Developing resources to alleviate muscle atrophy in FSHD by genome engineering

描述(申请人提供)：面肩肩周型肌营养不良症(FSHD)是继Duchenne和强直性肌营养不良之后的第三种最常见的遗传性肌营养不良症，每20,000名活产儿中就有一名受到影响。FSHD的主要特征是面部、肩部和上臂骨骼肌的进行性无力和萎缩，典型地表现在患者生命的第二或第三个十年。目前还没有治愈或有效治疗这种疾病的方法。FSHD是一种常染色体显性遗传病，在几乎所有病例中，疾病的遗传基础都涉及4Q染色体亚端粒区域的大卫星重复D4Z4的大小收缩。D4Z4由3.3kb序列的串联阵列组成，其中10-150个重复单元定义了健康个体中的单个等位基因。有趣的是，虽然D4Z4的大小减少到10个以下的重复单位与FSHD有关，但单靠收缩还不足以致病。事实上，在未受影响的个体中发现了大小与FSHD患者相似的D4Z4等位基因，而在其他人中，报告了在没有FSHD症状的情况下完全丧失了大卫星。相反，FSHD只与人类染色体4Q亚端粒区域特定变体上的D4Z4收缩有关。最近的研究表明，允许染色体共享一个典型的多腺苷酸化信号，该信号在收缩的等位基因上稳定来自最远端D4Z4重复单位的转录本，导致肌肉功能的有毒获得。这项提议的目的是产生必要的资源，以使致病的D4Z4等位基因无效。最近的技术进步使人类细胞通过定制的锌指核酸酶(ZFN)和TAL效应核酸酶(TALEN)进行基因组工程成为可能。这两种方法都涉及到产生序列特异的DNA结合蛋白，这些蛋白与核酸酶结合，在所需的序列上诱导双链断裂。将ZFN和TALEN与靶部位修饰的同源序列一起导入细胞，可在内源性基因座引入所需的变化，因为所提供的模板用于修复损伤。在目标1中，我们将开发针对D4Z4阵列近端序列的ZFN和TALEN。这些将与修复模板结合使用，修复模板引入端粒种子序列来移除大卫星，导致缺失D4Z4的截断染色体，与在未受影响的个体中观察到的类似。在目标2中，我们将开发ZFN和TALEN，这些ZFN和TALEN设计为紧邻D4Z4远端的序列，以将多聚腺苷信号转化为在不允许的染色体上发现的序列。这种资源的产生将使人们能够探索一种新的疗法来治疗这种衰弱的进行性疾病。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited form of muscular dystrophy after Duchenne and Myotonic dystrophy, affecting 1 in 20,000 live births. FSHD is primarily characterized by progressive weakness and atrophy of skeletal muscle of the face, shoulders and upper arms that typically manifest in a patients second or third decade of life. Currently there is no cure or effective treatment for this disease. FSHD is an autosomal dominant disorder and in almost all cases the genetic basis for disease involves a contraction in the size of the macrosatellite repeat D4Z4 in the subtelomeric region of chromosome 4q. D4Z4 is composed of a tandem array of a 3.3kb sequence with between 10-150 repeat units defining a single allele in healthy individuals. Intriguingly, whereas reduction in the size of D4Z4 to fewer than 10 repeat units is associated with FSHD, contraction alone is not sufficient to cause disease. Indeed, D4Z4 alleles comparable in size to those observed in FSHD patients have been detected in unaffected individuals, whereas in others complete loss of the macrosatellite has been reported in the absence of FSHD symptoms. Instead, FSHD is exclusively associated with contraction of D4Z4 on specific variants of the subtelomeric region of human chromosome 4q. Recent studies have demonstrated that permissive chromosomes share a canonical polyadenylation signal that upon a contracted allele stabilizes transcripts originating from the most distal D4Z4 repeat unit resulting in a toxic gain of function in muscle. The objective of this proposal is to generate the resources necessary to nullify pathogenic D4Z4 alleles. Recent technological advances have made possible genome engineering in human cells through custom-built zinc finger nucleases (ZFN) and TAL effector nucleases (TALEN). Both approaches involve generation of sequence specific DNA binding proteins tethered to nuclease enzymes that induce double strand breaks at a desired sequence. Introduction of ZFNs and TALENs into cells along with a modified homologous sequence to the target site results in the introduction of desired changes into the endogenous locus as the supplied template is used to repair the damage. In Aim 1, we will develop ZFNs and TALENs directed to sequences proximal to the D4Z4 array. These will be used in combination with a repair template that introduces a telomere seeding sequence to remove the macrosatellite, resulting in a truncated chromosome lacking D4Z4 similar to that observed in unaffected individuals. In Aim 2, we will develop ZFNs and TALENs designed to sequences immediately distal to D4Z4 to convert the polyadenylation signal to a sequence found on non-permissive chromosomes. Generation of such resources will permit exploration of a novel therapy for individuals afflicted with this debilitating progressive disorder.