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 ß-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.
