Motoneuron-selective Rescue of SMA Model Mice

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，可能是儿童中最著名的遗传性疾病。肌肉失神经支配和萎缩是运动神经元丧失的结果。目前还没有治愈SMA的方法。运动神经元1 （SMN1）基因存活的突变是导致SMA的原因。体内所有细胞都产生SMN，其主要功能是剪接体组装。在临床上，SMA表现出从致命到轻度的严重程度。这可以通过人类基因组中第二个SMN基因（SMN2）的存在来解释。SMN2基本上与SMN1相同，除了一个突变导致90% SMN2 RNA剪接中的外显子跳变，导致产生不稳定的、功能最低的蛋白质（smn7）。只有10%的SMN2转录本编码一个功能性的SMN蛋白。SMN2可以存在多个拷贝，因此SMN2拷贝越多，SMA的严重程度越低。虽然小鼠只携带一个SMN基因，但它们已被用于通过引入人类SMN2的多个拷贝来模拟人类SMA。小鼠SMN基因纯合缺失（SMN -/-）是胚胎致死的。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。为SMN添加cDNA ？7对基因型ⅰ型小鼠，提高生存期至平均14天（ⅱ型小鼠）。在新型II型小鼠中，目标Smn等位基因可以在cre重组后恢复为功能等位基因。它们表现出与标准II型小鼠相似的存活率和表型。通过将I型小鼠与泛神经元启动子驱动的表达正常SMN的转基因小鼠杂交，在基因上拯救了它们。肌纤维特异性表达正常的SMN不足以挽救I型小鼠。因此，这些结果表明神经元是I型SMA小鼠中SMN缺乏的目标，但它们并没有区分是否像在人类疾病中一样，这种缺乏主要发生在运动神经元中。如果是这样，有选择地恢复运动神经元中正常的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，我们将测试这一预测。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的表达由运动神经元选择性Hb9启动子驱动，来测试将它们杂交到I型小鼠中是否可以延长它们的生存时间并挽救它们的sma样表型。在Aim 2中，我们将使用一种补充方法，通过将新型II型小鼠与Hb9-Cre动物杂交来挽救它们，以便在这些动物的特殊SMN靶向等位基因的Cre反转后，在运动神经元中选择性地恢复内源性SMN表达。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）验证使用纯化运动神经元培养物进行高通量筛选以增加人类SMN水平的分子的治疗方法。

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.