GENE THERAPY FOR RESCUE OF REPRODUCTIVE FUNCTION

描述(申请人提供)：下丘脑、垂体和性腺之间协调和整合的信号是成功的哺乳性性成熟和繁殖所必需的。促性腺激素释放激素(GnRH)是下丘脑的一种十肽，对这种生殖激素级联的启动和控制起着中心作用。GnRH基因缺失的性腺功能低下的HPG小鼠说明了GnRH在这一调控途径中的重要性。这些小鼠提供了一个有用的模型系统，可以更好地研究GnRH的神经元功能和神经内分泌对GnRH产生和释放的调节。挽救HPG小鼠生殖完整性的一个潜在途径是使用病毒载体系统将完整的GnRH基因传递到这些小鼠的GnRH神经元中。基因治疗对神经系统的挑战是开发能够在有丝分裂后细胞中实现稳定转基因表达的无毒载体。单纯疱疹病毒扩增载体是一种独特的载体，具有DNA容量高，对啮齿动物和人类神经系统细胞高度感染，包装无辅助病毒基本无毒，理论上可以实现所传递基因的稳定和长期表达。将GnRH基因转移到HPG小鼠下丘脑的神经元，并评估随后GnRH的表达和生殖功能，可以作为评估HSV扩增子对神经系统进行基因治疗的成功的工具。本项目的总体目标是使用HSV扩增病毒载体系统将促性腺激素释放激素(GnRH)基因传递到性腺功能低下的成年雌性HPG小鼠的GnRH神经元中。本建议的具体目的是：(1)确定能否在注射了HSV、GnRH扩增子的HPG成年雌性小鼠中实现GnRH的长期调控表达；以及(2)表征HPG小鼠在基因传递后的生殖功能。本实验系统将作为一个模型，帮助我们实现两个特定目标：(1)评估HSV扩增子用于延长基因替代疗法在治疗中枢神经系统(CNS)神经元疾病中的作用；以及(2)更好地了解下丘脑GnRH在体内的作用和调节的生理学。

DESCRIPTION (provided by applicant): Coordinated and integrated signals between the hypothalamus, the pituitary and the gonads are necessary for successful mammalian sexual maturation and reproduction. Gonadotropin-releasing hormone (GnRH), a hypothalamic decapeptide, is central to the initiation and control of this reproductive hormone cascade. The importance of GnRH in this regulatory pathway is illustrated by the hypogonadal, GnRHdeficient hpg mouse, which have a deletion within the GnRH gene. These mice provide a useful model system that can be studied to better understand GnRH neuronal function and neuroendocrine regulation of GnRH production and release. One potential avenue for rescue of reproductive integrity of the hpg mice is to use a viral vector system to deliver the intact GnRH gene to GnRH neurons in these mice. The challenge of gene therapy to the nervous system is to develop nontoxic vectors that can achieve stable transgene expression in post-mitotic cells. The HSV amplicon vector is a unique vector that has high DNA capacity, is highly infectious to cells of the nervous system in rodents and humans, has essentially no toxicity when packaged free of helper virus, and theoretically can achieve stable and prolonged expression of the delivered gene. Delivery of the GnRH gene to neurons in the hypothalamus of the hpg mouse and evaluation of subsequent GnRH expression and reproductive function can serve as a tool to evaluate the success of the HSV amplicon for gene therapy to the nervous system. The overall goal of this project is to use the HSV amplicon viral vector system to deliver the gonadotropin-releasing hormone (GnRH) gene to GnRH neurons in hypogonadal, GnRH-deficient hpg adult female mice. The Specific Aims of this proposal are: (1) to determine whether prolonged, regulated expression of GnRH can be achieved in hpg adult female mice injected with HSV, GnRH amplicon; and (2) to characterize the reproductive function of hpg mice after gene delivery This experimental system will serve as a model to help us attain two specific objectives: (1) to assess the utility of the HSV amplicon for prolonged gene replacement therapy in the treatment of central nervous system (CNS) neuronal disorders; and (2) to better understand the physiology of hypothalamic GnRH action and regulation in vivo.