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Nanocarriers Designed to Deliver Nucleic Acids to Brain

旨在将核酸输送到大脑的纳米载体

基本信息

批准号：
9338335
负责人：
JUAN R SANCHEZ-RAMOS
金额：
$ 38.17万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9338335
关键词：
Applications Grants Behavior Behavioral Blood - brain barrier anatomy Brain Brain Diseases Brain region Caliber Cell Culture Techniques Cells Cerebral cortex Chelating Agents Chitosan Chronic Clinical Corpus striatum structure Culture Media DNA Data Development Diphosphates Dose Drug Delivery Systems Electron Microscopy Extracellular Fluid Fibroblasts Fluorescence Fluorescence Microscopy Formulation Gene Silencing Genes Goals Green Fluorescent Proteins Hour Huntington Disease Image Incubated Infusion procedures Injection of therapeutic agent Intranasal Administration Intrathecal Space Label Life Magnetic Resonance Imaging Manganese Measures Messenger RNA Metals Methods Motor Activity Mouse Cell Line Mus Neurodegenerative Disorders Neurons Nose Nucleic Acids Olfactory Mucosa Operative Surgical Procedures Pathologic Patients Pharmaceutical Preparations Plasmids Play Process Research Proposals Residual state Role Series Signal Transduction Small Interfering RNA System Technology Testing Therapeutic Therapeutic Agents Time Transfection Transgenic Mice Transgenic Organisms Viral Vector Western Blotting base clinically significant comparative crosslink cytotoxicity design ds-DNA exosome experimental study extracellular gene therapy in vivo light scattering mRNA Expression mouse model nanocarrier nanoparticle novel therapeutics olfactory bulb protein expression public health relevance red fluorescent protein

项目摘要

DESCRIPTION (provided by applicant): Gene therapy of brain diseases is hampered by the requirement for invasive stereotaxic injection into brain, or infusion of therapeutic agents into te intrathecal space. The surgical approach is not optimal for neurodegenerative diseases that require treatments throughout life. This obstacle has given impetus to the design of a new nose-to-brain delivery system for nucleic acids or drugs that cannot pass the blood-brain-barrier. The overall goal of this project is to refine and test a nanocarrier system consisting of chitosan-based, manganese-containing nanoparticles (mNPs) loaded with therapeutic nucleic acids: small interfering RNA (siRNA) and dsDNA. The experiments are designed to assess the mechanisms and extent to which the nanocarriers transport their payload from the olfactory mucosa to olfactory bulb and other brain regions to suppress marker genes in mouse models of rapid onset Huntington's Disease. Specific Aim 1: Testing optimized nanocarriers loaded with siRNA against a marker gene expressed in transgenic "green" mice that constitutively express green fluorescent protein (GFP) in brain neurons. mNPs will be packaged with siRNA directed against GFP. Primary end-points will be a) the extent and time-course of dissemination of mNPs from olfactory bulb to other regions of brain assessed by MRI T1-weighted imaging; b) GFP mRNA and GFP protein expression by comparative quantitiative PCR and Western blot analysis, respectively. Specific Aim 2: Study of gene-silencing in a mouse model of HD. siRNA directed against htt will be loaded into NPs and administered intra-nasally or microinjected directly into striatum in rapid-onset mouse models of Huntington's Disease. Primary-end points will be a) extent and time-course of dissemination of mNPs from olfactory bulb (or from striatum) to other regions of brain assessed by microbore MRI, b) quantitative analysis of htt mRNA expression by quantitative PCR and htt protein expression by Western blot in various brain regions, c) effects on behavior and locomotor activity. Specific Aim 3 a) Iterative optimization of the nanocarrier system in cell cultures expressing a marker gene guided by results from concurrent in vivo experiments in Aim 1 and b) studies of cellular extrusion of exosomes containing mNPs as an hypothesized mechanism for cell-to-cell dissemination. Specific Aim 4: Testing capacity of mNPs to deliver DNA (gene encoding a red fluorescent protein) in vivo. mNPs will be loaded with plasmid dsDNA encoding red fluorescent protein (RFP) and intranasally instilled into normal C57BL6 mice. End-points will be the same as those in Aim 2. Clinical Significance: The ability to dose patients chronically and non-invasively via intra-nasal administration of nanocarriers of gene-silencing agents or other large therapeutic molecules will have a dramatic impact in the therapeutics of brain disorders.