Enhanced APOE2 Expression into Brain for Therapeutic Strategy for Alzheimer's Disease - SUMMARY/ABSTRACT: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that has emerged as the most prevalent form of late-life dementia in humans. The accumulation, aggregation, and deposition of amyloid-β (Aβ) in the brain are central events in AD pathogenesis. Despite intense effort, an effective therapy for AD has yet to be established. While multiple genetic and environmental factors are involved in AD pathogenesis, the ε4 allele of the APOE gene encoding apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset AD among the three human APOE genotypes (ε2, ε3, ε4). In humans, Aβ deposition is more pronounced in APOE4 carriers compared with non-carriers in both AD patients and aged healthy individuals. APOE plays a critical role in maintaining synaptic plasticity and neuronal function by controlling lipid homeostasis, with the APOE2 allele having a superior function. The ε2 allelic variant has been found to be more prevalent among centenarians and associated with decreased susceptibility to AD. Studies on the role of the APOE2 in relation to AD suggest that APOE2 is neuroprotective and positively associated with cognitive functions in aging. Therefore, increasing APOE2 levels in the brain is predicted to be an effective therapeutic strategy for AD. Development of successful strategies for treating these disorders is limited due to the protective function of blood brain barrier (BBB). Gene therapy possesses a broad potential for the treatment of numerous neurological diseases, including AD. However, the major challenge in the field of gene therapy is the design of safe non-viral vectors that can cross the BBB. The transferrin (Tf) receptors are present on the surface of brain endothelial cells. The lipid nanoparticles can be surface modified with Tf protein for targeting the brain endothelial receptors and conjugated to brain specific cell penetrating peptide (CPP) for improving their internalization into brain by overcoming receptor saturation. Therefore, we propose to design near neutral, PEGylated liposomal nanoparticles encapsulating gene and modifying the surface of nanoparticles with Tf and CPP. Furthermore, the transfection properties of chitosan will be utilized for improving the transfection of gene by facilitating endosomal escape via the proton-sponge mechanism inside the cells. The long-term goal of the proposed research is to design a non- viral gene delivery carrier for efficient delivery of plasmid DNA encoding APOE2 (pAPOE2) to brain for prevention and treatment of AD. We propose three specific aims to accomplish the long-term goal of the proposed research: Aim 1. Synthesize and characterize liposomal nanoparticles loaded with chitosan-pAPOE2 polyplexes: The brain specific CPP-liposomes will be synthesized using thin film hydration technique followed by insertion of Tf coupled micelles using post-insertion technique. We propose to use five BBB specific CPPs: (i) CGN (d- CGNHPHLAKYNGT); (ii) RDP (KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR; (iii) Rabies Virus Glycoprotein RVG-9R, (iv) a non-toxic fragment of tetanus toxin, tetanus toxin C fragment (TTC), and (v) penetratin. The liposomal nanoparticles will be evaluated for particle size, zeta potential, encapsulation efficiency, cell uptake and uptake mechanism(s), transfection efficiency, cell cytotoxicity, and hemolysis assay. The transport efficacy of APOE2 loaded liposomal nanoparticles will be evaluated across an in vitro BBB model designed by combining human cerebral microvascular endothelial cells (hCMEC/D3), human astrocytes and APP Swe/Ind- or MAPT P301L-overexpressing human neuroblastoma cells (SHSY5Y). We will also determine the effect of liposomal nanoparticles on Aβ levels and Tau phosphorylation in the medium and cell lysates from the co-culture system. Aim 2. Evaluate the in vivo biocompatibility, organ toxicity, pharmacokinetics and APOE2 expression in mice of varying ages: To establish successful gene therapies fo
