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

项目摘要/摘要 面肩肱骨营养不良症(FSHD)是最常见的遗传性肌营养不良症之一，影响 全球多达87万人。目前还没有批准的治疗FSHD的方法和疗法 发展仍然是一个未得到满足的需要。从历史上看，FSHD分为两种形式，一种常见的类型， 称为FSHD1(95%的病例)和罕见类型，称为FSHD2(5%的病例)。这些形式在临床上是 无法区分，FSHD1和FSHD2最终都是由肌肉的异常抑制引起的 野生型基因，称为双同源盒4(DUX4)，它编码一种转录因子(DUX4)，对 肌肉。FSHD中DUX4的下调是由DUX4基因的染色质变化引起的。具体而言，在 在健康肌肉中，DUX4 DNA通常嵌入异染色质并被抑制；在FSHD肌肉中，基因 与FSHD相关的因素改变了DUX4基因座的表观遗传状态，使其更像常染色质 并允许有毒的DUX4表达。FSHD1和FSHD2的不同之处在于 引起FSHD表观遗传损害，包括正常促进的染色质修饰基因的突变 DUX4基因座异染色质沉积。其中一个基因的突变，称为结构维持 染色体铰链域1(Smchd1)，导致DUX4 DNA低甲基化，使DUX4 表情。来自FSHD1和FSHD患者的FSHD患者成肌细胞的最新体外数据表明 Smchd1的过表达可以挽救FSHD相关的表观遗传学病变，而不考虑潜在的原因。 因此，我们假设肌肉中的DUX4基因可以被Smchd1过表达抑制，从而 通过染色质重塑为FSHD提供一种新的治疗方法。我们的目标是在活体内使用一种 两种互补人源化FSHD小鼠模型的新基因治疗策略。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 包装容量有限，全长Smchd1开放阅读框(ORF)太大，无法放入单个 AAV载体。为了绕过这个大小问题，我们创建了一个AAV.SMCHD1拆分向量系统，其中一个向量 包含启动子和Smchd1基因的5‘半部分，第二个载体包含Smchd1基因的3’半部分 和聚A信号。这两个载体共享数百个碱基对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 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Smchd1拆分载体的功能影响 基因治疗纠正两种不同类型FSHD相关的表观遗传学损害和基因表达缺陷 人源化小鼠模型，表达人FSHD允许DNA片段(D4Z4-2.5)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 AAV.SMCHD1裂解载体基因治疗作为FSHD潜在新疗法的基础

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.