Developing resources to alleviate muscle atrophy in FSHD by genome engineering

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是继杜氏肌营养不良症和肌强直性营养不良症之后第三常见的遗传性肌肉营养不良症，每20,000例活产婴儿中就有1例受到影响。FSHD的主要特征是面部、肩部和上臂的骨骼肌进行性无力和萎缩，通常在患者生命的第二或第三个十年出现。目前，这种疾病没有治愈或有效的治疗方法。FSHD是一种常染色体显性疾病，在几乎所有病例中，疾病的遗传基础涉及染色体4q亚端粒区大卫星重复D4Z4大小的收缩。D4Z4由3.3kb序列的串联阵列组成，在健康个体中具有10-150个重复单元，定义单个等位基因。有趣的是，虽然D4Z4的大小减少到少于10个重复单位与FSHD有关，但仅收缩并不足以引起疾病。事实上，在未受影响的个体中发现了与FSHD患者中观察到的D4Z4等位基因大小相当的等位基因，而在没有FSHD症状的其他个体中，有报道称大卫星基因完全缺失。相反，FSHD只与人类染色体4q亚端粒区特定变体上D4Z4的收缩有关。最近的研究表明，允许染色体共享一个典型的多腺苷化信号，在一个收缩的等位基因上稳定起源于最远端D4Z4重复单元的转录本，导致肌肉功能的毒性增加。本提案的目标是产生必要的资源来消除致病性D4Z4等位基因。近年来的技术进步使得通过定制锌指核酸酶（ZFN）和TAL效应核酸酶（TALEN）在人类细胞中进行基因组工程成为可能。这两种方法都涉及到序列特异性DNA结合蛋白的产生，这些蛋白与核酸酶结合，在所需的序列上诱导双链断裂。将ZFNs和TALENs与修饰的靶位点同源序列一起引入细胞，当提供的模板用于修复损伤时，会导致内源性位点发生所需的变化。在Aim 1中，我们将开发针对D4Z4阵列近端的序列的zfn和TALENs。这些将与引入端粒播种序列的修复模板结合使用，以去除大卫星，导致缺乏D4Z4的染色体截短，与未受影响的个体相似。在Aim 2中，我们将开发设计成D4Z4远端序列的ZFNs和TALENs，以将聚腺苷化信号转化为非允许染色体上的序列。这些资源的产生将允许探索一种新的治疗方法，用于患有这种使人衰弱的进行性疾病的个体。

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.