ADENO ASSOCIATED VIRUS GENE THERAPY FOR THE CNS IN MUCO

大多数影响中枢神经系统（CNS）的遗传性疾病会在整个大脑中产生代谢异常和病变。在许多遗传疾病中，体细胞基因转移有可能阻止或逆转疾病过程。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能将外源基因传递到有限数量的细胞或特定结构中。影响中枢神经系统的一大类人类遗传疾病是溶酶体贮积障碍，其特征是智力迟钝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以一种可以纠正这组典型的神经退行性疾病的弥散性病变的方式传递到大脑中。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-葡糖苷酶（GUSB）在粘多糖病（MPS） VII型（Sly病）（一种典型的神经退行性储存病）GUSB缺陷动物模型大脑中的转移特性。初步研究表明，该载体可以表达持续治疗水平的转移GUSB，并在转导位点周围的大范围内分泌酶，这表明该载体可能适合长期纠正遗传缺陷。具体目的是：1)确定对在大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)表征AAV载体GUSB在大脑不同细胞类型和结构中的表达；3)定量测定GUSB在MPS VII脑中的表达量和寿命，并评估可能增加总转导和酶传递的方法；4)评估长期大脑病理变化。大部分的开发和测试将在MPS VII小鼠大脑中进行，以优化载体设计和递送策略。然后，这些方法将在MPS VII犬脑中进行测试，以评估载体性能和酶在更大体积脑组织中的扩散。在人类神经退行性疾病的真实临床模型中进行评估，将有助于确定这种治疗溶酶体贮积性疾病的中枢神经系统疾病的方法的潜力和局限性。

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.