Conditional Genetics Rescue of Angelman Syndrome

描述(申请人提供)：当母系遗传的15q11-13缺失或印记15q11-13基因Ube3a突变时，会导致Angelman综合征。当母亲的15q11-13是三重时，就会导致自闭症。父母的遗传模式可以用Ube3a来解释，Ube3a是唯一一个在神经元中只表达母系等位基因的15q11-13基因。相反的15Q障碍表现出一些截然不同的行为变化。安杰曼综合征表现为智力和运动障碍，但也是一种超社交行为，而15q三联反应显示出社交行为受损。有趣的是，除了以前的其他特征缺陷外，我们最近建立了Angelman综合征小鼠表现出更多的社会互动和超声波发声。相比之下，Ube3a基因剂量增加两倍，减少了社交互动和发声，提供了一个自闭症模型。这一结果证实了Ube3a基因剂量和社会行为的金发效应。互惠的基因剂量依赖效应还认为，一些Ube3a效应可能是由持续的调控缺陷而不是远程发育缺陷引起的。UBE3A作为E3泛素蛋白连接酶促进蛋白质降解，并作为核受体共激活剂。为了验证安杰曼综合症中Ube3a缺陷通过持续的调节缺陷而不是发育缺陷导致行为和回路功能缺陷的假设，我们将测试当Ube3a在成年后遗传重新激活时行为和回路缺陷的逆转。为了实现这一目标，我们产生了携带一个额外的Ube3a-on转基因拷贝的小鼠，该转基因被一个loxP侧翼的Stop卡带沉默。Ube3a-on转基因在loxP侧翼的终止盒被Cre重组酶删除之前是无效的。将Ube3a-on转基因小鼠与Cre-ER“融合蛋白转基因小鼠杂交，可使他莫昔芬诱导的Ube3a基因表达。ERTM发生突变，对他莫昔芬有反应，但对雌激素没有反应。Cre重组酶通过ERTM与细胞质中的热休克蛋白结合。他莫昔芬穿透中枢神经系统并结合ERTM，允许Cre进入细胞核并删除loxP侧翼的终止盒。如果没有成人救援，我们将进行出生后或胚胎发育期的时间映射。我们还创建了一个Ube3a-off转基因，其中loxP序列位于Ube3a外显子2的两侧，因此他莫昔芬导致Cre-ERTM灭活Ube3a。将Ube3a-Off/Cre-ERTM纳入Angelman模型，我们将确定在成年期或特定发育时期灭活Ube3a是否会重现这种障碍。这一结果将确立抢救Angelman综合征的可行性，并为该疾病的病理生理基础和治疗方案提供见解。

DESCRIPTION (provided by applicant): Angelman syndrome results when maternally-inherited 15q11-13 is deleted or imprinted 15q11-13 gene Ube3a is mutated. Autism results when maternal 15q11-13 is triplicated. The parental inheritance patterns are explained by Ube3a the only 15q11-13 gene expressed solely from the maternal allele in neurons. The countervalent 15q disorders display some contrasting behavioral changes. Angelman syndrome presents with intellectual and motor deficits, but also a hypersocial demeanor, while 15q triplication displays impaired social behavior. Interestingly, in additional to the other previousl characterized deficits, we recently established the Angelman syndrome mice display increased social interaction and ultrasonic vocalization. By contrast, tripling Ube3a gene dosage reduced social interaction and vocalization providing a model of autism. The results establish a Goldilock effect for Ube3a gene dosage and social behavior. Reciprocal gene dose-dependent effects also argue some Ube3a effects could arise from ongoing regulatory rather than remote developmental defects. Ube3a acts as an E3 ubiquitin protein ligase to promote protein degradation and as a nuclear receptor co-activator. To test the hypothesis that Ube3a deficiency in Angelman syndrome causes deficits in behavior and circuit function via ongoing regulatory rather than developmental defects, we will test for reversal of the behavioral and circuit deficits when Ube3a is genetically reactivated in adulthood. To achieve this goal, we generated mice carrying a single extra copy of Ube3a-ON transgene that is silenced by a loxP-flanked STOP cassette. The Ube3a-ON transgene is inactive until the loxP-flanked STOP cassette is deleted by Cre recombinase. Crossing Ube3a-ON transgenic to Cre-ER" fusion protein transgenic mice enables tamoxifen- induced Ube3a gene expression. ERTM is mutated, responding to tamoxifen but not estrogen. Cre recombinase is tethered to heat shock proteins in the cytoplasm by ERTM. Tamoxifen penetrates the CNS and binds ERTM, permitting Cre to enter the nucleus and delete the loxP-flanked STOP cassette. If adult rescue is not achieved, we will perform temporal mapping to postnatal or embryonic developmental periods. We also created an Ube3a-OFF transgene where loxP sequences flank Ube3a exon 2 so tamoxifen causes Cre-ERTM to inactivate Ube3a. Crossing Ube3a-OFF/Cre-ERTM into the Angelman model, we will determine if inactivating Ube3a in adulthood or at specific developmental times recreates the disorder. The result will establish the feasibility of rescuing Angelman syndrome and provide insights into the pathophysiological basis and treatment options for the disorder.