Gene therapy for ataxia

小脑是大脑的一个区域，在控制运动和认知方面起着重要作用。脊髓小脑性共济失调1型（SCA 1）是一种致命的人类疾病，是至少五种遗传性共济失调之一，总共至少八种神经退行性疾病，每种疾病都是由含有扩展的多聚谷氨酰胺区域的突变蛋白的表达引起的。发病机制是由于突变型共济失调蛋白-1蛋白赋予的功能获得，并已在转基因小鼠模型中重现，导致小脑中神经元浦肯野细胞变性。 作为SCA 1基因治疗的一种潜在方法，我们在目前的研究期间证明：（i）腺相关病毒载体（AAV）是体内基因转移到小脑浦肯野细胞中的有效工具，在单次小脑内注射后在小脑半球产生高达3%的转导;（ii）靶向共济失调蛋白信息中特定位置的核酶和反义RNA分别在体外和体内组织培养细胞中使共济失调蛋白信息不稳定。在该竞争性更新申请中，我们提出通过执行4个具体目标来扩展这些研究：（i）进一步表征和优化体内AAV介导的浦肯野细胞转导，包括碱性成纤维细胞生长因子受体1和硫酸肝素蛋白聚糖在病毒结合中的分子作用， 以及AAV血清型1、2和5在介导小脑浦肯野细胞转导中的相对有效性;（ii）进一步测试用于下调或校正扩增的共济失调蛋白-1信使的分子方法，包括反义、核酶、反式剪接和RNA干扰;（iii）抗共济失调蛋白转基因的建立，随后与SCA 1转基因小鼠杂交以评价这些方法在控制浦肯野细胞变性和共济失调发展中的有效性;和（iv）使用AAV载体将抗共济失调蛋白或共济失调蛋白校正序列递送至SCA 1转基因小鼠中的小脑浦肯野细胞， SCA 1基因治疗的直接模型。因此，这些研究的结果将提供优化的分子工具和递送条件，其不仅适用于SCA 1的治疗，而且也适用于其他显性作用和其他神经系统疾病的治疗。

The cerebellum is a region of the brain that plays a major role in the control of movement and cognition. Spinocerebellar Ataxia Type 1 (SCA1) is a fatal human disorder that is one of at least five inherited ataxias, and a total of at least eight neurodegenerative diseases, each caused by the expression of a mutant protein containing an expanded polyglutamine region. Pathogenesis is due to a gain of function conferred by mutant ataxin-1 protein and has been recapitulated in a transgenic mouse model resulting in degeneration of neuronal Purkinje cells in the cerebellum. As a potential approach toward gene therapy for SCA1, we demonstrated during the current period of study: (i) That adeno-associated virus vectors (AAV) are an effective tool for in vivo gene transfer into cerebellar Purkinje cells, yielding up to 3% transduction in a cerebellar hemisphere after a single intracerebellar injection; (ii) That ribozymes and antisense RNAs targeting specific positions in the ataxin message destabilize ataxin message in vitro and in tissue culture cells in vivo, respectively. In this competing renewal application, we propose to extend these studies through the execution of 4 specific aims: (i) Further characterization and optimization of AAV-mediated Purkinje cell transduction in vivo, including the molecular role of basic fibroblast growth factor receptor 1 and heparin sulfate proteoglycan in virus binding, and the relative effectiveness of AAV serotypes 1, 2 and 5 in mediating transduction of cerebellar Purkinje cells; (ii) Further testing of molecular approaches for downregulating or correcting expanded ataxin-1 message, including antisense, ribozyme, trans-splicing and RNA interference; (iii) Establishment of anti-ataxin transgenes, subsequently crossing with SCA1 transgenic mice to evaluate the effectiveness of these approaches in controlling Purkinje cell degeneration and development of ataxia; and (iv) Use of AAV vectors to deliver anti-ataxin or ataxin correcting sequences to cerebellar Purkinje cells in SCA1 transgenic mice, as a direct model of gene therapy for SCA1 in humans. Results from these studies will thus provide optimized molecular tools and delivery conditions which will be applicable to treatment not only of SCA1 but other dominant-acting and other neurologic disorders as well.