A Novel Therapeutic Agent for Postmenopausal Bone Loss

描述（由申请人提供）：绝经后骨质疏松症是由雌激素（E2）缺乏引起的骨重塑缺陷引起的。我们最近报道，E2缺乏期间的骨质流失主要是由T细胞来源的细胞因子分泌上调引起的，包括关键的破骨细胞因子、NFkB配体受体激活因子（RANKL）和肿瘤坏死因子（TNF）。诱导T细胞活化和细胞因子分泌的一个关键事件是IL-7产生的上调，这是雌激素缺乏的结果。我们最近报道，使用中和抗体抑制小鼠卵巢切除术（ovx）诱导的IL-7水平升高，可有效预防卵巢切除术诱导的小鼠体内骨质流失。这提示IL-7中和可能是预防和治疗骨质疏松症的一种新手段。然而，尽管这项实验取得了成功，但在人体中长期使用抗体治疗往往会导致严重的临床并发症和长期疗效差。为了克服基于抗体治疗的局限性，我们现在提出了一种新的策略来体内中和IL-7并改善绝经后骨质疏松症的骨质流失。该策略涉及设计一种新型肽类治疗剂，该治疗剂包含小鼠IL-7受体α (sll - 7rα)的可溶性细胞外配体结合域。为了延长体内半衰期，该肽将作为与人IgG1-Fc区域（sll - 7rα - fc）的融合蛋白表达。我们假设这种药物将在体内作为可溶性IL-7诱骗受体发挥作用，并通过中和过量的IL-7来防止E2缺乏时的骨质流失。我们提出了两个具体目标来进一步研究我们的假设：具体目标1：证明可溶性IL-7诱饵受体是一种有效的治疗辅助剂，可以预防体内雌激素缺乏引起的骨质流失。这将通过将纯化的重组sll - 7rα - fc注射到卵巢切除后的小鼠体内，并检测卵巢切除后骨转换的物理、生化和组织形态学指标来实现。通过测定血清中游离IL-7和sll - 7rα - fc浓度随时间的变化，评估sll - 7rα - fc的长期稳定性和生物半衰期。具体目标2：通过基因转移的方法在体内表达可溶性IL-7诱饵受体，证明雌激素缺乏后保留骨量的可行性。通过采用两种不同的基因转移程序，即针对骨骼肌的肌肉注射和针对肝脏的流体动力注射，将编码slL-7Ralpha-Fc的哺乳动物表达载体引入ovx小鼠，从而实现这一特定目的。这些具体目标将展示两个重要的“原理证明”：(1)一种新的治疗剂sll - 7rα - fc，是预防雌激素缺乏后骨质流失的有效疗法。(2)基因转移是体内引入和表达sll - 7rα - fc的有效机制。

DESCRIPTION (provided by applicant): Postmenopausal osteoporosis results from defective bone remodeling stemming from estrogen (E2) deficiency. We have recently reported that bone loss during E2 deficiency is driven principally by upregulated T cell-derived cytokine secretion including the key osteoclastogenic cytokines, Receptor-activator of NFkB Ligand (RANKL) and Tumor necrosis Factor alpha (TNF). A key event in inducing T cell activation and cytokine secretion is the upregulation of IL-7 production, a consequence of estrogen deficiency. We have recently reported that inhibiting the elevated levels of IL-7 induced by ovariectomy (ovx) in mice using a neutralizing antibody, is efficacious in preventing ovx-induced bone loss in mice in vivo. This suggests that IL-7 neutralization may be a novel means to prevent and treat osteoporosis. However, despite the success of this experiment, long-term therapeutic treatment with antibodies in humans often lead to serious clinical complications and poor long-term efficacy. In order to overcome the limitations of antibody-based therapies, we now propose a novel strategy to neutralize IL-7 in vivo and ameliorate bone loss in postmenopausal osteoporosis. This strategy involves the design of a novel peptide based therapeutic agent comprising the soluble extracellular ligand-binding domain of the mouse IL-7 receptor alpha (slL-7Ralpha). To extend in vivo half-life this peptide will be expressed as a fusion protein with the human IgG1-Fc region (slL-7Ralpha-Fc). We hypothesize that this agent will function in vivo as a soluble IL-7 decoy receptor and prevent bone loss during E2 deficiency, by neutralizing excess IL-7. We propose two Specific Aims to further investigate our hypothesis: Specific Aim 1: To demonstrate that a soluble IL-7 decoy receptor is an efficacious therapeutic adjunct in preventing estrogen deficiency-induced bone loss in vivo. This will be achieved by injecting purified recombinant slL-7Ralpha-Fc into mice following ovx and examining physical, biochemical and histomorphometric indices of bone turnover following ovariectomy. Long-term stability and biological half-life of slL-7Ralpha-Fc will be evaluated by measuring levels of free IL-7 and slL-7Ralpha-Fc concentrations in blood serum as a function of time. Specific Aim 2: To demonstrate the feasibility of preserving bone mass following estrogen deficiency by expressing a soluble IL-7 decoy receptor in vivo, using a gene transfer approach. This Specific Aim will be achieved by the introduction into ovx mice of a mammalian expression vector encoding slL-7Ralpha-Fc by application of two different gene transfer procedures, intramuscular injection targeting skeletal muscle and hydrodynamic injection targeting the liver. These Specific Aims will demonstrate two important "Proofs of Principle": (1) a novel therapeutic agent slL-7Ralpha-Fc, is an efficacious therapy for the prevention of bone loss following estrogen deficiency. (2) Gene transfer is an effective mechanism for introducing and expressing slL-7Ralpha-Fc in vivo.