Gene Therapy in the Senile Brain and Hypophysis

描述(申请人提供)：阿根廷城市地区老年人口的增长与北美和欧洲的许多城市相当。因此，与年龄相关的神经病理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申请者和阿根廷申请者之间建立长期合作，他们都对基因疗法治疗神经退行性疾病的潜力感兴趣。提出了三个具体目标：1)评价自调型腺相关病毒(AAV)载体在老年大鼠脑内的作用。这些载体含有一个报告基因，编码人源化绿色荧光蛋白(HGFP)，受tet-off或tet-on启动子系统的控制，分别可被四环素(Tet)或某些Tet衍生物关闭或开启。上述载体将被立体定向注射到脑室旁、脑室周围和弓状核中，所有这些核都含有已知易老化的多巴胺(DA)神经元。2)评价携带神经保护分子人胶质细胞源性神经营养因子(HGDNF)基因的自调式端粒型AAV载体的治疗效果。该载体将被立体定向注射到24月龄大鼠的弓状核内，6个月后，将通过DA神经元的定量免疫组织化学和hGDNF的ELISA法来评估其防止正常的下丘脑结节漏斗DA(TIDA)神经元的年龄相关性丢失的能力。在整个实验过程中，将监测动物的血清PRL水平(TIDA神经元功能的一个指标)。3)构建TET-OFF系统控制下携带自杀基因的两个自调型腺病毒载体(Rad)。其中一株RAD将携带一种混合自杀基因，编码hGFP和HSV1胸苷激酶(HSV1-TK)之间的融合蛋白，该融合蛋白保留荧光和HSV1-TK自杀活性。另一个Rad将在双向Tet-off调控元件的控制下包含HSV1-TK和hGFP的单独基因。这两种载体预计比不可调控的载体更安全，将用于治疗实验性的大鼠催乳素瘤。除了上述具体目标外，还计划与美国和阿根廷的基础和临床神经学家进行接触，以制定一项关注老年性大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.