Delivery of Soluble FGFR3 as a Treatment for Achondroplasia

描述（由申请人提供）：软骨发育不全是最常见的短肢侏儒症，大约每30000个活产儿中就有1个。患病儿童长骨发育异常，脊椎骨和颅骨变形。软骨发育不全是无法治愈的，现有的治疗方法只能解决一些并发症。软骨发育不全是由成纤维细胞生长因子受体-3 （FGFR3）跨膜区突变引起的。野生型FGFR3是软骨内骨生长的众多抑制因子之一，与FGF配体相互作用后，对生长板软骨细胞的增殖和终末分化均产生负向作用。在98%的软骨发育不全患者中，这种表型是由FGFR3的特定点突变引起的，导致380位的精氨酸取代甘氨酸（Gly380Arg）。这种突变是一种“功能获得”，即增加配体介导的FGFR3信号传导，导致骨生长过度抑制。与通过全身给药可溶性肿瘤坏死因子1受体治疗类风湿关节炎类似，我们假设全身递送可溶性FGFR3分子同样会滴定受体特异性FGF配体，从而减少异常FGFR3信号以挽救骨生长。通过转基因插入突变FGFR3基因的小鼠同源基因（FGFR3G374R）产生的小鼠软骨发育不全模型的初步实验显示出很大的希望。天然存在的FGFR3分泌亚型的基因传递(FGFR3？（TM）对新生软骨发育不全小鼠股四头肌的作用被发现可提供FGFR3？TM进入循环。适当剂量的基因传递载体产生循环FGFR3水平？TM足以恢复骨骼生长，因此，在四周时，治疗小鼠的大小与正常窝鼠基本没有区别。转基因模型在我们的概念验证研究中非常有用。然而，某些特征可能不自然地影响FGFR3治疗的生物反应幅度。TM。因此，为了探索可溶性FGFR3抑制剂在与人类软骨发育不全突变基因型和表型更相关的背景下的治疗潜力，我们将扩展这些研究，包括小鼠FGFR3G374R敲入软骨发育不全模型。我们将讨论与支持骨骼生长恢复机制相关的问题，以及这种治疗方法的潜在临床应用。我们将解决以下具体目标：(1)确定FGFR3的容量？TM结合fgf配体，从而抑制转基因和敲入软骨发育不全模型生长板软骨细胞中异常的FGFR3G374R信号。(2)确定FGFR3长期递送的影响？TM对FGR3G374R敲入软骨发育不全模型骨骼生长和生理的影响。(3)确定重组可溶性FGFR3对FGFR3G374Rneo-/+小鼠骨生长的恢复能力。(4)确定干预年龄与可溶性FGFR3治疗后骨骼反应程度之间的关系。

DESCRIPTION (provided by applicant): Achondroplasia, the most common form of short-limb dwarfism, occurs in approximately 1 in 30,000 live births. Affected children suffer from abnormal long bone development and deformations of the vertebrae and bones in the skull. There is no cure for achondroplasia, and existing treatments only address some of the complications. Achondroplasia is caused by a mutation in the transmembrane region of fibroblast growth factor receptor-3 (FGFR3). Wild type FGFR3 is one of many inhibitory regulators of endochondral bone growth, and following interaction with FGF ligand acts negatively on both proliferation and terminal differentiation of growth plate chondrocytes. In 98% of those with achondroplasia, the phenotype is caused by a specific point mutation in FGFR3, resulting in the substitution arginine for glycine at position 380 (Gly380Arg). This mutation is one of "gain-of-function," i.e. increased ligand-mediated signaling of FGFR3, which leads to excessive inhibition of bone growth. Similar to the treatment of rheumatoid arthritis through the systemic administration of soluble receptors for tumor necrosis factor 1, we hypothesized that systemic delivery of a soluble FGFR3 molecule would likewise titrate receptor-specific FGF ligands and thereby reduce aberrant FGFR3 signaling to rescue bone growth. Initial experiments in a murine model of achondroplasia generated by transgenic insertion of the murine orthologue of the mutant FGFR3 gene (FGFR3G374R), have shown great promise. Gene delivery of a naturally-occurring, secreted isoform of FGFR3 (FGFR3?TM) to the quadriceps of neonatal achondroplastic mice was found to provide sustained release of FGFR3?TM into the circulation. Appropriate dosing of the gene delivery vector generated levels of circulating FGFR3?TM sufficient to rescue bone growth, such that treated mice were essentially indistinguishable in size from normal littermates at four weeks. The transgenic model has been extremely useful in our proof-of-concept studies. However, certain features may unnaturally influence the amplitude of the biological response to treatment with FGFR3?TM. Therefore, to explore the treatment potential of soluble FGFR3 inhibitors in a context more relevant to the human achondroplasia mutant genotype and phenotype, we will extend these investigations to include the murine FGFR3G374R knock-in achondroplasia model. We will address issues relevant to the mechanisms supporting rescue of skeletal growth and to the potential clinical application of this treatment approach. We will address the following Specific Aims: (1) To determine the capacity of FGFR3?TM to bind FGF-ligand and thereby inhibit aberrant FGFR3G374R signaling in growth plate chondrocytes from transgenic and knock-in models of achondroplasia. (2) To determine the effects of long-term delivery of FGFR3?TM on the skeletal growth and physiology of the FGR3G374R knock-in achondroplasia model. (3) To determine the capacity of administration of a recombinant soluble FGFR3 to rescue bone growth in FGFR3G374Rneo-/+ mice. (4) To determine the relationship between age of intervention and the magnitude of the skeletal response following treatment with soluble FGFR3.