A Novel Therapeutic Agent for Postmenopausal Bone Loss

描述(申请人提供)：绝经后骨质疏松症是由雌激素(E2)缺乏引起的骨质重塑缺陷所致。我们最近报道，雌激素缺乏时的骨丢失主要是由T细胞来源的细胞因子分泌上调所致，这些细胞因子包括关键的破骨细胞因子、NFkB受体激活剂配体(RANKL)和肿瘤坏死因子α(TNF)。诱导T细胞活化和细胞因子分泌的一个关键事件是上调IL-7的产生，这是雌激素缺乏的结果。我们最近报道，使用中和抗体抑制卵巢切除(OVX)引起的小鼠IL-7水平的升高，在体内预防OVX诱导的小鼠骨丢失是有效的。提示IL-7中和可能是防治骨质疏松症的一种新手段。然而，尽管这项实验取得了成功，但在人类体内长期使用抗体进行治疗往往会导致严重的临床并发症和较差的长期疗效。为了克服基于抗体的治疗的局限性，我们现在提出了一种新的策略，在体内中和IL-7，并改善绝经后骨质疏松症的骨丢失。这一策略包括设计一种新的基于多肽的治疗剂，该治疗剂包含小鼠IL-7受体α(SLL-7Rpha)的可溶性细胞外配体结合域(SLL-7Rpha)。为了延长体内半衰期，该肽将被表达为与人IgG1-Fc区(SLL-7Rpha-Fc)的融合蛋白。我们推测，该制剂将在体内作为可溶性IL-7诱饵受体发挥作用，并通过中和过量的IL-7来防止E2缺乏时的骨丢失。我们提出了两个特定的目标来进一步验证我们的假设：特定的目标1：证明可溶性IL-7诱骗受体在体内预防雌激素缺乏引起的骨丢失方面是有效的治疗辅助工具。这将通过将纯化的重组SLL-7Rpha-Fc注射到去势后的小鼠体内，并检测卵巢切除后骨转换的物理、生化和组织形态计量学指标来实现。SLL-7Rpha-Fc的长期稳定性和生物半衰期将通过测量血清中游离IL-7和SLL-7Rpha-Fc浓度随时间的变化来评估。具体目的2：通过基因转移的方法，在体内表达可溶性IL-7诱骗受体，以证明在雌激素缺乏后保存骨量的可行性。通过应用两种不同的基因转移程序，将编码SLL-7Rpha-Fc的哺乳动物表达载体导入OVX小鼠，以骨骼肌为靶点的肌肉注射和以肝脏为靶点的流体动力注射，将达到这一特定目的。这些特定的目的将证明两个重要的“原则证明”：(1)新型治疗剂SLL-7Rpha-Fc，是预防雌激素缺乏所致骨丢失的有效疗法。(2)基因转移是体内导入和表达sIL-7Rpha-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.