Gene Therapy in the Senile Brain and Hypophysis

描述（由申请人提供）：阿根廷城市地区老年人口的增长与许多北美和欧洲城市相当。 因此，与年龄有关的神经系统疾病的发病率正在成为对该国产生重大医疗和经济影响的问题。 在这方面，本提案的总体目标是在美国和阿根廷申请人之间建立长期合作，这两个申请人对基因疗法治疗神经变性疾病的潜力有共同的兴趣。 提出了三个具体目标：1）评估自我调节腺相关病毒（AAV）载体在老年大鼠脑中的性能。 这些载体含有编码人源化绿色荧光蛋白（hGFP）的报告基因，该报告基因在tet-off或tet-on启动子系统的控制下，所述启动子系统可分别通过施用四环素（泰特）或某些泰特衍生物而被关闭或打开。 将上述载体立体定位注射到室旁核、室周核和弓状核中，所有这些核都含有已知易老化的多巴胺能（DA）神经元。 2)评估携带人胶质细胞源性神经营养因子（hGDNF）基因（一种有效的神经保护分子）的自调节tet-off AAV载体的治疗效果。 将载体立体定位注射到24月龄大鼠的弓状核中，6个月后，通过DA神经元的定量免疫组织化学和hGDNF的ELISA评估其预防下丘脑结节漏斗DA（TIDA）神经元的正常年龄相关损失的能力。 在整个实验期间，将监测动物的血清PRL水平（TIDA神经元功能的指标）。 3)目的构建两种tet-off系统控制的自杀基因自调控腺病毒载体。 其中一个RAd将携带编码hGFP和HSV 1胸苷激酶（HSV 1-TK）之间的融合蛋白的杂合自杀基因，其保留荧光和HSV 1-TK自杀活性。 另一个RAd将在双向Tet-off调节元件的控制下携带HSV 1-TK和hGFP的单独基因。 这两种载体，这是预计将比不可调节的同行更安全，将用于治疗实验大鼠泌乳素瘤。 除上述具体目标外，还计划与美国和阿根廷的基础和临床神经科学家进行接触，以制定一项重点关注老年脑疾病的后续研究计划。

DESCRIPTION (provided by applicant): The increase of the elderly population in the urban areas of Argentina is comparable to that of many North American and European cities. Consequently, the incidence of age-related neurological pathologies is becoming a problem of significant medical and economic impact for the country. In this context, the overall goal of the present proposal is to establish a long-term collaboration between the American and Argentine applicants, who share an interest in the potential of gene therapy for the treatment of neurodegenerative diseases. Three specific objectives are proposed: 1) To assess the performance of self-regulating adeno associated viral (AAV) vectors in the brain of old rats. These vectors harbor a reporter gene encoding humanized green fluorescent protein (hGFP) under the control of the tet-off or tet-on promoter system which can be turned off or on, respectively by administration of tetracycline (Tet) or certain Tet derivatives. The above vectors will be stereotaxically injected in the paraventricular, periventricular and arcuate nuclei, all of which contain dopaminergic (DA) neurons of known vulnerability to aging. 2) To assess the therapeutic efficacy of a self-regulating tet-off AAV vector harboring the gene for human glial cell line-derived neurotrophic factor (hGDNF), a potent neuroprotective molecule. The vector will be stereotaxically injected in the arcuate nucleus of 24-month old rats and 6 months later, its ability to prevent the normal age-related loss of hypothalamic tuberoinfundibular DA (TIDA) neurons will be assessed by quantitative immuno-histochemistry for DA neurons and ELISA for hGDNF. Serum PRL levels (an index of TIDA neuron function) will be monitored in the animals throughout the experiment. 3) To construct two self-regulating adenoviral vectors (RAd) harboring suicide genes under the control of the tet-off system. One of the RAds will harbor a hybrid suicide gene coding for a fusion protein between hGFP and HSV1 thymidine kinase (HSV1-TK), which retains fluorescence and HSV1-TK suicide activity. The other RAd will harbor the separate genes for HSV1-TK and hGFP under the control of a bi-directional Tet-off regulatory element. These two vectors, which are expected to be safer than non-regulatable counterparts, will be employed to treat experimental rat prolactinomas. Further to the above specific objectives, contacts with basic and clinical neuroscientists are planned in the US and Argentina in order to assemble a follow up research program focused on senile brain disorders.