Motoneuron-selective Rescue of SMA Model Mice

SMA 模型小鼠的运动神经元选择性拯救

• 批准号: 8114311
• 负责人: MENDELL RIMER
• 金额: $ 6.94万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8114311
• 关键词: Accounting; Address; Adenovirus Vector; Affect; Alleles; Animals; Birth; Cells; Cessation of life; Child; Childhood; Code; Complement; Complementary DNA; Cystic Fibrosis; Defect; Disease; Embryo; Embryonic Development; Engineering; Exhibits; Exons; Genes; Genetic Recombination; Genotype; Goals; Hereditary Disease; Human; Human Genome; Infection; Inherited; Knock-out; Mediating; Messenger RNA; Modeling; Motor Neurons; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle denervation procedure; Muscular Atrophy; Mutation; Neonatal; Nervous system structure; Neuromuscular Diseases; Neurons; Organism; Phenotype; Positioning Attribute; Prions; Production; Proteins; RNA; RNA Splicing; Role; SMN2 gene; Screening procedure; Severities; Spinal Muscular Atrophy; Spliceosome Assembly Pathway; Testing; Therapeutic; Time; Transcript; Transgenes; Transgenic Mice; Transgenic Organisms; Type II Spinal Muscular Atrophy; Werdnig-Hoffmann Disease; cell type; high throughput screening; human disease; improved; induced pluripotent stem cell; loss of function mutation; mouse model; nerve supply; novel; offspring; postnatal; prevent; promoter; recombinase; research study; restoration; survival motor neuron gene

DESCRIPTION (provided by applicant): Lower motoneuron death is believed to be the primary defect in spinal muscular atrophy (SMA), a childhood hereditary neuromuscular disease almost as prevalent as cystic fibrosis, perhaps the best-known genetic disease in children. Muscle denervation and atrophy ensue as a result of motoneuron loss. There is no current cure for SMA. Mutations in the survival of motoneuron 1 (SMN1) gene account for SMA. All cells in the body produce SMN and its major function is in spliceosome assembly. Clinically, SMA exhibits several degrees of severity from lethal to mild. This is explained by the presence of a second SMN gene in the human genome (SMN2). SMN2 is essentially identical to SMN1 except for a mutation that causes exon skipping in the splicing of 90% SMN2 RNA, leading to the production of an unstable, minimally functional protein (SMN 7). Only 10% of SMN2 transcripts code for a functional SMN protein. SMN2 can exist in multiple copies, hence the more SMN2 copies the less severe SMA. Although mice only harbor one SMN gene, they have been used to model human SMA by introducing multiple copies of human SMN2. Mouse homozygous for a deletion in their SMN gene (Smn-/-) are embryonic lethal. Smn-/- mice with two copies of SMN2 die around 4-5 days after birth and show features of the most severe human disease (i.e. type I SMA). Addition of a cDNA for SMN?7 to the genotype of type I mice, improves survival to 14 days in average (type II mice). Novel type II mice, in which the targeted Smn allele can be reverted to a functional one following Cre-recombination have been generated. They show similar survival and phenotype as standard type II mice. Type I mice were genetically rescued by crossing them with transgenic mice expressing normal SMN driven by a pan-neuronal promoter. Muscle fiber-specific expression of normal SMN was insufficient to rescue type I mice. Thus, these results indicate that neurons are the targets of SMN deficiency in type I SMA mice but they do not distinguish whether, like in the human disease, the deficiency occurs primarily in motoneurons. If so, restoration of normal SMN levels selectively in motoneurons should have great positive impact on the survival and phenotype of the model SMA mice. Here, we will test this prediction. In Aim 1, we will use transgenic mice that we have generated, in which human SMN expression is driven by the motoneuron-selective Hb9 promoter, to test whether their crossing into type I mice can extend their survival and rescue their SMA-like phenotype. In Aim 2, we will use a complementary approach that will attempt rescuing the novel type II mice by crossing them to Hb9-Cre animals, so that endogenous SMN expression will be restored selectively in motoneurons following Cre inversion of the special Smn targeted allele in these animals. Results from the experiments proposed here will: (i) clarify the role of motoneurons in SMA mouse models, (ii) validate therapeutic approaches that use purified motoneuron cultures in high throughput screening for molecules that increase SMN levels in humans. PUBLIC HEALTH RELEVANCE: Human SMA results from motoneuron loss that is accompanied by muscle atrophy, caused by low levels of SMN protein. Although mouse models of SMA recapitulate many features of the human disease, it is still unclear whether their phenotypes are primarily due to motoneuron deficits. Results from the experiments proposed will clarify the role of motoneurons in SMA mouse models, and validate therapeutic approaches that use purified motoneuron cultures in screening for agents that increase SMN levels in humans.

描述（由申请人提供）：下运动神经元死亡被认为是脊髓性肌萎缩症（SMA）的主要缺陷，SMA是一种儿童遗传性神经肌肉疾病，几乎与囊性纤维化一样普遍，可能是儿童中最知名的遗传性疾病。肌肉失神经支配和萎缩是运动神经元丢失的结果。SMA目前没有治愈方法。运动神经元1（SMN 1）基因的存活突变导致SMA。体内所有细胞都产生SMN，其主要功能是剪接体组装。临床上，SMA表现出从致死到轻度的几种严重程度。这是由于人类基因组中存在第二个SMN基因（SMN 2）。SMN 2与SMN 1基本相同，除了在90% SMN 2 RNA剪接中引起外显子跳跃的突变，导致产生不稳定的功能最低的蛋白质（SMN 7）。只有10%的SMN 2转录物编码功能性SMN蛋白。SMN 2可以以多个拷贝存在，因此SMN 2拷贝越多，SMA的严重程度就越低。尽管小鼠仅携带一个SMN基因，但它们已通过引入多个拷贝的人SMN 2用于模拟人SMA。SMN基因缺失的纯合子小鼠（Smn-/-）具有胚胎致死性。具有两个SMN 2拷贝的Smn-/-小鼠在出生后约4-5天死亡，并显示出最严重的人类疾病（即I型SMA）的特征。添加SMN的cDNA？7与I型小鼠的基因型相比，将存活期平均提高至14天（II型小鼠）。新的II型小鼠，其中靶向的Smn等位基因可以恢复到一个功能性的Cre重组后已经产生。它们显示出与标准II型小鼠相似的存活率和表型。通过将I型小鼠与表达由泛神经元启动子驱动的正常SMN的转基因小鼠杂交，对I型小鼠进行遗传拯救。正常SMN的肌纤维特异性表达不足以拯救I型小鼠。因此，这些结果表明神经元是I型SMA小鼠中SMN缺乏的靶点，但它们不能区分是否与人类疾病一样，缺乏主要发生在运动神经元中。如果是这样的话，选择性恢复运动神经元中的正常SMN水平应该对SMA模型小鼠的存活和表型有很大的积极影响。在这里，我们将测试这个预测。在目标1中，我们将使用我们已经产生的转基因小鼠，其中人SMN表达由运动神经元选择性Hb 9启动子驱动，以测试它们与I型小鼠的杂交是否可以延长它们的生存期并挽救它们的SMA样表型。在目标2中，我们将使用一种互补的方法，该方法将尝试通过将新的II型小鼠与Hb 9-Cre动物杂交来拯救它们，使得在这些动物中的特殊Smn靶向等位基因的Cre反转后，内源性SMN表达将在运动神经元中选择性地恢复。本文提出的实验结果将：（i）阐明运动神经元在SMA小鼠模型中的作用，（ii）验证在高通量筛选中使用纯化的运动神经元培养物的治疗方法，以寻找增加人类SMN水平的分子。 公共卫生相关性：人类SMA是由运动神经元丢失引起的，伴有低水平SMN蛋白引起的肌肉萎缩。尽管SMA的小鼠模型概括了人类疾病的许多特征，但仍不清楚其表型是否主要归因于运动神经元缺陷。所提出的实验结果将阐明运动神经元在SMA小鼠模型中的作用，并验证使用纯化的运动神经元培养物筛选增加人类SMN水平的药物的治疗方法。