Therapeutic Strategy to Treat Alzheimer's Disease by VGF Delivery into Brain

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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, in which the formation and accumulation of hyperphosphorylated tau protein and amyloid-β (Aβ) are believed to play key roles in AD pathogenesis. Of note, the recent multiscale causal network analysis in Accelerated Medicines Partnership for Alzheimer’s Disease (AMP-AD) cohort identified that VGF is the only downregulated key driver for AD. VGF is synthesized by neurons in the brain where it promotes growth and survival of neurons, and is involved in neurogenesis, synaptogenesis and energy homeostasis. VGF plays a critical role in learning, memory, and pathophysiology of neurodegenerative diseases. Therefore, this proposal aims to develop a novel effective gene therapy for AD by targeting VGF. The major challenge in the field of gene therapy for AD is to design a safe vector that can cross the blood brain barrier (BBB) and target the desired cells. We propose to develop innovative and targeted nanoparticles conjugated with human VGF cDNA plasmid (pVGF) for the treatment of AD by delivering into brain after intravenous and intranasal administration. Intranasal route provides a direct entrance of CNS therapeutics to the brain and therefore this is a promising non-invasive pathway for gene to reach the brain parenchyma by bypassing the BBB. We would synthesize two types of nanoparticles- liposomal nanoparticles and ω-3 fatty acid grafted chitosan based nanomicelles. Both types of nanoparticles will be grafted with targeting ligands [transferrin (Tf), mannose (MAN), and brain and neuron specific cell penetrating peptide (CPP)]. It has been found that the Tf and GLUT-1 receptors are present on the surface of brain endothelial cells as well as on neurons. MAN is a substrate for GLUT1. In addition, the CPP will further improve the penetration of nanoparticles/nanomicelles into brain. Therefore, we propose to design liposomal nanoparticles encapsulating gene and modifying the surface of nanoparticles with Tf, MAN and CPP. Similarly, ω-3 fatty acid grafted chitosan will be also modified by grafting with Tf, MAN and CPP. These graft polymers will form self-assembled cationic nanomicelles in aqueous environment to provide selective targeting of complexed pVGF to brain. The long-term goal of the proposed research is to design a non-viral gene delivery carrier for efficient delivery of pVGF to brain through intravenous and intranasal administrations for prevention and treatment of aging-related cognitive decile including AD. We propose three specific aims to accomplish the long-term goal of the proposed research. Aim 1. Synthesize and characterize nanoparticles/nanomicelles loaded with pVGF: The CPP-liposomal nanoparticles will be synthesized using thin film hydration technique followed by insertion of Tf- and MAN- coupled micelles using post-insertion technique. We propose to use three BBB and neuron specific CPPs: (i) a non-toxic fragment of tetanus toxin known as tetanus toxin C fragment (TTC), (ii) penetratin, and (iii) rabies virus glycoprotein (RVG-9R containing a nerve binding region). For nanomicelles, we will synthesize graft polymer (GP) of chitosan with ω-3 fatty acid. The GP will be further grafted with MAN, Tf and CPP, and characterize by infrared (IR) and NMR spectroscopy. The GP will self-assemble in aqueous media to form nanomicelles. The nanoparticles/nanomicelles 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 pVGF loaded nanoparticles/nanomicelles will be evaluated across an in vitro BBB model designed by combining primary human epithelial cells (HBMECs) and primary human astrocytes (HA). We will evaluate the effect of nanoparticles/nanomicelles on transfection efficiency, Aβ levels and tau-phosphorylation in the cell culture BBB model by seeding the APP Swe/Ind or MAPT P301L-overexpressing SHSY5Y cells in 24-well plates. Secretion of Aβ40 and Aβ42 in the culture supernatant, as well as intracellular accumulation in cell lysates, will be determined by ELISA. Total tau and phosphorylated tau levels in the cell lysates and culture medium will be measured by Western blot assay/ELISA. Aim 2. Evaluate the in vivo biocompatibility, organ toxicity, pharmacokinetics and VGF expression in wild type mice of varying ages: To establish successful gene therapies for AD, we will validate the nanoparticles/nanomicelles for their biocompatibility, organ toxicity, and pharmacokinetics (biodistribution) after administering intravenously or intranasally into wild type mice at 3 months of age. In addition, the VGF gene delivery will be further validated in wild-type mice at 3 and 24 months of ages. Aim 3. Assess the therapeutic effects of the nanoparticle/nanomicelle-mediated VGF gene delivery on cognitive impairment and Aβ pathology: To establish successful gene therapies for AD-related phenotypes and age-related cognitive decline, we will examine effects of VGF gene therapy through the functionalized-nanoparticles/nanomicelles on neurobehaviors, synaptic functions and/or amyloid pathology. The nanoparticles will be administered intravenously or intranasally into amyloid model 5xFAD mice and aged wild- type mice, and the effects will be assessed. For human relevance, we will also use iPSC-derived neurons and cerebral organoids from AD patients and assess the effects on neurodegeneration and Aβ/tau pathologies. Collectively, we anticipate that the proposed study will contribute towards the development of high efficiency non-viral gene delivery system to deliver pVGF into brain for successful gene therapy for AD and other neurodegenerative diseases.