmental Grants (R21)
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Therapeutic Approaches to Genetic Diseases Study Section (TAG)
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Zou, Sige
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Jackson Laboratory
Bar Harbor
United States
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