Motoneuron-selective Rescue of SMA Model Mice

描述(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，可能是儿童中最著名的遗传病。运动神经元丢失会导致肌肉失神经和萎缩。目前还没有治疗SMA的方法。运动神经元1(SMN1)基因的生存突变是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。临床上，SMA表现出从致命到轻度的几种严重程度。这是因为人类基因组中存在第二个SMN基因(SMN2)。SMN2与SMN1基本相同，除了一个突变导致90%SMN2RNA剪接中的外显子跳过，导致产生一种不稳定的、功能最低的蛋白(SMN 7)。只有10%的SMN转录本编码具有功能的SMN蛋白。SMN2可以存在于多个拷贝中，因此SMN2拷贝越多，SMA就越不严重。虽然小鼠只有一个SMN基因，但它们已经被用来通过引入多个拷贝的人SMN来建立人类SMA的模型。纯合子小鼠的SMN基因缺失(SMN-/-)是胚胎致死的。携带两个SMN副本的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。在I型小鼠的基因中添加SMN？7的cDNA，可将平均存活时间提高到14天(II型小鼠)。已经产生了新的II型小鼠，在这些小鼠中，目标SMN等位基因可以在Cre重组后恢复到功能上的等位基因。它们的存活率和表型与标准的II型小鼠相似。通过将I类小鼠与由泛神经元启动子驱动的表达正常SMN的转基因小鼠杂交，I类小鼠得到了遗传拯救。正常SMN的肌纤维特异性表达不足以挽救I型小鼠。因此，这些结果表明，在I型SMA小鼠中，神经元是SMN缺陷的目标，但它们没有区分是否像人类疾病一样，这种缺陷主要发生在运动神经元。如果是这样的话，选择性地恢复运动神经元中正常的SMN水平将对SMA模型小鼠的生存和表型产生巨大的积极影响。在这里，我们将检验这一预测。在目标1中，我们将使用我们已经建立的转基因小鼠，在其中，人SMN的表达是由运动神经元选择性HB9启动子驱动的，以测试它们与I型小鼠的杂交是否可以延长它们的生存时间，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，试图通过将新的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定的SMN靶向等位基因Cre倒置后，内源性SMN在运动神经元中的表达将有选择地恢复。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)验证使用纯化运动神经元培养在高通量筛选增加人类SMN水平的分子中的治疗方法。 公共卫生相关性：人类SMA是由于运动神经元丢失并伴有肌肉萎缩，由低水平的SMN蛋白引起。尽管SMA小鼠模型概括了人类疾病的许多特征，但尚不清楚它们的表型是否主要是由于运动神经元缺陷所致。拟议的实验结果将阐明运动神经元在SMA小鼠模型中的作用，并验证使用纯化运动神经元培养筛选提高人类SMN水平的药物的治疗方法。

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.