Conditional Genetics Rescue of Angelman Syndrome

天使综合症的条件遗传学拯救

• 批准号: 8860218
• 负责人: MATTHEW P ANDERSON
• 金额: $ 20.36万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8860218
• 关键词: 15q; Adult; Age; Alleles; Angelman Syndrome; Autistic Disorder; Bacterial Artificial Chromosomes; Behavior; Behavioral; Binding; Brain; Cell Nucleus; Chimeric Proteins; Chromosomes, Human, Pair 15; Cognitive; Conceptions; Cytoplasm; Defect; Development; Disease; Dose; Embryo; Estrogens; Event; Exons; Eye; Functional disorder; Future; Gene Dosage; Gene Expression; Genes; Genetic; Goals; Health; Heat shock proteins; ILK gene; Individual; Inheritance Patterns; Inherited; Isodicentric Chromosome; Knock-out; Laughter; Length; Location; Maps; Modeling; Motor; Mus; Mutate; Neurons; Nuclear Receptors; Phenotype; Physiological; Proteins; RNA Splicing; Smiling; Social Behavior; Social Interaction; Synapses; Tamoxifen; Testing; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Ultrasonics; base; imprint; insight; motor deficit; mouse model; multicatalytic endopeptidase complex; neurobehavioral; neuronal circuitry; neurophysiology; neuropsychiatry; novel; postnatal; protein degradation; recombinase; social communication; therapy development; trait; ubiquitin-protein ligase; vector; vocalization

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.

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，会导致天使综合征。当母亲 15q11-13 重复三倍时，就会导致自闭症。 Ube3a 解释了亲本遗传模式，Ube3a 是神经元中唯一仅由母体等位基因表达的 15q11-13 基因。相反的 15q 疾病表现出一些对比鲜明的行为变化。 Angelman 综合征表现为智力和运动缺陷，但也表现出过度社交行为，而 15q 三倍体则表现出社交行为受损。有趣的是，除了之前描述的其他缺陷之外，我们最近发现天使综合征小鼠表现出社交互动和超声波发声的增加。相比之下，将 Ube3a 基因剂量增加三倍会减少社交互动和发声，从而提供了自闭症模型。结果建立了 Ube3a 基因剂量和社会行为的金发姑娘效应。相互基因剂量依赖性效应也认为一些 Ube3a 效应可能是由持续的调节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。为了检验天使综合征中 Ube3a 缺陷通过持续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 Ube3a 在成年后基因重新激活时行为和回路功能缺陷的逆转。为了实现这一目标，我们培育了携带一个额外的 Ube3a-ON 转基因拷贝的小鼠，该转基因拷贝被 loxP 侧翼的 STOP 盒沉默。 Ube3a-ON 转基因处于非活性状态，直至 loxP 侧翼的 STOP 盒被 Cre 重组酶删除。将 Ube3a-ON 转基因小鼠与 Cre-ER”融合蛋白转基因小鼠杂交，可实现他莫昔芬诱导的 Ube3a 基因表达。ERTM 发生突变，对他莫昔芬有反应，但对雌激素无反应。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 CNS 并与 ERTM 结合，使 Cre 进入 核并删除 loxP 侧翼的 STOP 盒。如果成人救援未能实现，我们将进行出生后或胚胎发育时期的时间映射。我们还创建了 Ube3a-OFF 转基因，其中 loxP 序列位于 Ube3a 外显子 2 侧翼，因此他莫昔芬会导致 Cre-ERTM 灭活 Ube3a。将 Ube3a-OFF/Cre-ERTM 交叉到 Angelman 模型中， 我们将确定在成年期或特定发育时期使 Ube3a 失活是否会重现该疾病。该结果将确立拯救天使综合征的可行性，并为该疾病的病理生理学基础和治疗方案提供见解。