Optimization and preclinical evaluation of brain permeant SE-CRISPR to edit ApoE4 after IV infusion in AD models

PROJECT SUMMARY/ABSTRACT This proposal is based on our successful SE-CRISPR editing of the APOE4 gene at the codon for amino acid 112 distinguishing it from APOE3 (1), in the brain E4-5XFAD AD model mice (2) by a single IV infusion of microfluidic synthetic exosome (SE)-encapsulated CRISPR sgRNA+CBE mRNA leading to increased E3 mRNA production in brain. The editing of neurotoxic major genetic risk factor for sporadic AD ApoE4 (E4) to benign E3 represents a powerful novel therapeutic approach for Alzheimer’s disease (AD) (3-6). Compared to E3, E4 exacerbates the two predominant hallmarks of AD brain, Aβ amyloid plaques and tau pathology (7-14). Utilization of SE-CRISPR base-editing technology that allows editing in both dividing and non-dividing cells to edit E4 to E3 even in a subset of cells may decrease the risk for AD conferred by E4. SE-CRISPR IV infusion therapy could compliment the currently approved antibodies for AD, aducanumab and lecanemab, which reduce Aβ pathology but only modestly reduce cognitive decline. For effective gene editing in AD, CRISPR components must be delivered across the blood-brain barrier (BBB). Our lab has developed a brain delivery platform comprising microfluidic reactor-synthesized deformable lipid nanovesicles the size of natural exosomes (<150 nm), called ‘synthetic exosomes’ (SEs), that can encapsulate therapeutic macromolecules. Our preliminary data demonstrate that SE-CRISPR crosses the BBB and edits E4 to E3 in the mouse brain. In this proposal, we plan to optimize E4 to E3 editing in brain and determine alterations in the E4 phenotype of AD models in which E4 has been edited. In Aim 1, the dosage/dose frequency of the current promising SE-CRISPR candidate for editing of E4 in E4-5XFAD mice would be optimized with a goal of >50% editing in brain. In Aim 2, synthesis of SE- CRISPR would be optimized by use of ionizable cationic lipids and surface modification with glycol(PEG)ylated lipids and transferrin (Tf)-peptides to enhance plasma half-life and BBB permeability, as assessed by determination of editing efficiency in E4-5XFAD mouse brain using NGS. In Aim 3, chronic efficacy and safety of prioritized SE-CRISPR candidate(s) will be assessed in SE-CRISPR vs. SE-empty IV-injected E4-5XFAD AD model mice (14-16 mice/cohort) 5 days and 3 and 6 months after dosing, including memory behavior at 3 and 6 months, using SE-empty injected mice as controls. Editing efficiency will determined by brain region (cortex, hippocampus, etc.) and cell type (neurons, astrocytes, etc.), and in liver and WBCs. Biochemical analyses will include brain/liver E3 and E4 mRNA and protein, Aβ40/42, phospho-tau, and inflammatory markers, and plasma neurodegeneration biomarker Nfl. In Aim 4, to show species translation, the effects of prioritized SE-CRISPR candidate(s) will be assessed in E4-knockin (KI) rats using the established dose/delivery schedule, compared to SE-empty controls (6-8 rats/group). Rats will be euthanized 5 days and 3 months post final IV tail vein injection, and E4 editing assessed by brain region/cell type, and biochemical analyses as described for E4-5XFAD mice to identify a SE-CRISPR candidate for further clinical development as IV infusion therapy for AD.