Towards Gene Therapy for Angelman Syndrome

描述(由申请人提供)：这项建议将开发一种潜在的安杰曼综合征(AS)的基因疗法。AS的根本原因是UBE3A基因母体拷贝的丢失。在大脑的大多数区域，父亲的等位基因是完整的，但在表观遗传上是沉默的。我们的目标是激活沉默的UBE3A等位基因，以改善AS的临床表现。我们正在开发基于锌指的人工转录因子(ATF)，旨在激活培养的小鼠神经细胞中小鼠Ube3a的表达。在特定目标1中，我们将创建直接激活小鼠Ube3a转录或抑制小鼠Ube3a反义转录(ATS)表达的ATF。里程碑式的目标将是创建一个或多个ATF，可以激活至少2倍的Ube3a表达。已经构建并测试了几个ATF，我们的初步数据显示，其中几个能够上调内源性Ube3a蛋白水平。在特定的目标2中，ATF将被包装成AAV病毒载体，并被输送到AS小鼠的大脑中。我们将检测ATF在脑内的分布和表达，以及它对Ube3a表达的影响。具有里程碑意义的目标是实现ATF在注射的脑结构中的广泛神经元转导和表达(&gt；10%的海马细胞，以及一些周围组织的转导)。一个或多个ATF预计会导致父亲等位基因Ube3a的表达至少增加2倍。这些里程碑的成功实现将证明扩大临床前研究和开发ATF到人类UBE3A是合理的。 与公共卫生相关：安杰曼综合征(AS)是一种神经遗传性疾病，会导致发育迟缓和神经损伤，如缺乏语言。目前，没有治愈的方法，也没有特效的药物治疗。ATF激活沉默的父亲UBE3A等位基因可以解决导致AS的根本遗传缺陷，因此是一种潜在的治愈基因疗法。这项提议将在小鼠模型中研究这种疗法。

DESCRIPTION (provided by applicant): This proposal will develop of a potential gene therapy for Angelman syndrome (AS). The root cause of AS is the loss of the maternal copy of the gene UBE3A. In most regions of the brain, the paternal allele is intact, but is epigenetically silenced. Our goal is to activate of the silenced UBE3A allele to ameliorate the clinical manifestations of AS. We are developing zinc finger-based artificial transcription factors (ATFs) designed to activate the expression of the murine Ube3a in cultured mouse neuronal cells. In Specific Aim 1, we will create ATFs that directly activate the transcription of murine Ube3a or repress the expression of murine Ube3a-Antisense Transcript (ATS). The milestone goal will be to create one or more ATFs that can activate Ube3a expression at least 2-fold. Several ATFs have been constructed and tested, and our preliminary data show that several are able to upregulate endogenous Ube3a protein levels. In Specific Aim 2, the ATFs will be packaged into AAV viral vectors and delivered into the brains of AS mice. We will examine the distribution and expression of the ATF in the brain, its effect on Ube3a expression. The milestone goal is to achieve widespread neuronal transduction and expression of the ATF in injected brain structures (>10% of hippocampal cells, and transduction of some surrounding tissue). One or more ATFs are expected to cause at least a 2-fold increase in Ube3a expression from the paternal allele. Successful achievement of these milestones would justify expanded pre-clinical studies and the development of ATFs to the human UBE3A. PUBLIC HEALTH RELEVANCE: Angelman Syndrome (AS) is a neurogenetic disorder causing developmental delays and neurological impairments such as lack of speech. Currently, there is no cure and no specific drug therapy. Activation of the silenced paternal UBE3A allele by an ATF could address the fundamental genetic deficiency causing AS, and thus be a potentially curative gene therapy. This proposal will investigate such a therapy in a mouse model.