Conditional Genetics Rescue of Angelman Syndrome

描述（由申请人提供）：当母系遗传的15 q11 -13缺失或印迹15 q11 -13基因Ube 3a突变时，会导致Angelman综合征。当母亲的15 q11 -13是三重的时候，自闭症就会出现。父母的遗传模式是由Ube 3a解释的，Ube 3a是唯一的15 q11 -13基因，在神经元中仅从母亲等位基因表达。相反的15 q紊乱则表现出一些截然不同的行为变化。安格尔曼综合征表现为智力和运动缺陷，但也有超社会行为，而15 q三联体则表现为社会行为受损。有趣的是，除了其他先前表征的缺陷之外，我们最近确定了Angelman综合征小鼠显示出增加的社会互动和超声波发声。相比之下，将Ube 3a基因剂量增加两倍会减少社交互动和发声，从而提供自闭症模型。结果建立了Ube 3a基因剂量和社会行为的Goldilock效应。相互基因剂量依赖效应也表明，一些Ube 3a效应可能来自持续的调控，而不是远程发育缺陷。Ube 3a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Angelman综合征中Ube 3a缺陷通过持续的调节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Ube 3a在成年期被基因重新激活时行为和回路缺陷的逆转。为了实现这一目标，我们产生了携带单个额外拷贝的Ube 3a-ON转基因的小鼠，该转基因被loXP侧翼的STOP盒沉默。Ube 3a-ON转基因是无活性的，直到loxP侧翼的STOP盒被Cre重组酶删除。将Ube 3a-ON转基因小鼠与Cre-ER-融合蛋白转基因小鼠杂交能够实现他莫昔芬诱导的Ube 3a基因表达。ERTM是突变的，对他莫昔芬有反应，但对雌激素无反应。Cre重组酶通过ERTM与细胞质中的热休克蛋白连接。他莫昔芬穿透CNS并结合ERTM，允许Cre进入细胞核并删除loxP侧翼的STOP盒。如果没有达到成人拯救，我们将进行时间映射到出生后或胚胎发育期。我们还创建了Ube 3a-OFF转基因，其中loxP序列位于Ube 3a外显子2的侧翼，因此他莫昔芬导致Cre-ERTM抑制Ube 3a。将Ube 3a-OFF/Cre-ERTM与Angelman模型交叉，我们将确定在成年期或特定发育时期失活Ube 3a是否会重新产生疾病。该结果将确立挽救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.