Viral vector-based RNAi therapy for Alexander Disease

描述（由申请人提供）： 亚历山大病（AlxD）是一种常染色体显性遗传的白色物质神经系统破坏性疾病，影响儿童。AlxD没有特异性治疗，但其发病机制使其成为利用RNA干扰进行基因治疗的理想候选者。本项目的目标是开发一种有效的AlxD基因治疗方法。AlxD是由胶质细胞酸性蛋白（GFAP）突变引起的，GFAP是一种仅在脑内星形胶质细胞中表达的蛋白质。AlxD的病理标志是Rosenthal纤维，一种由GFAP和几种其他蛋白质聚集体组成的包涵体。Rosenthal纤维在AlxD中大量存在，并且仅在星形胶质细胞中发现。该疾病是由毒性功能获得引起的，其中突变的GFAP诱导异常丝状组装体的形成，所述丝状组装体是罗森塔尔纤维。我们的合作者已经建立了一个AlxD的动物模型，其中小鼠表达突变的GFAP基因。异常基因含有点突变，其在与导致人类AlxD的位点相同的位点诱导氨基酸取代。来自突变小鼠的星形胶质细胞在体外和体内发育罗森塔尔纤维。RNA干扰（RNAi）是选择性抑制特定基因的有力工具。我们将利用RNAi抑制突变GFAP基因的表达，从而阻断并可能逆转AlxD的神经病理学。为了将RNAi传递到星形胶质细胞，我们将用淋巴细胞性脉络丛脑膜炎病毒（LCMV）设计一种假型慢病毒，LCMV是一种选择性靶向发育中大脑中星形胶质细胞的病毒。这种含有针对突变型GFAP的短发夹RNA（shRNA）的病毒基因治疗载体将选择性地抑制星形胶质细胞中突变型GFAP的表达。目的一是将突变基因对应的小干扰RNA（siRNA）用于转染野生型和突变型GFAP的Cos细胞中，以实现等位基因特异性抑制。在目标二中，携带相应的shRNA的慢病毒载体将用于在来自AlxD小鼠的星形胶质细胞培养物中实现选择性RNA干扰。目的三：将病毒载体注射到新生AlxD小鼠脑内，选择性抑制突变型GFAP mRNA和蛋白的表达，阻断罗森塔尔纤维的形成。这项研究与公共卫生高度相关，因为它可能导致开发针对AlxD的特异性和有效的治疗方法，AlxD是一种致命的儿童退行性疾病，目前尚无特异性治疗方法。亚历山大病是一种进行性和破坏性的儿科脑部疾病。这种疾病是由于基因异常引起的。本研究旨在开发一种基因治疗的形式来预防和逆转亚历山大病的脑功能障碍。

DESCRIPTION (provided by applicant): Alexander Disease (AlxD) is an autosomal dominantly inherited neurologically devastating disorder of white matter that affects children. AlxD has no specific therapy, but its pathogenesis makes it an ideal candidate for gene therapy utilizing RNA interference. The goal of this project is to develop an effective gene therapy for AlxD. AlxD is caused by a mutation of glial fibrillary acidic protein (GFAP), a protein expressed exclusively in astrocytes within the brain. The pathologic hallmark of AlxD is the Rosenthal fiber, an inclusion body composed of aggregates of GFAP and several other proteins. Rosenthal fibers are present in large numbers in AlxD and found exclusively in astrocytes. The disease is caused by a toxic gain of function in which the mutated GFAP induces formation of abnormal filamentous assemblies, which are the Rosenthal fibers. Our collaborator has generated an animal model of AlxD in which mice express mutated GFAP genes. The abnormal genes contain point mutations inducing amino acid substitutions at the same sites as those causing AlxD in humans. Astrocytes from the mutant mice develop Rosenthal fibers in vitro and in vivo. RNA interference (RNAi) is a powerful tool for selectively suppressing specific genes. We will utilize RNAi to suppress expression of the mutant GFAP gene, thus blocking and perhaps reversing the neuropathology of AlxD. To deliver the RNAi to astrocytes, we will engineer a lentivirus pseudotyped with lymphocytic choriomeningitis virus (LCMV), a virus that selectively targets astrocytes within the developing brain. This viral gene therapy vector containing short hairpin RNA (shRNA) against mutant GFAP will selectively suppress expression of mutant GFAP in astrocytes. In Aim One, small interfering RNA (siRNA) corresponding to the mutated genes will be used in Cos cells transfected with wild type and mutant GFAP to achieve allele-specific suppression. In Aim Two, the lentiviral vector, carrying corresponding shRNA, will be used to achieve selective RNA interference in astrocyte cultures derived from AlxD mice. In Aim Three, the viral vector will be injected into the brains of neonatal AlxD mice, to selectively inhibit expression of mutant GFAP mRNA and protein and block the formation of Rosenthal fibers. This research is highly relevant to public health because it may lead to development of a specific and effective treatment for AlxD, a fatal degenerative disease of childhood for which there is currently no specific treatment. Alexander Disease is a progressive and devastating pediatric brain disease. The disease is due to an abnormal gene. This research aims to develop a form of gene therapy to prevent and reverse the brain dysfunction of Alexander Disease.