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Gene editing in the brain with CRISPR-PEG

使用 CRISPR-PEG 对大脑进行基因编辑

基本信息

批准号：
10378044
负责人：
Hye Young Lee
金额：
$ 64.61万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10378044
关键词：
Address Advanced Development Beds Brain Brain Diseases Brain region CGG repeat CRISPR therapeutics Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Corpus striatum structure DNA Damage Diffuse Diffusion Disease Etiology Exposure to FMR1 FMRP Formulation Fragile X Syndrome Gene Expression Genes Genetic Genetic Diseases Genomic DNA Gold Hypermethylation Inherited Injections Intellectual functioning disability Intranasal Administration Knock-in Mouse Knockout Mice Length Morphology Motor Cortex Mus Neurons Neurosciences Other Genetics Patients Phenotype Polyethylene Glycols Promoter Regions Proteins Publishing Reagent Ribonucleoproteins Signal Transduction Solubility Specificity Testing Therapeutic Time Tissues Toxic effect Transfection Variant Vertebral column Viral base base editing behavior test behavioral phenotyping biomaterial compatibility brain tissue brain volume delivery vehicle design experimental study immunogenicity improved in vivo in vivo evaluation induced pluripotent stem cell innovation knock-down mouse model nanoGold nanoparticle neurotoxicity novel olfactory bulb promoter repetitive behavior restoration tool translational impact translational potential

项目摘要

CRISPR-based gene editing has the potential to revolutionize the treatment of genetic brain disorders. However, complications with brain delivery have limited the utility of CRISPR-based therapeutics. To address this critical need, we have developed a new gene editing delivery vehicle, termed CRISPR-PEG, which is composed of Cas9 RNP conjugated to polyethylene glycol (PEG). CRISPR-PEG has tremendous promise as a delivery vehicle because of its excellent biocompatibility, the well-established clinical track record of PEG, and its enhanced tissue diffusion capability in comparison to nanoparticles. Our preliminary results demonstrate that CRISPR-PEG delivers and edits neurons efficiently in the motor cortex or striatum in mice; after an intracranial injection, neurons were edited with a high specificity (45~85%). Notably, CRISPR-PEG also edited neurons in the olfactory bulb after intranasal administration. These exciting results demonstrate that CRISPR-PEG has great potential as bio-tool, and as a platform for developing therapeutics. In this proposal we will test our novel delivery vehicle CRISPR-PEG in fragile X syndrome (FXS). We have selected FXS as a test bed for CRISPR-PEG because it is the most common inherited cause of intellectual disability with no treatment available. In addition, FXS has a monogenic cause, namely expanded CGG repeats>200 and hypermethylation in the FMR1 promoter region, which causes silencing of the fragile X mental retardation 1 (FMR1) gene. Therefore, the central objectives of this proposal are (1) to test CRISPR-PEG in brain disorders by targeting FXS-associated genes, and (2) to develop new CRISPR-PEG variants with improved diffusion and efficiency. The central hypothesis is: the novel non-viral delivery vehicle CRISPR- PEG will deliver Cas9 RNPs into the brain, efficiently edit FXS-associated genes in neurons, and rescue mice from multiple FXS-associated phenotypes. The central objective will be accomplished by completing the following specific aims. Specific Aim 1. Knock down mGluR5 using CRISPR-PEG in the mouse model of FXS as proof of principle. Specific Aim 2. Reactivate FMR1 gene expression using CRISPR-PEG. Specific Aim 3. Develop CRISPR-PEGs that diffuse throughout the brain and edit brain tissue efficiently. At the completion of this proposed study, we will have developed an efficient strategy for gene editing neurons using a novel non-viral delivery vehicle CRISPR-PEG. Our proposed studies are significant because the results will provide the basis for developing therapeutics for FXS and fragile X-associated disorders caused by FMR1 deficiency. Moreover, we will develop a non-viral-based vehicle that can edit large volumes of brain tissue after a single injection. The experiments in this proposal are highly innovative because we will have developed an efficient and safe non-viral delivery vehicle, which will greatly advance the field of neuroscience and CRISPR-based therapeutics.

基于crispr的基因编辑有可能彻底改变遗传性脑部疾病的治疗。然而,