Molecular Pathphysiology of FSHD muscular dystrophy via genome-wide approaches

通过全基因组方法研究 FSHD 肌营养不良症的分子病理生理学

• 批准号: 7817383
• 负责人: YI-WEN CHEN
• 金额: $ 38.77万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-28 至 2011-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7817383
• 关键词: 4q35; Animals; Anterior; Apoptosis; Binding Sites; Candidate Disease Gene; Cell Culture Techniques; Cell Proliferation; Cell model; Cells; Chromosomes; D4Z4; Data; Development; Facioscapulohumeral Muscular Dystrophy; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Goals; Grant; Homeodomain Proteins; Human; Immunoblotting; Injection of therapeutic agent; Intramuscular Injections; Laboratories; Leg; Link; Location; Manuscripts; Mediating; Modeling; Molecular; Molecular Target; Mus; Muscle; Muscular Atrophy; Muscular Dystrophies; Myoblasts; Occupations; Oxidative Stress; Parents; Pathology; Pathway interactions; Phenotype; Placebos; Postdoctoral Fellow; Preclinical Drug Evaluation; Predisposition; Preparation; Promoter Regions; Proteins; RNA; Regulatory Pathway; Repression; Research; Reverse Transcriptase Polymerase Chain Reaction; Rhabdomyosarcoma; Rotation; Skeletal Muscle; System; Technology; Testing; Text; Therapeutic; Toxic effect; Transgenes; Transgenic Animals; Transgenic Mice; Transgenic Model; Translations; Update; base; disease phenotype; efficacy testing; gene function; genome-wide; graduate student; homeobox protein PITX1; in vivo; insight; intravenous injection; mouse model; public health relevance; research study; small molecule; wasting

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.

描述（由申请人提供）：Faciosculohumeral肌肉营养不良症（FSHD）与4Q35染色体的亚电体区域上的3.3 kb D4Z4重复阵列相关。据信D4Z4阵列的缩短对D4Z4区域内或附近的基因具有去抑制作用。在母体R01中，我们提议测试一个假设的FSHD模型，该模型涉及4Q35缺失，双同子蛋白4（DUX4）和成对的同源域转录因子1（PITX1）之间的直接调节关系。在AIM 1中，我们建议通过表征PITX1基因启动子区域中假定的DUX4结合位点来确定PITX1是否是DUX4的直接转录靶标。在AIM 2中，我们建议生成和表征有条件的肌肉特异性PITX1转基因小鼠模型。在AIM 3中，我们建议使用PITX1转基因模型定义PITX1表达下游的分子转录途径。在母体R01的支持下，我们能够证明Dux4确实是PITX1的转录调节剂（Dixit等，2007）。此外，在AIM2中产生的PITX1转基因小鼠模型显示出与FSHD相似的病理学的肌肉萎缩表型。在这项修订中，我们建议检验以下假设：抑制FSHD涉及的基因的表达将挽救该疾病的病理和表型。在新目标4中，我们提议使用Morpholino反义寡寡做寡素来使用细胞培养和体内系统抑制DUX4，DUX4C和PITX1的表达。目的是评估使用吗啡作为治疗FSHD的潜在治疗平均值的可行性。在AIM 4A中，我们将首先确定一个合适的细胞模型，以测试形态学的功效。在AIM 4B中，我们将确定吗啡的疗效以及靶基因在体内通过肌电内注入吗啡的作用。在AIM 4C中，我们将系统地向Pitx1转基因小鼠提供针对PITX1的体内 - 甲酚。目的是确定体内 - 多磷脂是否可以抑制转基因并逆转表型，并确定全身递送的体内 - 莫尔苯甲酸的毒性。研究结果将提供有关使用反义技术治疗FSHD的重要信息。 公共卫生相关性：Facioscapulohumeral肌肉营养不良（FSHD）与缩短的3.3 kb D4Z4重复阵列从11-100到1-10份在4q35染色体的亚电体区域上的副本。据信D4Z4阵列的缩短对D4Z4区域内或附近的基因具有去抑制作用。根据我们的初步数据，我们开发了FSHD的病理生理模型，涉及4Q35缺失，双同子蛋白4（DUX4）和成对的同源域转录因子1（PITX1）之间的直接调节关系。在母体R01中，我们提出了以下假设：Dux4是PITX1和PITX1的转录调节剂，激活了骨骼肌肌肉萎缩的基因。在AIM 1中，我们建议通过表征PITX1基因启动子区域中推定的DUX4结合位点来确定PITX1是否是DUX4的直接靶标。我们还建议在目标中确定DUX4的其他下游分子靶标。在AIM 2中，我们提议生成和表征有条件的肌肉特异性PITX1转基因小鼠模型，并测试诱导参与肌肉浪费的下游基因是否取决于转基因小鼠肌肉的解剖位置。在AIM 3中，我们建议使用PITX1转基因模型来定义PITX1表达下游的分子转录途径，并确定PITX1过表达的影响是否可逆。这是赠款的第三年，我们证明了Dux4是Pitx1的转录调节剂（Dixit等，2007）。此外，我们已经生成并表征了PITX1转基因小鼠模型（制备中的手稿）。 PITX1转基因动物显示出类似于FSHD（初步数据）的肌肉萎缩表型和病理。在这项修订中，我们建议检验以下假设：抑制FSHD涉及的基因的表达将挽救该疾病的病理和表型。 在新的目标4中，我们建议使用摩托哥反义寡素寡素寡素，以使用细胞培养和体内系统抑制Dux4，dux4c和pitx1的表达。目的是评估使用吗啡作为治疗FSHD的潜在治疗平均值的可行性。在AIM 4A中，我们将首先确定一个合适的细胞模型，以测试形态学和其他小分子在开发治疗手段的功效。在AIM 4B中，我们将确定吗啡的疗效以及靶基因在体内通过肌电内注入吗啡的作用。在AIM 4C中，我们将系统地向Pitx1转基因小鼠提供针对PITX1的体内 - 甲酚。目的是确定体内 - 多磷脂是否可以抑制转基因并逆转表型，并确定全身递送的体内 - 莫尔苯甲酸的毒性。 有效治疗手段的发展需要深入了解介导FSHD中肌肉萎缩的细胞和分子机制。由父R01支持生成的数据支持涉及DUX4异常表达的调节途径，DUX4激活PITX1和涉及FSHD的PITX1下游基因。在新目标中，我们提出使用形态学来抑制Dux4，Dux4c和Pitx1基因，以鉴定有可能用于抑制FSHD中这些基因翻译的分子。细胞培养物和体内系统都将用于确定该方法的功效，毒性和可行性。这些研究还将提供有关这些基因分子功能的见解，并有助于开发和评估用于FSHD药物筛查的潜在细胞模型。 在此提案中，不包括原始提案中的旧目标和一些初步数据，以释放新文本的空间。所有其他部分都以粗体括号标记的添加进行了更新。修订将如何加速科学研究的速度并允许创造就业和保留。拟议的研究将有助于：1。开发和评估一个细胞模型，以研究在FSHD和药物筛查中过表达的基因。 2。测试使用吗啡治疗FSHD的功效，毒性和可行性。 3。通过抑制细胞和肌肉中的蛋白质翻译来增加对FSHD候选基因功能的理解。为了完成这些目标，将保留一名研究生（Vishakha Sharma女士），并将雇用一名研究助理。夏尔马女士在轮换期间进行了初步研究，将能够留在我的实验室中，以完成她在AIM 4A和4B中提议的项目。将雇用研究助理，以协助目标4C的目标中提出的实验。