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: 8581873
• 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 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8581873
• 关键词: Motoneuron; selective; Rescue; SMA; Model

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.

描述（由申请人提供）：据信较低的运动神经元死亡是脊柱肌肉萎缩（SMA）的主要缺陷，这是一种童年的遗传神经肌肉疾病，几乎与囊性纤维化一样普遍，也许是儿童中最著名的遗传病。由于运动神经元丧失而导致肌肉神经神经支配和萎缩。 SMA目前没有治疗方法。运动神经元1（SMN1）基因存活中的突变为SMA。体内的所有细胞都会产生SMN，其主要功能在剪接体组装中。在临床上，SMA表现出从致命到轻度的几个严重程度。这是通过人类基因组中的第二个SMN基因的存在来解释的（SMN2）。 SMN2基本与SMN1相同，除了导致外显子跳过90％SMN2 RNA的突变，导致产生不稳定的，最小功能的蛋白质（SMN 7）。功能性SMN蛋白的SMN2转录物代码中只有10％。 SMN2可以以多个副本存在，因此SMN2的副本越严重。尽管小鼠只有一个SMN基因，但它们已通过引入人类SMN2的多个副本来对人类SMA进行建模。小鼠在其SMN基因（SMN - / - ）中缺失的纯合子是胚胎致死的。 SMN - / - 带有两份SMN2副本的小鼠在出生后4-5天左右死亡，并显示出最严重的人类疾病的特征（即I型SMA）。在I型小鼠的基因型中添加了SMN？7的cDNA，将生存率提高到平均14天（II型小鼠）。新型II型小鼠，可以将靶向的SMN等位基因恢复为功能性的CRE重组后的功能性。它们显示出与标准II型小鼠相似的生存和表型。 I型小鼠是通过与表达由泛神经元启动子驱动的正常SMN的转基因小鼠跨越基因挽救的。正常SMN的肌肉纤维特异性表达不足以营救I型小鼠。因此，这些结果表明，神经元是I型SMA小鼠中SMN缺乏症的靶标，但它们没有区分像在人类疾病中，缺乏主要发生在运动神经元中。如果是这样，在运动神经元中有选择性地恢复正常的SMN水平应对模型SMA小鼠的存活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AIM 1中，我们将使用我们已经产生的转基因小鼠，其中人类SMN表达是由运动神经元选择性HB9启动子驱动的，以测试它们进入I型小鼠是否可以扩展其存活并营救其SMA样表型。在AIM 2中，我们将使用一种补充方法，该方法将尝试通过将它们越过HB9-CRE动物挽救新型II型小鼠，以便在这些动物的特殊SMN靶向后，内源性SMN表达将在运动神经元中有选择地恢复。此处提出的实验的结果将：（i）阐明运动神经元在SMA小鼠模型中的作用，（ii）验证在高吞吐量筛选中使用纯化的运动神经元培养物的治疗方法，以增加增加人类SMN水平的分子。