GENE THERAPY FOR CEREBELLAR ATAXIA

小脑是大脑的一个区域，在控制运动和认知方面发挥着重要作用。脊髓小脑共济失调1型(SCA1)是一种致命的人类疾病，是至少五种遗传性共济失调之一，总共至少有八种神经退行性疾病，每一种疾病都是由含有扩展的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的ataxin-1蛋白赋予的功能获得，并已在转基因小鼠模型中概述，导致小脑中神经元浦肯野细胞的退化。 作为一种潜在的SCA1基因治疗方法，我们在本研究期间证明：(I)腺相关病毒载体(AAV)是一种有效的体内基因转移工具，可将基因转移到小脑浦肯野细胞中，单次小脑注射后，小脑半球的转导效率高达3%；(Ii)针对ataxin信息中特定位置的核酶和反义RNA在体外和体内组织培养细胞中分别破坏了ataxin信息的稳定性。在这一竞争性的更新应用中，我们建议通过执行四个特定目标来扩展这些研究：(I)进一步鉴定和优化AAV介导的体内Purkinje细胞转导，包括碱性成纤维细胞生长因子受体1和硫酸肝素蛋白多糖在病毒结合中的分子作用， 以及AAV血清型1、2和5型在介导小脑浦肯野细胞转导中的相对有效性；(Ii)进一步测试下调或纠正扩大的ataxin-1信息的分子方法，包括反义、核酶、反式剪接和RNA干扰；(Iii)建立抗ataxin转基因，随后与SCA1转基因小鼠杂交，评估这些方法在控制Purkinje细胞退化和共济失调发展方面的有效性；以及(Iv)使用AAV载体将抗ataxin或ataxin校正序列传递到SCA1转基因小鼠的小脑Purkinje细胞中，作为 人类SCA1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给药条件，不仅适用于SCA1的治疗，也适用于其他显性作用和其他神经疾病的治疗。

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.