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）：面肩肱型肌营养不良症（FSHD）与染色体 4q35 亚端粒区域上从 11-100 到 1-10 个拷贝的缩短的 3.3 kb D4Z4 重复阵列相关。 D4Z4 阵列的缩短被认为对 D4Z4 区域内或附近的基因具有去抑制作用。在亲本 R01 中，我们建议测试 FSHD 的假设模型，该模型涉及 4q35 缺失、双同源盒蛋白 4 (DUX4) 和配对样同源域转录因子 1 (PITX1) 之间的直接调控关系。在目标 1 中，我们提出通过表征 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。在目标 2 中，我们建议生成并表征条件肌肉特异性 Pitx1 转基因小鼠模型。在目标 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 等，2007）。此外，在aim2中生成的Pitx1转基因小鼠模型显示出肌肉萎缩表型，其病理学类似于FSHD。在本次修订中，我们建议检验以下假设：抑制 FSHD 相关基因的表达将挽救该疾病的病理学和表型。在新目标 4 中，我们建议使用吗啉代反义寡核苷酸通过细胞培养和体内系统来抑制 DUX4、DUX4c 和 PITX1 的表达。目的是评估使用吗啉作为治疗 FSHD 的潜在治疗手段的可行性。在目标 4A 中，我们将首先确定合适的细胞模型来测试吗啉代的功效。在目标4B中，我们将通过肌肉注射吗啉来确定吗啉的功效以及体内靶基因抑制的效果。在目标 4C 中，我们将系统地将针对 Pitx1 的体内吗啉递送至 Pitx1 转基因小鼠。目标是确定体内吗啉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吗啉的毒性。研究结果将为利用反义技术治疗FSHD提供重要信息。 公共健康相关性：面肩肱型肌营养不良症 (FSHD) 与染色体 4q35 亚端粒区域上缩短的 3.3 kb D4Z4 重复序列（从 11-100 到 1-10 个拷贝）有关。 D4Z4 阵列的缩短被认为对 D4Z4 区域内或附近的基因具有去抑制作用。基于我们的初步数据，我们开发了 FSHD 的病理生理学模型，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。在母体 R01 中，我们提出检验以下假设：DUX4 是 PITX1 的转录调节因子，并且 PITX1 激活参与骨骼肌萎缩的基因。在目标 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 的直接靶标。我们还建议在该目标中确定 DUX4 的其他下游分子靶标。在目标 2 中，我们建议生成并表征条件肌肉特异性 Pitx1 转基因小鼠模型，并测试参与肌肉萎缩的下游基因的诱导是否取决于转基因小鼠肌肉的解剖位置。在目标 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，并确定 Pitx1 过度表达的影响是否可逆。这是该资助的第三年，我们已经证明 DUX4 是 PITX1 的转录调节因子（Dixit 等，2007）。此外，我们还生成并表征了 Pitx1 转基因小鼠模型（手稿正在准备中）。 Pitx1转基因动物表现出与FSHD相似的肌肉萎缩表型和病理学（初步数据）。在本次修订中，我们建议检验以下假设：抑制 FSHD 相关基因的表达将挽救该疾病的病理学和表型。 在新目标 4 中，我们建议利用细胞培养和体内系统，使用 mopholino 反义寡核苷酸来抑制 DUX4、DUX4c 和 PITX1 的表达。目的是评估使用吗啉作为治疗 FSHD 的潜在治疗手段的可行性。在目标 4A 中，我们将首先确定合适的细胞模型，用于测试吗啉代和其他潜在小分子的功效，以开发治疗手段。在目标4B中，我们将通过肌肉注射吗啉来确定吗啉的功效以及体内靶基因抑制的效果。在目标 4C 中，我们将系统地将针对 Pitx1 的体内吗啉递送至 Pitx1 转基因小鼠。目标是确定体内吗啉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吗啉的毒性。 开发有效的治疗手段需要深入了解介导 FSHD 肌肉萎缩的细胞和分子机制。亲本 R01 支持产生的数据支持涉及 FSHD 的 DUX4 异常表达、DUX4 激活 PITX1 以及 PITX1 下游基因的调控途径。在新的目标中，我们建议使用吗啉抑制 DUX4、DUX4c 和 PITX1 基因，以鉴定可潜在用于抑制 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的分子功能的见解，并帮助开发和评估用于 FSHD 药物筛选的潜在细胞模型。 在该提案中，原提案中的旧目标和一些初步数据未包括在内，以便为新案文腾出空间。所有其他部分均已更新，添加内容以粗体括号标记。此次修订将如何加快科学研究的节奏并创造和保留就业机会。拟议的研究将有助于： 1. 开发和评估用于研究 FSHD 和药物筛选中过度表达的基因的细胞模型。 2. 检验吗啉类药物治疗FSHD的疗效、毒性及可行性。 3. 通过抑制细胞和肌肉中的 FSHD 候选基因的蛋白质翻译来增加对 FSHD 候选基因功能的了解。为了实现这些目标，将保留一名研究生（Vishakha Sharma 女士）并聘用一名研究员。在轮换期间进行初步研究的 Sharma 女士将能够留在我的实验室完成她在目标 4A 和 4B 中提出的项目。将聘请一名研究助理来协助目标中提出的实验，重点关注目标 4C。