ADENO ASSOCIATED VIRUS GENE THERAPY FOR THE CNS IN MUCO

大多数影响中枢神经系统(CNS)的遗传性疾病会导致整个大脑的代谢异常和损伤。体细胞基因转移有可能阻止或逆转许多遗传性疾病的发病过程。然而，目前可用的将基因转移到中枢神经系统的方法通常只将外来基因传递到有限数量的细胞或特定的结构。影响中枢神经系统的一大类人类遗传病是溶酶体储存障碍，其特征是智力低下和其他进行性退行性异常。我们研究的长期目标是开发一种方法，将正常的溶酶体酶基因输送到大脑中，以纠正这组神经退行性疾病的典型播散性病变。在这项资助中，我们将研究腺相关病毒(AAV)载体介导的治疗性基因--β-葡萄糖醛酸苷酶(GUSB)在粘多糖病(MPS)VII型(Sly病)动物模型中的作用。初步研究表明，该载体可以表达GUSB的持续治疗水平，并在转导部位周围的大范围校正范围内分泌酶，提示该载体可能适合于长期修复遗传缺陷。其具体目的是：1)确定在脑中广泛表达或在神经元中高水平表达的载体基因表达启动子的影响；2)鉴定AAV载体中GUSB在脑中各种细胞类型和结构中的表达；3)定量GUSB在MPS VII脑中的表达数量和寿命，并评估可能增加总转导和酶输送的方法；以及4)评估长期脑内的病理变化。大部分的开发和测试将在MPS VII小鼠的大脑中完成，以优化载体设计和传递策略。然后，这些方法将在MPS VII狗的大脑中进行测试，以评估载体性能和酶在更大体积的脑组织中的扩散。在人类神经退行性疾病的真实临床模型中进行评估将有助于确定这种方法在治疗溶酶体储存障碍的中枢神经系统疾病方面的潜力和局限性。

Most genetic diseases that affect the central nervous system (CNS) produce metabolic abnormalities and lesions throughout the brain. Somatic gene transfer is potentially capable of arresting or reversing the disease process in many genetic disorders. However, the methods presently available to transfer genes into the central nervous system generally only deliver a foreign gene to a limited number of cells or to specific structures. A large class of human genetic diseases affecting the CNS are the lysosomal storage disorders, which are characterized by mental retardation and other progressive degenerative abnormalities. The long-term goal of our studies is to develop a method to deliver normal lysosomal enzyme genes into the brain in a manner that can correct the disseminated lesions that are typical of this group of neurodegenerative diseases. In this grant we will study the properties of adeno-associated virus (AAV) vector-mediated transfer of a therapeutic gene, beta-glucuronidase (GUSB), in the brains of GUSB-deficient animal models of mucopolysaccharidosis (MPS) type VII (Sly disease), a representative neurodegenerative storage disease. Preliminary studies demonstrate that the vectors can express sustained therapeutic levels of the transferred GUSB, and secrete the enzyme in a large sphere of correction surrounding the transduction site, suggesting that this vector may be suitable for long-term correction of genetic deficiencies. The Specific Aims are to: 1) Determine the effects on vector gene expression promoters that are expressed widely in the brain or at high levels in neurons; 2) Characterize the expression of GUSB from AAV vectors in various cell types and structures of the brain; 3) Quantitate the amount and longevity of expression of GUSB in the MPS VII brain and evaluate methods which may increase total transduction and enzyme delivery; and 4) Evaluate changes in pathology in the brain over the long-term. Most of the development and testing will be done in the MPS VII mouse brain to optimize the vector designs and delivery strategies. Then, the methods will be tested in the MPS VII dog brain to evaluate vector performance and enzyme diffusion in a larger volume of brain tissue. Evaluation in true clinical models of a human neurodegenerative disease will help define both the potential and limitations of this approach to treating the CNS disease in lysosomal storage disorders.