Molecular Pathphysiology of FSHD muscular dystrophy via genome-wide approaches

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）与染色体4q35亚端粒区域缩短的3.3 kb D4Z4重复序列有关，从11-100个拷贝到1-10个拷贝。D4Z4序列的缩短被认为对D4Z4区域内或附近的基因具有去抑制作用。在亲本R01中，我们提出测试一个假设的FSHD模型，该模型涉及4q35缺失、双同源框蛋白4 （DUX4）和配对样同源域转录因子1 （PITX1）之间的直接调控关系。在aim 1中，我们提出通过表征Pitx1基因启动子区域推测的DUX4结合位点来确定Pitx1是否是DUX4的直接转录靶点。在目标2中，我们提出了一种条件肌肉特异性Pitx1转基因小鼠模型的产生和表征。在aim 3中，我们提出使用Pitx1转基因模型来定义Pitx1表达下游的分子转录途径。在亲本R01的支持下，我们能够证明DUX4确实是PITX1的转录调节因子（Dixit et al., 2007）。此外，在aim2中生成的Pitx1转基因小鼠模型显示肌肉萎缩表型，病理类似于FSHD。在本次修订中，我们提出验证抑制FSHD相关基因的表达将拯救疾病的病理和表型的假设。在新的目标4中，我们提出使用morpholino反义寡核苷酸在细胞培养和体内系统中抑制DUX4， DUX4c和PITX1的表达。目的是评估使用morpholinos作为治疗FSHD的潜在治疗手段的可行性。在目标4A中，我们将首先确定一个合适的细胞模型来测试morpholinos的功效。在目的4B中，我们将通过肌肉注射morpholinos来确定morpholinos的有效性和体内靶基因抑制的效果。在目标4C中，我们将系统地向Pitx1转基因小鼠体内传递抗Pitx1的morpholinos。目的是确定体内morpholinos是否可以抑制转基因并逆转表型，以及确定全身递送的体内morpholinos的毒性。研究结果将为利用反义技术治疗FSHD提供重要信息。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is linked to a shortened 3.3 kb D4Z4 repeat array from 11-100 to 1-10 copies on the subtelomeric region of chromosome 4q35. The shortening of the D4Z4 array is believed to have a de-repression effect on genes in or near the D4Z4 region. In the parent R01, we proposed to test a hypothesized model of FSHD involving direct regulatory relationships between 4q35 deletions, double homeobox protein 4 (DUX4), and Paired-like homeodomain transcription factor 1 (PITX1). In aim 1, we proposed to determine whether Pitx1 was a direct transcriptional target of DUX4 by characterizing a putative DUX4 binding site in the promoter region of Pitx1 gene. In aim 2, we proposed to generate and characterize a conditional muscle-specific Pitx1 transgenic mouse model. In aim 3, we proposed to define molecular transcriptional pathways downstream of Pitx1 expression using the Pitx1 transgenic model. Supported by the parent R01, we were able to showed that DUX4 indeed is a transcriptional regulator of the PITX1 (Dixit et al., 2007). In addition, the Pitx1 transgenic mouse model generated in aim2 showed muscle atrophy phenotype with pathology similar to FSHD. In this revision, we propose to test the hypothesis that repressing the expression of genes involved in FSHD will rescue the pathology and phenotype of the disease. In the new aim 4, we proposed to use morpholino antisense oligos to repress the expression of DUX4, DUX4c and PITX1 using both cell culture and in vivo systems. The goal is to evaluate the feasibility of using morpholinos as a potential therapeutic mean for treating FSHD. In aim 4A, we will first identify a suitable cell model for testing the efficacy of the morpholinos. In aim 4B, we will determine the efficacy of the morpholinos and the effects of target gene suppressing in vivo by intramuscular injection of the morpholinos. In aim 4C, we will systematically deliver vivo-morpholinos against Pitx1 to the Pitx1 transgenic mice. The goal is to determine if the vivo-morpholinos can suppress the transgene and reverse the phenotype, as well as to determine the toxicity of systemic delivered vivo-morpholinos. The findings of the studies will provide important information of using anti-sense technology in treating FSHD. PUBLIC HEALTH RELEVANCE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to a shortened 3.3 kb D4Z4 repeat array from 11-100 to 1-10 copies on the subtelomeric region of chromosome 4q35. The shortening of the D4Z4 array is believed to have a de-repression effect on genes in or near the D4Z4 region. Based on our preliminary data, we developed a pathophysiological model of FSHD involving direct regulatory relationships between 4q35 deletions, double homeobox protein 4 (DUX4), and paired-like homeodomain transcription factor 1 (PITX1). In the parent R01, we proposed to test the hypothesis that DUX4 is a transcription regulator of PITX1 and PITX1 activates genes involved in muscle atrophy in skeletal muscles. In aim 1, we proposed to determine if Pitx1 was a direct target of DUX4 by characterizing a putative DUX4 binding site in the promoter region of Pitx1 gene. We also proposed to identify additional downstream molecular targets of DUX4 in the aim. In aim 2, we proposed to generate and characterize a conditional muscle-specific Pitx1 transgenic mouse model and to test whether the induction of downstream genes involved in muscle wasting depends on the anatomical location of the muscle of the transgenic mice. In aim 3, we proposed to define molecular transcriptional pathways downstream of Pitx1 expression using the Pitx1 transgenic model and to determine whether the effect of Pitx1 over-expression is reversible. This is the third year of the grant and we have demonstrated that DUX4 is a transcriptional regulator of PITX1 (Dixit et al., 2007). In addition, we have generated and characterized the Pitx1 transgenic mouse model (manuscript in preparation). The Pitx1 transgenic animals showed muscle atrophy phenotype and pathology similar to FSHD (preliminary data). In this revision, we propose to test the hypothesis that repressing the expression of genes involved in FSHD will rescue the pathology and phenotype of the disease. In the new aim 4, we propose to use mopholino antisense oligos to repress the expression of DUX4, DUX4c and PITX1 using both cell culture and in vivo systems. The goal is to evaluate the feasibility of using morpholinos as a potential therapeutic mean for treating FSHD. In aim 4A, we will first identify a suitable cell model for testing the efficacy of the morpholinos and potentially other small molecules for developing therapeutic means. In aim 4B, we will determine the efficacy of the morpholinos and the effects of target gene suppressing in vivo by intramuscular injection of the morpholinos. In aim 4C, we will systematically deliver vivo-morpholinos against Pitx1 to the Pitx1 transgenic mice. The goal is to determine if the vivo-morpholinos can suppress the transgene and reverse the phenotype, as well as to determine the toxicity of systemic delivered vivo-morpholinos. The development of effective therapeutic means necessitates an in depth understanding of the cellular and molecular mechanisms mediating muscle atrophy in FSHD. The data generated by the support of the parent R01 supported that a regulatory pathway involving aberrant expression of DUX4, activation of PITX1 by DUX4 and PITX1 downstream genes involved in FSHD. In the new aim, we proposed to suppress the DUX4, DUX4c and PITX1 genes using morpholinos to identify molecules that can potentially used to suppress the translation of these genes in FSHD. Both cell cultures and in vivo systems will be used to determine the efficacy, toxicity, and feasibility of the approach. The studies will also provide insights on molecular functions of these genes and help develop and evaluate a potential cell model for drug screening for FSHD. In this proposal, the old aims and some preliminary data in the original proposal were not included to free up space for the new texts. All the other sections were updated with the additions marked in bold brackets. How the revision will accelerate the tempo of scientific research and allow for job creation and retention. The proposed study will help: 1. To develop and evaluate a cellular model for studying genes over-expressed in FSHD and drug screening. 2. To test the efficacy, toxicity and feasibility of using morpholinos to treat FSHD. 3. To increase understanding of functions of the FSHD candidate genes by suppressing their protein translation in cells and in muscles. To complete these aims, one graduate student (Ms. Vishakha Sharma) will be retained and one research associate will be hired. Ms. Sharma who performed the preliminary studies during her rotation will be able to stay in my laboratory to complete her project proposed in aim 4A and 4B. A research associate will be hired to assist the experiments proposed in the aims with focus on aim 4C.