GENE THERAPY FOR RESCUE OF REPRODUCTIVE FUNCTION

描述（申请人提供）：下丘脑、垂体和性腺之间的协调和综合信号是哺乳动物成功性成熟和生殖所必需的。促性腺激素释放激素（GnRH）是一种下丘脑十肽，是启动和控制这种生殖激素级联的核心。GnRH在这一调节途径中的重要性通过性腺功能低下、GnRHdeficient hpg小鼠（GnRH基因缺失）得到证实。这些小鼠提供了一个有用的模型系统，可以更好地研究GnRH神经元功能和GnRH产生和释放的神经内分泌调节。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。神经系统基因治疗面临的挑战是开发无毒载体，使其能够在有丝分裂后的细胞中实现稳定的转基因表达。HSV扩增子载体是一种独特的载体，具有高DNA容量，对啮齿动物和人类神经系统细胞具有高度传染性，在没有辅助病毒的包装下基本上没有毒性，理论上可以实现所传递基因的稳定和长时间表达。将GnRH基因传递到hpg小鼠下丘脑神经元，并评估随后的GnRH表达和生殖功能，可以作为评估HSV扩增子用于神经系统基因治疗成功的工具。该项目的总体目标是利用HSV扩增子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、GnRH缺陷的成年雌性hpg小鼠的GnRH神经元。本研究的具体目的是：(1)确定注射HSV、GnRH扩增子的hpg成年雌性小鼠能否实现GnRH的长时间调控表达；该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扩增子在延长基因替代疗法治疗中枢神经系统（CNS）神经元疾病中的效用；(2)更好地了解下丘脑GnRH的生理作用和体内调节。

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.