Engineered human CD34+ hematopoietic stem cells as an innovative approach to modulate the immunoinflammatory response during atherosclerosis

Background: Atherosclerosis is one of the leading factors for cardiovascular diseases, driven by increased plasma cholesterol levels and activation of immune-inflammatory responses. Despite effective lipid-lowering therapies, some patients still exhibit residual immuno-inflammatory cardiovascular risk. Working hypothesis: The emerging crosstalk between immunometabolic pathways and immunomodulatory therapies underscores the need for experimental models to translate molecular insights into targeted interventions for atherosclerosis. Main objective: To profile the impact of immune system humanization in an atherosclerosis-prone setting on vascular inflammation and disease progression with a focus on the manipulation of immunometabolic genes as a strategy for CVD. Strategy (type of approach, main technology, innovative aspects): Humanized TKO-LDLr KO mice (generated by crossing LDLr-KO with immunodeficient Rag2-KO/IL2rg-KO/CD47-KO mice, HuTKOL) were generated after low-dose irradiation (250 cGy) of 2-3 days old pups followed by hepatic injection of human hematopoietic stem cells w/o CRISPR/Cas editing (CD34, 250.000-300.000 cells/mouse). After 12 weeks, human cell engraftment was evaluated by FACS. Mice were then fed 12-weeks high-cholesterol diet to investigate immunometabolic phenotype in circulation and in bone marrow, together with the development of atherosclerosis. Expected results: Control and engineered CD34+ cells should differently impact immune response and atherosclerosis development, unveiling novel immunometabolic molecular mechanisms and therapeutic approaches against vascular inflammation. Optional - Preliminary results: Engraftment of HuTKOL mice with standard hCD34+ results in 1:4 human circulating leucokocytes, with Bcells representing the most abundant population at 12 wks, but decreasing over time, whereas Tcells increase over time, being the most represented human subset after 12-wks of WTD. On WTD, HuTKOL mice develop dyslipidemia (plasma cholesterol levels: 1211,96mg/dl, SE±63,72) and atherosclerosis (% aortic sinus plaque occlusion: 18,35%, SE±1,90%) with activation of B and Tcells directed toward atherosclerosis-related antigens. scRNAseq profiling of bone marrow, revealed gender dependent effects on lymphocyte’s engraftment and human cell activation. Scientific outlook (plausible impact of the expected results): hCD34+ humanized TKO-LDLR mice represent a valuable platform for studying the immunometabolic response during atherosclerosis and could represent a useful tool for testing immunotherapies for CVD.