Technical Neurodegenerative disorders affect over 40 million people worldwide and most, if not all, are mediated by inflammation. Even though several drugs have recently been approved by the FDA for treating neurological disorders including Alzheimer's disease (AD), their efficacy as AD therapeutics has not been realized as extremely low levels of the drug reach the brain. The goal of this Phase I project is to develop an effective targeted delivery system for siRNA against NFκB expression (siNFκB) to mitigate neuroinflammatory diseases. We have recently shown that siRNA entrapped in bovine colostrum-derived exosomes and polyethyleneimine matrix (EPM) can effectively deliver siRNA to reduce target gene expression. We will apply our extensive experience in exosomes, siRNA therapeutics and inflammatory responses for efficient, targeted delivery of siNFκB to the microenvironment of the brain where progressive increase in inflammation ultimately leads to manifestation of neuroinflammatory diseases including AD. We hypothesize that EPM-siNFκB formulations administered via a novel trans-spinal (t.s.) route can effectively deliver siNFκB to the brain and knockdown the inflammatory regulator NFκB and attenuate corresponding disease markers. We also hypothesize that use of smaller exosomes (smExo) (average size 45 nm) coupled with folic acid (FA)-functionalization can enhance the brain delivery presumably due to more efficient crossing of the blood brain barrier and high expression of folate receptor-a (FRa) in the brain, respectively. Our hypotheses are supported by high siRNA entrapment (>90%) in the EPM, protection of EPM-siRNA from enzymatic degradation, higher knockdown of NFκB in murine cells than conventional reagents, higher brain accumulation of smEPM-siRNA in mice treated via t.s. route compared with the traditional routes, intravenously and intranasally, and FA-functionalization of exosomes for brain targeting. Innovation in this project lies in the use of bovine colostrum powder - an abundant and cost-effective source of native exosomes, use of smaller exosomes and FA-functionalization coupled with t.s. administration. Investigators experienced in exosomes, drug delivery, siRNA therapeutics and animal models, leveraging extensive experience of Dr. Robert Friedland in AD research, will pursue the following specific aims: Aim 1. Optimize delivery of siNFκB using FA-functionalized exosomal vector in vitro and in vivo. Murine cell lines bearing WT sequences of NFκB will be used to identify effective siNFκB sequences based on NFκB knockdown. Aim 2. In vivo efficacy of brain-targeted FA-smEPM-siNFκB. The lead formulation from Aim 1 will be tested for efficacy in murine models of AD. Therapeutic benefit will be based on knockdown of NFκB in the target (cortex and hippocampus) and non-target tissues, related histopathological and AD biomarker changes, and assessment of behavioral endpoints. This project will provide proof-of-principle for inhibition of neuroinflammation by targeted t.s. delivery of siNFκB. In Phase II, we will compare inhibition of other targets such as BACE1, a key target for AD, alone and in combination with NFκB using multiple rodent AD models to identify the optimal gene target(s) for this disease.
Public Health Relevance Statement: Narrative (Public Health Relevance Statement) In the proposed project, we will use a novel nano formulation of small interfering siRNA to mitigate neuroinflammation and attenuate neurological diseases including Alzheimer's disease (AD). In our approach, siRNA of the key inflammation regulator, NFκB (siNFκB) is sequestered in exosomes (native nanoparticles) isolated from standardized bovine colostrum powder, the exosomes are attached to folic acid to target the brain sub tissues such as cortex and hippocampus, and the formulation is delivered trans-spinally by topical application for efficient brain targeting. The novel formulation will be optimized using mouse cell lines and the trans-spinal delivery system tested using mouse models for inflammation and AD. Terms: AD dementia; AD model; Adult Folate Receptor 1; Adult Folate-Binding Protein; Affect; Alzheimer Type Dementia; Alzheimer disease dementia; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimer's biomarker; Alzheimer's disease biological marker; Alzheimer's disease model; Alzheimer's disease therapeutic; Alzheimer's therapeutic; Alzheimers Dementia; Alzheimer's biological marker; Alzheimer's disease biomarker; Ammon Horn; Animal Model; Animal Models and Related Studies; Animals; Area; Attenuated; BACE; BACE1; BBB crossing; Base Sequence; Behavior assessment; Behavioral; Biodistribution; Biological Markers; Body Tissues; Bovine Species; Brain; Brain Inflammation; Brain Nervous System; Brain region; Cattle; Cell Body; Cell Communication and Signaling; Cell Line; Cell Signaling; CellLine; Cells; Coloring Agents; Colostrum; Cornu Ammonis; Coupled; Data; Degenerative Neurologic Disorders; Disease; Disease Marker; Disorder; Dose; Drug Delivery; Drug Delivery Systems; Drug Targeting; Drugs; Dryness; Dyes; Encephalitis; Encephalon; Euthanasia; FDA approved; FOLR; FOLR1; FOLR1 gene; Female; Fibroblasts; Fluorescence; Folate; Folate Receptor Alpha; Folic Acid; Formulation; Gene Expression; Gene Targeting; Genes; Goals; Hippocampus; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Intracellular Communication and Signaling; Intravenous; Investigators; Label; Lead; Liver; MOv18; Mediating; Medication; Mercy Killing; Mice; Mice Mammals; Mouse Cell Line; Murine; Mus; Nervous System Degenerative Diseases; Nervous System Diseases; Nervous System Disorder; Neural Degenerative Diseases; Neural degenerative Disorders; Neurodegenerative Diseases; Neurodegenerative Disorders; Neurologic Degenerative Conditions; Neurologic Disorders; Neurological Disorders; Nucleotide Sequence; Ovarian Cancer-Associated Antigen; Pb element; Persons; Pharmaceutical Preparations; Phase; Polyaziridine; Polyethyleneimine; Powder dose form; Powders; Primary Senile Degenerative Dementia; Pteroylglutamic Acid; Radiolabeled; Reagent; Regimen; Research; Research Personnel; Researchers; Rodent; Rodentia; Rodents Mammals; Route; Short interfering RNA; Signal Transduction; Signal Transduction Systems; Signaling; Small Interfering RNA; Source; Spinal; Standardization; Strains Cell Lines; Sum; System; Testing; Therapeutic; Tissue imaging; Tissues; Topical Drug Administration; Topical application; Toxic effect; Toxicities; Treatment Efficacy; Vitamin M; administer topically; alzheimer model; apply topically; attenuate; attenuates; behavioral assessment; beta-secretase 1; beta-site APP cleaving enzyme 1; beta-site amyloid precursor protein cleaving enzyme 1; bio-markers; biologic marker; biological signal transduction; biomarker; blood-brain barrier crossing; bloodbrain barrier crossing; bovid; bovine; candidate identification; cost effective; cow; cultured cell line; degenerative diseases of motor and sensory neurons; degenerative neurological diseases; deliver short interfering RNA; deliver siRNA; deliver small interfering RNA; deliver topically; delivery system for siRNA; delivery system for small interfering RNA; delivery vectors for siRNA; drug/agent; efficacy study; efficacy testing; ex vivo imaging; exosome; experience; folate carrier; folate receptor; folate-binding protein; folate-methotrexate transporter; folic acid binding protein; folic acid receptor; heavy metal Pb; heavy metal lead; hepatic body system; hepatic organ system; hippocampal; homopolymer Aziridine; imager; immunogenicity; immunotoxicity; in vivo; inflammation marker; inflammatory marker; inflammatory modulation; innovate; innovation; innovative; intervention efficacy; knock-down; knockdown; memapsin 2; methotrexate-binding protein; model of animal; mouse model; murine model; nano formulation; nano particle; nano-sized particle; nanoformulation; nanoparticle; nanosized particle; neural inflammation; neurodegenerative illness; neuroinflammation; neuroinflammatory; neurological disease; novel; nucleic acid sequence; primary degenerative dementia; public health relevance; radiolabeling; radiologically labeled; response to therapy; response to treatment; safety assessment; senile dementia of the Alzheimer type; short interfering RNA delivery; siRNA; siRNA delivery; siRNA therapy; siRNA-based therapeutic; siRNA-based therapy; site targeted delivery; small interfering RNA delivery; targeted delivery; therapeutic efficacy; therapeutic response; therapeutic siRNA; therapeutic small interfering RNA; therapy efficacy; therapy response; topical administration; topical delivery; topical drug application; topical treatment; topically administered; topically applied; topically delivered; topically treated; toxic reaction in immunology; treat topically; treatment response; treatment responsiveness; vector; vitamin Bc; ß-secretase 1; ß-site APP cleaving enzyme 1
