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Novel gene based therapy for nemaline myopathy

线状肌病的新型基因疗法

基本信息

批准号：
10020762
负责人：
JAMES J DOWLING
金额：
$ 34.72万
依托单位：
HOSPITAL FOR SICK CHLDRN (TORONTO)
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-18 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10020762
关键词：
Actins Back Behavior Biochemical Biological Models Biology CRISPR/Cas technology Cell Line Cell model Cells Childhood Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Contracture Dependence Development Disease Elements Ensure Enteral Feeding Event Excision Exons Generations Genes Genomics Germ Lines Goals Human Image Impairment Infusion procedures Interruption Intramuscular Injections Knowledge Length Mediating Modeling Molecular Mus Muscle Muscle Contraction Muscle Fibers Mutant Strains Mice Mutation Myoblasts Myopathy Myosin ATPase Nemaline Myopathies Neonatal Nonsense Mutation Organism Patients Phenotype Post-Translational Protein Processing Preclinical Drug Development Preclinical Testing Proteins RNA RNA Splicing Reading Frames Reagent Sarcomeres Series Severities Severity of illness Site Skeletal Muscle Specificity Splice-Site Mutation Structure Swimming System Technology Testing Therapeutic Thin Filament Transcript Translating Translations Wheelchairs Work Zebrafish base clinical translation disability efficacy testing exon skipping experimental study gene therapy imaging capabilities improved in vivo mouse model mutant nebulin novel novel therapeutic intervention photo switch physically handicapped prevent protein expression success therapy development treatment strategy

项目摘要

PROJECT SUMMARY/ABSTRACT Nemaline myopathy (NM) is a childhood-onset skeletal muscle disease that is characterized by severe disabilities, including (in many cases) wheelchair and feeding tube dependence. Mutations in more than a dozen genes can cause NM. Most of these genes encode components of the thin filament (a principle part of the sarcomere), and mutations associated in these genes alter the structure and/or function of the thin filament, resulting in impaired muscle contraction and generalized weakness. There are currently no treatments for NM. The overarching goal of this proposal is to develop therapies for this devastating disease. Recessive mutations in NEB are the most common cause of NM. NEB encodes the giant protein Nebulin, which functions to regulate the length of the thin filament. Many NEB mutations cause single exon skipping or single exon deletion. Such mutations do not alter the RNA reading frame of NEB; however, despite only removing a very small part of an otherwise giant protein, they unexpectedly result in significant reduction (or even complete loss) of the whole Nebulin protein. The reason for this is not known. We hypothesize that the reason for this surprising observation is that these mutations remove an incomplete portion of repeat elements within NEB, the consequence of which is to make the Nebulin protein out of register, thereby preventing it from incorporating into the thin filament. We will test this hypothesis in Aim 1. Aim 1: we will use cutting edge imaging and biochemical strategies to study the Nebulin protein in a series of in-frame nebulin mutants. To accomplish this, we will employ the zebrafish model system, which is ideal for this purpose because we can visualize the Nebulin protein in intact skeletal muscle in a living organism. Nebulin protein levels in patients with NEB mutations are correlated with disease severity (less protein = more severe disease). This fact means that a therapeutic strategy targeted at increasing Nebulin protein expression should be very effective. Based on our hypothesis above, we predict that we can accomplish this by removing more of the Nebulin RNA to take out complete repeats, the result of which should be to enable a shortened Nebulin to re-integrate into the thin filament and be stably maintained. We will test this idea, which we call “domain skipping”, in Aims 2 and 3. Aim 2: Using either morpholino mediated multi exon skipping or CRISPR/Cas9 genomic deletion, we will establish the feasibility and efficacy of “domain skipping” in zebrafish. Zebrafish allow us to rapidly and comprehensively examine this strategy across the entire nebulin gene. Aim 3: We will translate our findings from zebrafish to the mouse model and to patient cells, focusing specifically on two common Neb mutations (exon 55 deletion and a nonsense mutation in exon 61). The use of the mouse model will enable us to test efficacy in a mammalian system, and testing in human cells will provide vital proof of concept and reagent development necessary for clinical translation.

项目总结/文摘