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

项目总结/摘要 面肩肱营养不良（FSHD）是最常见的遗传性肌营养不良症之一， 全世界有87万人。目前还没有批准的FSHD治疗方法和治疗方法 发展仍然是一个未得到满足的需求。历史上，FSHD被细分为两种形式，一种是常见类型， 称为FSHD 1（95%的病例）和罕见类型，称为FSHD 2（5%的病例）。这些形式在临床上 FSHD 1和FSHD 2最终都是由肌肉中的异常去抑制引起的， 一种野生型基因，称为双同源框4（DUX 4），它编码一种转录因子（DUX 4），对 肌肉. FSHD中DUX 4去阻遏是由DUX 4基因座的染色质变化引起的。具体到 在健康肌肉中，DUX 4 DNA通常嵌入异染色质中并被抑制;在FSHD肌肉中，遗传 与FSHD相关的因素改变了DUX 4基因座的表观遗传状态，使其更像常染色质 并允许毒性DUX 4表达。FSHD 1和FSHD 2的区别在于遗传机制， 引起FSHD表观遗传损伤，包括通常促进FSHD的染色质修饰基因突变， 在DUX 4 DNA位点处的异染色质沉积。其中一个基因的突变，称为结构维持 染色体铰链结构域1（SMCHD 1）的基因，导致DUX 4 DNA低甲基化，使DUX 4 表情来自FSHD 1和FSHD患者的FSHD患者成肌细胞的最新体外数据表明， SMCHD 1过表达可以挽救FSHD相关的表观遗传病变，无论潜在的原因。 因此，我们假设肌肉中的DUX 4位点可以被SMCHD 1过表达抑制，从而 通过染色质重塑为FSHD提供了一种新的疗法。我们的目标是在体内测试这一假设， 在两种互补的人源化FSHD小鼠模型中的新基因治疗策略。我们的基因疗法 一种方法涉及使用腺相关病毒载体（AAV）将SMCHD 1递送至肌肉，但AAV具有 有限的包装容量，并且全长SMCHD 1开放阅读框（ORF）太大而不能装入单个 AAV载体。为了规避这个大小问题，我们创建了AAV.SMCHD1分裂载体系统，其中一个载体 含有启动子和SMCHD 1基因的5 ′端，第二个载体含有SMCHD 1基因的3 ′端 和多聚腺苷酸信号两个载体共享SMCHD 1序列的几百个碱基对， 体内同源重组。我们的初步数据支持该系统的重组效率， 老鼠的肌肉在这里，我们协同两个实验室的专业知识来测试SMCHD 1分裂载体的功能影响 基因治疗，以纠正与FSHD相关的表观遗传病变和基因表达缺陷，在两个不同的 人源化小鼠模型，表达人FSHD允许DNA片段（D4 Z4 -2.5）的转基因系 和人FSHD肌肉异种移植模型。成功完成我们的具体目标将提供一个 为将AAV.SMCHD1分裂载体基因治疗作为FSHD的前瞻性新治疗方法奠定了基础。

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.