GENE THERAPY FOR RESCUE OF REPRODUCTIVE FUNCTION

描述（由申请人提供）：下丘脑、垂体和性腺之间的协调和整合信号是哺乳动物性成熟和生殖成功所必需的。促性腺激素释放激素（GnRH），下丘脑十肽，是中央的启动和控制这一生殖激素级联反应。GnRH在这一调节途径中的重要性通过性腺功能减退的GnRH缺陷型hpg小鼠得到了说明，该小鼠的GnRH基因内存在缺失。这些小鼠提供了一个有用的模型系统，可以进行研究，以更好地了解GnRH神经元功能和GnRH产生和释放的神经内分泌调节。拯救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基因递送到这些小鼠中的GnRH神经元。基因治疗对神经系统的挑战是开发无毒载体，可以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，其具有高DNA容量，对啮齿动物和人类的神经系统细胞具有高度感染性，当包装时不含辅助病毒时基本上没有毒性，并且理论上可以实现递送基因的稳定和延长的表达。将GnRH基因递送至hpg小鼠下丘脑中的神经元并评价随后的GnRH表达和生殖功能可以作为评价HSV扩增子用于神经系统基因治疗的成功的工具。本项目的总体目标是使用HSV扩增子病毒载体系统将促性腺激素释放激素（GnRH）基因递送至性腺功能减退、GnRH缺陷的hpg成年雌性小鼠的GnRH神经元。本提案的具体目的是：（1）确定是否可以在注射HSV（GnRH扩增子）的hpg成年雌性小鼠中实现GnRH的延长的、受调节的表达;以及（2）表征基因递送后hpg小鼠的生殖功能。本实验系统将作为帮助我们实现两个具体目标的模型：（1）评估HSV扩增子在中枢神经系统（CNS）神经元疾病治疗中用于延长基因替代疗法的效用;（2）更好地了解体内下丘脑GnRH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.