Project Summary Alzheimer's disease (AD) affects 6.7 million Americans. Extracellular deposition of E-amyloid (AE) and accumulation of hyperphosphorylated-tau inside neurons are two primary culprits of AD pathology and targets for emerging AD therapies. Recent clinical trials of anti-AE monoclonal antibodies (mAbs) in AD patients showed AE deposition can be reversed resulting in significant slowing of cognitive decline. Yet, anti-AE immunotherapy comes with several shortcomings. These include need for recurring mAb infusions, staggering costs, and serious side effects in the form of brain bleeds and/or edema, and contraindications disqualifying a large number of patients. Chimeric antigen receptor (CAR) therapies have recently revolutionized several areas of oncology and hold high promise for adaptation in other medical fields including neurodegeneration, where CAR-engineered macrophages (Mĭs) can be used for targeted clearance of disease specific misfolded proteins and modulating neuroinflammation. A key issue in CAR-based therapies are their high costs resulting from in vitro engineering of patient-derived T cells or Mĭs and subsequent autologous implantation. This can be addressed using HLA- compatible, “off-the-shelf” human induced pluripotent stem cells (hiPSCs) as a platform for expressing disease specific CARs. This R61/R33 application proposes to develop a unique CAR-Mĭ therapy for AD based on “off- the-shelf” hiPSCs, which will be genetically programmed for fast and robust transdifferentiation into self- sustainable, macrophages/microglia (iMĭ) expressing CARs targeting AD specific proteins. Our CAR-iMĭs will be engineered to withstand CSF1R inhibition , which will be used to facilitate their brain homing. They also will be modified for attenuated inflammatory response and equipped with “kill switches” for therapy control. Our preliminary work for this grant application includes identification of five novel anti-AE clones binding AE deposits and effecting their clearance in APP/H3 mice by Fc-mediated microglia phagocytosis. We also have engineered a doxycycline-inducible genetic circuit expressing SPI1 and CEBPĮ transcription factors allowing for robust trans- differentiation of hiPSCs into iMĭ. Aims of the R61 phase are as follows: 1) to optimize iMĭ transdifferentiation protocol and characterize resulting iMĭ cells; 2) to engineer CARAE using scFv sequences from our novel anti- AE clones and express them in iMĭs; 3) to engineer an orthogonal CSF1 receptor, which will be constitutively active and resilient to pharmacological inhibition; 4) to reprogram hiPSC HLA to “cloak” the cells against the intact murine immune system permitting in vivo testing. Aims of the R33 phase will be as follows: 1) to produce CARAE-iMĭs by combining all singular genetic elements tested in the R61 phase and characterize CARAE-iMĭ biodistribution, brain homing and survival in vivo; 2) to test engagement and clearance of AE plaques by CARAE- iMĭs in APP/H3 and APP/H4 mice; 3) to characterize the transcriptomic profile of CARAE-iMĭs in APP/H3 and APP/H4 mice and explore effects of knocking-out genes, which are key to acquisition of MGnD phenotype and propagating inflammatory response; 4) to engineer and test “kill switches” for on demand activity control.

Project Narrative This R61/R33 proposal outlines development of a novel, cost-efficient, CAR Macrophage/Microglia therapy for Alzheimer's disease, which will lead to targeted clearance of disease specific proteins. It will carry a low- inflammatory signature and attenuate neuroinflammation, which inherently co-exists with Alzheimer pathology.