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Principal Investigator: Takahisa Kanekiyo
Organization: NORTH DAKOTA STATE UNIVERSITY
Fiscal Year: 2024
Award: $525,712
Funding agency: National Institute on Aging
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 for AD, we will validate
the Tf-CPP-liposomal nanoparticles for their biocompatibility, organ toxicity, and pharmacokinetics
(biodistribution) in wild type mice at 3 months of age. In addition, the APOE2 gene delivery will be further
validated in APOE-knockout mice at 3 and 24 months of ages. Aim 3. Assess the therapeutic effects of the
functionalized-liposome-mediated APOE2 gene delivery on cognitive impairment and Aβ and tau
pathology in vivo: To establish successful gene therapies for AD-related phenotypes and age-related cognitive
decline, we will examine effects of APOE2 gene therapy through the functionalized-liposomes on
neurobehaviors, synaptic functions and/or amyloid and tau pathology. The liposomes will be injected into amyloid
model APP/PS1 mice (3 and 6 months old), tau model PS19 mice (3 and 6 months old) and aged wild-type mice
(12 and 24 months old), and the effects will be assessed 3 months after the administration. For clinical relevance,
we will measure neurofilament light chain (NfL) levels in their plasma samples to assess effects on
neurodegeneration. In addition, we will also measure plasma concentrations of Aβ and p-tau in respective mouse
models. In addition, interstitial fluid Aβ clearance will be analyzed using in vivo microdialysis in the APP/PS1
mice at 3 months of age 1 month after the administration. Collectively, we anticipate that the proposed study will
contribute towards the development of high efficiency non-viral gene delivery system to cross the BBB for
successful gene therapy for neurological disorders and determine protective effects of increasing brain APOE2
on AD-related conditions.
Terms: <100+ years old><AD dementia><APOE e4><APOE-ε4><APOEε4><APP-PS1><APP/PS1><Abeta clearance><Age><Age Months><Age-associated cognitive decline><Age-associated memory impairment><Age-related cognitive decline><Aging><Alleles><Allelomorphs><Alzheimer Type Dementia><Alzheimer beta-Protein><Alzheimer disease dementia><Alzheimer sclerosis><Alzheimer syndrome><Alzheimer's><Alzheimer's Amyloid beta-Protein><Alzheimer's Disease><Alzheimer's amyloid><Alzheimer's disease patient><Alzheimer's disease therapy><Alzheimer's patient><Alzheimer's therapy><Alzheimers Dementia><Amentia><American><Amyloid><Amyloid Alzheimer's Dementia Amyloid Protein><Amyloid Beta-Peptide><Amyloid Protein A4><Amyloid Substance><Amyloid beta-Protein><Amyloid β><Amyloid β clearance><Amyloid β-Peptide><Amyloid β-Protein><Apo-E><ApoE><ApoE protein><Apolipoprotein E><Assay><Astrocytes><Astrocytus><Astroglia><Autoregulation><Aβ><Aβ clearance><BBB crossing><BBB function><Benign senescent forgetfulness><Bioassay><Biodistribution><Biological Assay><Blood - brain barrier anatomy><Blood Plasma><Blood-Brain Barrier><Brain><Brain Nervous System><CD71><Cell Body><Cells><Centenarian><Cerebral endothelium><Chitosan><Clostridium tetani Toxin><Co-culture><Cocultivation><Coculture><Coculture Techniques><Cognitive><Cognitive Disturbance><Cognitive Impairment><Cognitive decline><Cognitive function abnormal><Coupled><DNA Therapy><Degenerative Neurologic Disorders><Dementia><Development><Disease><Disorder><Disturbance in cognition><Drug Kinetics><Elderly><Encapsulated><Encephalon><Endosomes><Environmental Factor><Environmental Risk Factor><Event><Film><Gene Delivery><Gene Transfer Clinical><Gene variant><Genes><Genetic><Genetic Intervention><Genetic predisposing factor><Genotype><Goals><H(C) fragment, TeNT><H+ element><Hemato-Encephalic Barrier><Hemolysis><Homeostasis><Human><Hydration><Hydration status><Hydrogen Ions><Impaired cognition><In Vitro><Individual><Intercellular Fluid><Interstitial Fluids><KO mice><Knock-out><Knock-out Mice><Knockout><Knockout Mice><Late Onset Alzheimer Disease><Leanness><Light><Lipids><Liposomal><Liposomes><Lytotoxicity><MT-bound tau><Measures><Mediating><Mice><Mice Mammals><Micelles><Microdialysis><Modeling><Modern Man><Murine><Mus><Nerve Cells><Nerve Degeneration><Nerve Unit><Nervous System Degenerative Diseases><Nervous System Diseases><Nervous System Disorder><Neural Cell><Neural Degenerative Diseases><Neural degenerative Disorders><Neurocyte><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Neurologic Disorders><Neurological Disorders><Neuron Degeneration><Neurons><Non-Viral Vector><Null Mouse><Organ><Particle Size><Pathogenesis><Pathology><Pathway interactions><Penetration><Peptides><Pharmacokinetics><Phenotype><Photoradiation><Physiological Homeostasis><Plasma><Plasma Serum><Play><Poliglusam><Porifera><Predisposition><Prevention><Primary Senile Degenerative Dementia><Property><Proteins><Protons><Receptor Protein><Receptosomes><Research><Reticuloendothelial System, Serum, Plasma><Role><Sampling><Siderophilin><Sponges><Surface><Susceptibility><Synaptic plasticity><System><TFR gene><TFR protein><TFR1><TFRC><TFRC gene><TRFR><Techniques><Tetanus Toxin><Therapeutic><Therapeutic Effect><Thinness><Toxic effect><Toxicities><Transfection><Transferrin><Transferrin Receptor><Transferrin Receptor 1><Wild Type Mouse><a beta peptide><a-beta peptide clearance><abeta><abeta accumulation><abeta aggregation><abeta deposition><abeta peptide clearance><advanced age><age associated memory decline><age related cognitive deficit><age related cognitive impairment><age related memory dysfunction><age-induced cognitive decline><age-related decline in cognition><age-related decline in cognitive function><aged><ages><aging associated><aging related><allele variant><allelic variant><amyloid beta><amyloid beta accumulation><amyloid beta aggregation><amyloid beta clearance><amyloid beta deposition><amyloid beta peptide clearance><amyloid β accumulation><amyloid β aggregation><amyloid β deposition><amyloid-b protein><apo E-4><apo E4><apo epsilon4><apoE epsilon 4><apoE-4><apoE4><apolipoprotein E epsilon 4><apolipoprotein E-4><apolipoprotein E4><astrocytic glia><aβ accumulation><aβ aggregation><aβ deposition><beta amyloid fibril><biocompatibility><biomaterial compatibility><blood-brain barrier crossing><blood-brain barrier function><bloodbrain barrier><bloodbrain barrier crossing><bloodbrain barrier function><brain endothelial cell><brain endothelium><brain microvascular endothelial cell><brain vascular endothelial cell><centenarian human (100+)><cerebral endothelial cell><cerebral microvascular endothelial cell><cerebral vascular endothelial cell><clinical applicability><clinical application><clinical relevance><clinically relevant><cognitive dysfunction><cognitive function><cognitive loss><cortical endothelium><cytotoxicity><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><design><designing><developmental><effective therapy><effective treatment><environmental risk><erythrolysis><gene delivery system><gene repair therapy><gene therapeutics><gene therapy><gene-based therapeutic><gene-based therapeutics><gene-based therapy><genes therapeutic><genes therapeutics><genetic risk factor><genetic therapy><genetic variant><genomic therapy><genomic variant><geriatric><improved><in vivo><inherited factor><late life><late onset alzheimer><lipid based nanoparticle><lipid nanoparticle><microtubule bound tau><microtubule-bound tau><model design><mouse model><murine model><nano particle><nano-sized particle><nanoparticle><nanosized particle><neural degeneration><neurobehavior><neuroblastoma cell><neurodegeneration><neurodegenerative><neurodegenerative illness><neurofilament><neurological degeneration><neurological disease><neuronal><neuronal degeneration><neuroprotection><neuroprotective><non-viral gene delivery><nonviral gene delivery><nonviral vector><overexpress><overexpression><p-tau><p-τ><pathway><patient living with Alzheimer's disease><patient suffering from Alzheimer's disease><patient with Alzheimer's><patient with Alzheimer's disease><penetratin><phospho-tau><phospho-τ><phosphorylated tau><plasmid DNA><post-translational modification of tau><posttranslational modification of tau><primary degenerative dementia><protective effect><rabies virus G protein><rabies virus glycoprotein><rabies virus glycoprotein G><receptor><senile dementia of the Alzheimer type><senior citizen><social role><soluble amyloid precursor protein><synapse function><synaptic function><tau><tau Proteins><tau factor><tau phosphorylation><tau posttranslational modification><tau-1><tetanus toxin H(C) fragment><tetanus toxin HC fragment><tetanus toxin fragment C><tetanus toxin heavy chain fragment C><therapeutic gene><therapeutically effective><transcytosis><uptake><vector><wildtype mouse><zeta potential><τ Proteins><τ phosphorylation>