In Vivo Production of CAR-Microglia for Treating Alzheimer’s Disease

University Of Chicago, Chicago IL

Investigators

Abstract

SUMMARY Despite the current prevalence of Alzheimer’s disease (AD), there are few effective disease-modifying therapies1. AD is characterized pathologically by amyloid-beta plaques (Aβ), neurofibrillary tau tangles, neuroinflammation, and neurodegeneration1. Microglia, the resident macrophages in the central nervous system (CNS), constantly patrol the CNS for damage2. In AD, microglia mount a strong pro-inflammatory immune response to Aβ4, which is partially effective at clearing Aβ in the early stages of AD but loses effectiveness as Aβ load increases4,5. Microglial deficiency in clearing Aβ is likely because microglia are not evolutionarily equipped with an optimal receptor to recognize and degrade Aβ3. A known microglial receptor recognizing Aβ is the triggering receptor expressed on myeloid cells 2 (TREM2), whose primary ligand is anionic phospholipids3,7 rather than Aβ. Aducanumab is an FDA approved anti-Aβ monoclonal antibody that has been shown to be effective at reducing Aβ plaques in AD.14,21. However, it also induces an excessive pro-inflammatory microglial phenotype that is associated with brain volume loss and amyloid-related imaging abnormalities in patients21-23. Chimeric antigen receptor (CAR) allows targeted recognition of specific antigens by immune cells and has been successfully used to target tumors8. Because of the lack of a specific anti-Aβ microglial receptor, CAR technology likely represents an important therapeutic strategy for AD. Traditional CAR therapy requires extensive resources as ex vivo engineering of immune cells is needed10. We propose to use blood brain barrier (BBB) crossing lipid nanoparticles (LNPs) to encapsulate mRNA encoding an anti-Aβ CAR and IL-10 to directly induce expression of anti-Aβ microglial CARs and IL-10 to promote an anti-inflammatory brain microenvironment in vivo. In Aim 1, we will develop two anti-Aβ CAR constructs that specifically detect Aβ via an aducanumab single chain variable fragment (scFv) and promote phagocytosis via the TREM2 or Fc receptor (FcR) signaling pathways. We will then package mRNA encoding the CARs and IL-10 in LNPs with anti-TREM2 antibody Fab on the LNP surface to promote preferential uptake by microglia, which upregulate TREM2 in the vicinity of plaques. We will transfect primary mouse microglia with these constructs or controls and measure phagocytosis of Aβ and microglial activation in vitro. We will proceed with the most effective construct for in vivo studies. In Aim 2, we will test whether the LNP-CAR can transfect microglia and reduce Aβ plaques in 5XFAD mice in vivo. We will administer LNP-CARs by intravenous injection into 5XFAD at several timepoints during amyloidosis. We predict LNP-CARs will effectively transfect microglia in vivo, will lead to increased microglial recognition and engulfment of Aβ plaques, and limit neuroinflammation, dystrophic neurites, neurodegeneration, and cognitive decline. In summary, we propose a strategy to treat AD by using cell-type specific LNPs containing mRNA encoding an anti-Aβ CAR construct and IL-10 to induce CAR microglia and an anti- inflammatory brain microenvironment in vivo.