Chromatin remodeling gene therapy for FSHD using split-vector AAV SMCHD1 vectors

使用分裂载体 AAV SMCHD1 载体进行 FSHD 染色质重塑基因治疗

基本信息

批准号：
10288435
负责人：
Scott Q Harper
金额：
$ 21.8万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
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：
2021-07-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10288435
关键词：
4q35 Affect Base Pairing Chromatin Chromatin Remodeling Factor Chromosome 18 Chromosomes Clinical Code Collaborations Complementary DNA D4Z4 DNA Data Defect Deposition Disease Epigenetic Process Euchromatin FDA approved Facioscapulohumeral Facioscapulohumeral Muscular Dystrophy Foundations Gene Expression Gene Transduction Agent Genes Genetic Genetic Diseases Goals Heterochromatin Homeobox Human Human Chromosomes In Vitro Industry Inherited Intramuscular Lead Length Lesion Maintenance Masks Molecular Mus Muscle Muscle Cells Muscular Dystrophies Mutation Myoblasts Open Reading Frames Patients Phenotype Poly A Proteins Repression Safety Serotyping Signal Transduction Structure System Testing Transgenes Transgenic Organisms Translating Xenograft Model Xenograft procedure adeno-associated viral vector chromatin remodeling gene therapy homologous recombination humanized mouse in vivo mouse model novel novel strategies novel therapeutics overexpression promoter prospective reconstitution therapeutic gene therapy development transcription factor vector

项目摘要

Project Summary/Abstract Facioscapulohumeral dystrophy (FSHD) is among the most commonly inherited muscular dystrophies, affecting up to 870,000 people worldwide. There are currently no approved treatments for FSHD and therapy development remains an unmet need. Historically, FSHD has been subdivided into two forms, a common type, called FSHD1 (95% of cases) and rare type, called FSHD2 (5% of cases). These forms are clinically indistinguishable, and both FSHD1 and FSHD2 are ultimately caused by aberrant de-repression in muscle of a wild-type gene, called double homeobox 4 (DUX4), which encodes a transcription factor (DUX4) that is toxic to muscle. DUX4 de-repression in FSHD is caused by chromatin changes at the DUX4 locus. Specifically, in healthy muscle, DUX4 DNA is normally embedded in heterochromatin and repressed; in FSHD muscle, genetic factors associated with FSHD change the epigenetic status of the DUX4 locus, making it more euchromatin-like and allowing toxic DUX4 expression. FSHD1 and FSHD2 are distinguished by the genetic mechanisms that give rise to the FSHD epigenetic lesion, including mutation in chromatin modifier genes that normally promote heterochromatin deposition at the DUX4 DNA locus. Mutations in one such gene, called structural maintenance of chromosomes hinge domain 1 (SMCHD1), lead to DUX4 DNA hypomethylation and enables DUX4 expression. Recent in vitro data in FSHD patient myoblasts from FSHD1 and FSHD patients suggested that SMCHD1 over-expression can rescue the FSHD-associated epigenetic lesion, regardless of underlying cause. Thus, we hypothesize that the DUX4 locus in muscles can be repressed by SMCHD1 over-expression, thereby offering a novel therapy for FSHD via chromatin remodeling. Our goal is to test this hypothesis in vivo using a novel gene therapy strategy in two complementary humanized FSHD mouse models. Our gene therapy approach involves using adeno-associated viral vectors (AAV) to deliver SMCHD1 to muscle, but AAV has a limited packaging capacity, and the full-length SMCHD1 open reading frame (ORF) is too large to fit into a single AAV vector. To circumvent this size problem, we created an AAV.SMCHD1 split-vector system, where one vector contains a promoter and the 5’ half of the SMCHD1 gene, and a second vector contains the 3’ half of SMCHD1 and a poly A signal. The two vectors share several hundred base pairs of SMCHD1 sequence to allow homologous recombination in vivo. Our preliminary data support the efficiency of this system to recombine in mouse muscle. Here we synergize expertise of two labs to test the functional impacts of SMCHD1 split-vector gene therapy to correct the epigenetic lesions and gene expression defects associated with FSHD in two different humanized mouse models, a transgenic line expressing a human FSHD-permissive DNA fragment (D4Z4-2.5) and a human FSHD muscle xenograft model. Successful completion of our Specific Aims will provide a foundation for translating AAV.SMCHD1 split vector gene therapy as a prospective new treatment for FSHD.

项目摘要/摘要 Facioscapulohumeral营养不良（FSHD）是最常见的肌肉营养不良，影响全球多达870,000人。目前尚无FSHD和治疗的批准治疗发展仍然是未满足的需求。从历史上看，FSHD被细分为两种形式，一种常见类型，称为FSHD1（占病例的95％）和稀有类型，称为FSHD2（占病例的5％）。这些形式在临床上是难以区分，FSHD1和FSHD2最终都是由A肌肉异常引起的野生型基因，称为Double Homeobox 4（DUX4），该基因编码转录因子（DUX4）对此有毒肌肉。 FSHD中的DUX4去抑制是由DUX4基因座的染色质变化引起的。具体来说，在健康的肌肉，DUX4 DNA通常嵌入异染色质中并再现。在FSHD肌肉中，通用与FSHD相关的因素改变了Dux4基因座的表观遗传状态，使其更类似于恒菌素并允许有毒Dux4表达。 FSHD1和FSHD2以遗传机制为特征引起FSHD表观遗传病变，包括通常促进的染色质修饰基因中的突变 DUX4 DNA基因座的异染色质沉积。一个这样的基因中的突变，称为结构维护染色体铰链结构域1（SMCHD1），导致DUX4 DNA低甲基化并启用DUX4 表达。 FSHD患者和FSHD患者的FSHD患者成肌细胞的最新体外数据表明， SMCHD1过表达可以挽救与FSHD相关的表观遗传病变，无论其根本原因如何。那就是我们假设肌肉中的Dux4基因座可以通过SMCHD1过表达来抑制通过染色质重塑为FSHD提供新颖的疗法。我们的目标是使用两种完整的人源化FSHD小鼠模型中的新型基因治疗策略。我们的基因疗法方法涉及使用腺相关病毒载体（AAV）将SMCHD1传递到肌肉，但AAV具有包装能力有限，全长SMCHD1开放阅读框（ORF）太大了，无法适应一个 AAV矢量。为了解决这个尺寸问题，我们创建了一个aav.smchd1拆分矢量系统，其中一个向量包含一个启动子和SMCHD1基因的5'一半，第二个向量包含SMCHD1的3英尺半和一个信号。这两个矢量共享几百个基对的SMCHD1序列，以允许体内同源重组。我们的初步数据支持该系统重组的效率小鼠肌肉。在这里，我们协同两个实验室的专业知识，以测试SMCHD1拆分矢量的功能影响基因疗法以纠正与FSHD相关的表观遗传病变和基因表达缺陷人源化小鼠模型，一种表达人FSHD腐蚀性DNA片段的转基因线（D4Z4-2.5）和人类FSHD肌肉特征模型。成功完成我们的特定目标将提供翻译AAV.SMCHD1分裂载体基因疗法的基金会作为FSHD的前瞻性新疗法。