Engineered AAV vectors for combinatorial treatment of rare genetic brain diseases

项目摘要 基于AAV的基因治疗需要开发安全、高效和靶向性的载体。AAV介导型 外周组织(血液和骨骼肌)的基因治疗已经取得了长足的进步，主要集中在基因上。 替代功能丧失的疾病。然而，关于显性遗传的中枢神经系统的研究很少 由有毒基因产品引起的疾病。我们聚集了一位世界级的、多学科的学者 由我们的工业合作伙伴支持的研究团队开发基于AAV的创新基因敲除和 罕见遗传性疾病的替代治疗，包括脊髓小脑性共济失调7型(SCA7)和Valosin- 含蛋白(VCP)多系统蛋白病。我们响应RFA项目目标并提出 三个具体目标。在目标1中，我们将设计和制造具有更高临界质量的新型AAV载体 基因治疗的属性(安全性、有效性、靶向性)。我们提出了创新的神经元特异性基因 传递，基因表达的时间控制和中枢神经系统中免疫反应的减少。我们已经建造了 表达EGFP和mRNA条形码的AAV载体用于改进筛查，以支持我们建议的AAV 治疗。在目标2中，我们将通过结合下一代测序来开发高级定量分析 以及条形码AAVs，用于在小鼠模型中有效地评估体内基因传递。我们将放映和 比较使用不同启动子/增强子的不同衣壳变异体AAV介导的基因表达。 单细胞RNAseq也将用于评估选定的AAV载体的免疫应答，通过不同的 管理路线。在目标3中，为了有效地治疗SCA7和VCP疾病，我们将开发新型AAV 同时敲除有毒基因产物同时取代密码子正常基因产物的载体 经过优化，不受击倒影响。这一提议的联合治疗将建立一个证据- 许多其他显性遗传性疾病的概念，其中正常等位基因表达的丧失是由于非特异性 沉默本身也会带来问题。我们已经为目标3建立了很好的SCA7和VCP疾病模型 学习。我们的团队在基础研究中开发、制造和应用AAV载体方面拥有丰富的专业知识 和临床前应用。我们已发表的工作和初步数据确立了可行性和关键 拟议研究的方法学。最重要的是，我们有成熟的病毒生产设施和 我们的UCI神经回路映射中心运行的分销平台支持病毒试剂设计， 验证和制造。

Project Summary AAV-based gene therapy requires the development of safe, efficient, and target-specific vectors. AAV-mediated gene therapy for peripheral tissues (blood and skeletal muscle) has made great strides, focused mainly on gene replacement for loss of function diseases. However, there has been little research on dominantly inherited CNS disorders that are caused by toxic gene products. We have assembled a world class, multi-disciplinary academic research team supported by our industrial partners to develop innovative AAV-based gene knockdown and replacement treatments for rare genetic diseases including Spinocerebellar Ataxia Type 7 (SCA7) and valosin- containing protein (VCP) multisystem proteinopathy. We respond to the RFA Project Objectives and propose three Specific Aims. In Aim 1, we will design and manufacture new AAV vectors with improved critical quality attributes (safety, efficacy, target specificity) for gene therapy. We propose innovative neuron specific gene delivery, temporal control of gene expression and reduced immune responses in the CNS. We have constructed AAV vectors that express EGFP and mRNA barcodes for improved screening to support our proposed AAV treatments. In Aim 2, we will develop advanced quantitative analytics by combining next-generation sequencing and bar-coded AAVs for efficient assessment of in vivo gene delivery in the mouse model. We will screen and compare AAV-mediated gene expression with different capsid variants using different promoters / enhancers. Single-cell RNAseq also will be used to assess immunological responses of selected AAV vectors by different administration routes. In Aim 3, to effectively treat the SCA7 and VCP diseases, we will develop novel AAV vectors that simultaneously knockdown toxic gene products while replacing normal gene products that are codon optimized to be unaffected by knockdown. This proposed combinatorial treatment will establish a proof-of- concept for many other dominant inherited diseases, where loss of normal allele expression due to non-specific silencing causes its own problems. We have well developed models of the SCA7 and VCP diseases for Aim 3 studies. Our team has great expertise in developing, manufacturing and applying AAV vectors in basic research and preclinical application. Our published work and preliminary data establish the feasibility and key methodologies for the proposed research. Critically, we have an established viral production facility and distribution platform operating out of our UCI Center for Neural Circuit Mapping that supports viral reagent design, validation, and manufacturing.