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，脊髓性肌萎缩症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，囊性纤维化可能是儿童中最著名的遗传病。由于运动神经元损失，导致肌肉去神经支配和萎缩。目前 SMA 尚无治愈方法。运动神经元 1 (SMN1) 基因存活突变导致 SMA。体内所有细胞都会产生 SMN，其主要功能是剪接体组装。临床上，SMA 表现出从致命到轻微的多种严重程度。这是通过人类基因组中存在第二个 SMN 基因 (SMN2) 来解释的。 SMN2 本质上与 SMN1 相同，只是有一个突变导致 90% SMN2 RNA 的剪接中外显子跳跃，从而导致产生不稳定的、功能最低限度的蛋白质 (SMN 7)。只有 10% 的 SMN2 转录本编码功能性 SMN 蛋白。 SMN2 可以存在多个拷贝，因此 SMN2 拷贝越多，SMA 的严重程度就越轻。尽管小鼠只携带一种 SMN 基因，但它们已通过引入多个人类 SMN2 拷贝来模拟人类 SMA。 SMN 基因 (Smn-/-) 缺失的纯合小鼠胚胎致死。携带两个 SMN2 拷贝的 Smn-/- 小鼠在出生后 4-5 天左右死亡，并表现出最严重的人类疾病（即 I 型 SMA）的特征。将 SMN?7 的 cDNA 添加到 I 型小鼠的基因型中，可将平均存活时间提高至 14 天（II 型小鼠）。新型 II 型小鼠已经诞生，其中目标 Smn 等位基因在 Cre 重组后可以恢复为功能性基因。它们表现出与标准 II 型小鼠相似的存活率和表型。 I 型小鼠通过与表达由泛神经元启动子驱动的正常 SMN 的转基因小鼠杂交而获得基因拯救。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 SMN 缺陷的目标，但它们没有区分是否像人类疾病一样，缺陷主要发生在运动神经元中。如果是这样，在运动神经元中选择性恢复正常的 SMN 水平应该会对 SMA 小鼠模型的生存和表型产生巨大的积极影响。在这里，我们将测试这个预测。在目标 1 中，我们将使用我们生成的转基因小鼠（其中人类 SMN 表达由运动神经元选择性 Hb9 启动子驱动）来测试它们与 I 型小鼠的杂交是否可以延长其生存并挽救其 SMA 样表型。在目标 2 中，我们将使用一种补充方法，尝试通过将新型 II 型小鼠与 Hb9-Cre 动物杂交来拯救它们，以便在这些动物中特殊 Smn 靶向等位基因发生 Cre 反转后，运动神经元中的内源 SMN 表达将选择性地恢复。这里提出的实验结果将：(i) 阐明运动神经元在 SMA 小鼠模型中的作用，(ii) 验证使用纯化的运动神经元培养物高通量筛选增加人类 SMN 水平的分子的治疗方法。 公共健康相关性：人类 SMA 是由运动神经元损失引起的，运动神经元损失伴有肌肉萎缩，而肌肉萎缩是由 SMN 蛋白水平低引起的。尽管 SMA 小鼠模型重现了人类疾病的许多特征，但仍不清楚它们的表型是否主要是由于运动神经元缺陷所致。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提高人类 SMN 水平的药物的治疗方法。