PDF(Sci-hub)
Abstract：
UNASSIGNED: Sclerosteosis, a severe autosomal recessive sclerosing skeletal dysplasia characterised by excessive bone formation, is caused by absence of sclerostin, a negative regulator of bone formation that binds LRP5/6 Wnt co-receptors. Current treatment is limited to surgical management of symptoms arising from bone overgrowth. This study investigated the effectiveness of sclerostin replacement therapy in a mouse model of sclerosteosis.
UNASSIGNED: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (μCT).
UNASSIGNED: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively.
UNASSIGNED: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis.
UNASSIGNED: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.
UNASSIGNED: Recombinant wild type mouse sclerostin (mScl) and novel mScl fusion proteins containing a C-terminal human Fc (mScl hFc), or C-terminal human Fc with a poly-aspartate motif (mScl hFc PD), were produced and purified using mammalian expression and standard chromatography methods. In vitro functionality and efficacy of the recombinant proteins were evaluated using three independent biophysical techniques and an in vitro bone nodule formation assay. Pharmacokinetic properties of the proteins were investigated in vivo following a single administration to young female wild type (WT) or SOST knock out (SOST-/-) mice. In a six week proof-of-concept in vivo study, young female WT or SOST-/- mice were treated with 10 mg/kg mScl hFc or mScl hFc PD (weekly), or 4.4 mg/kg mScl (daily). The effect of recombinant sclerostin on femoral cortical and trabecular bone parameters were assessed by micro computed tomography (μCT).
UNASSIGNED: Recombinant mScl proteins bound to the extracellular domain of the Wnt co-receptor LRP6 with high affinity (nM range) and completely inhibited matrix mineralisation in vitro. Pharmacokinetic assessment following a single dose administered to WT or SOST-/- mice indicated the presence of hFc increased protein half-life from less than 5 min to at least 1.5 days. Treatment with mScl hFc PD over a six week period resulted in modest but significant reductions in trabecular volumetric bone mineral density (vBMD) and bone volume fraction (BV/TV), of 20% and 15%, respectively.
UNASSIGNED: Administration of recombinant mScl hFc PD partially corrected the high bone mass phenotype in SOST-/- mice, suggesting that bone-targeting of sclerostin engineered to improve half-life was able to negatively regulate bone formation in the SOST-/- mouse model of sclerosteosis.
UNASSIGNED: These findings support the concept that exogenous sclerostin can reduce bone mass, however the modest efficacy suggests that sclerostin replacement may not be an optimal strategy to mitigate excessive bone formation in sclerosteosis, hence alternative approaches should be explored.
摘要：
■硬化，以过度骨形成为特征的严重常染色体隐性硬化性骨骼发育不良，是由于缺乏硬化蛋白引起的，结合LRP5/6Wnt共受体的骨形成的负调节物。目前的治疗仅限于由骨过度生长引起的症状的手术治疗。这项研究调查了硬化蛋白替代疗法在硬化病小鼠模型中的有效性。
■重组野生型小鼠硬化蛋白（mScl）和含有C末端人Fc（mSclhFc）的新型mScl融合蛋白，或具有聚天冬氨酸基序（mSclhFcPD）的C末端人Fc，使用哺乳动物表达和标准层析方法产生和纯化。使用三种独立的生物物理技术和体外骨结节形成测定来评估重组蛋白的体外功能和功效。在对年轻雌性野生型(WT)或SOST敲除(SOST-/-)小鼠单次施用后,在体内研究蛋白质的药代动力学性质。在为期六周的体内概念验证研究中，年轻的雌性WT或SOST-/-小鼠用10mg/kgmSclhFc或mSclhFcPD治疗（每周），或4.4mg/kgmScl（每日）。通过显微计算机断层扫描（μCT）评估重组硬骨素对股骨皮质和小梁骨参数的影响。
重组mScl蛋白以高亲和力(nM范围)与Wnt共受体LRP6的胞外结构域结合,并在体外完全抑制基质矿化。向WT或SOST-/-小鼠施用单剂量后的药代动力学评估表明hFc的存在使蛋白质半衰期从小于5分钟增加到至少1.5天。用mSclhFcPD治疗超过6周的时间导致小梁体积骨矿物质密度(vBMD)和骨体积分数(BV/TV)的适度但显著的降低,20%和15%，分别。
■施用重组mSclhFcPD部分纠正了SOST-/-小鼠的高骨量表型，提示在SOST-/-硬化病小鼠模型中,以提高其半衰期为目标的硬化素能够负向调节骨形成。
■这些发现支持外源性硬化素能减少骨量的概念，然而，适度的疗效表明，硬骨素替代可能不是缓解硬骨病中骨过度形成的最佳策略。因此，应该探索替代方法。
■重组野生型小鼠硬化蛋白（mScl）和含有C末端人Fc（mSclhFc）的新型mScl融合蛋白，或具有聚天冬氨酸基序（mSclhFcPD）的C末端人Fc，使用哺乳动物表达和标准层析方法产生和纯化。使用三种独立的生物物理技术和体外骨结节形成测定来评估重组蛋白的体外功能和功效。在对年轻雌性野生型(WT)或SOST敲除(SOST-/-)小鼠单次施用后,在体内研究蛋白质的药代动力学性质。在为期六周的体内概念验证研究中，年轻的雌性WT或SOST-/-小鼠用10mg/kgmSclhFc或mSclhFcPD治疗（每周），或4.4mg/kgmScl（每日）。通过显微计算机断层扫描（μCT）评估重组硬骨素对股骨皮质和小梁骨参数的影响。
重组mScl蛋白以高亲和力(nM范围)与Wnt共受体LRP6的胞外结构域结合,并在体外完全抑制基质矿化。向WT或SOST-/-小鼠施用单剂量后的药代动力学评估表明hFc的存在使蛋白质半衰期从小于5分钟增加到至少1.5天。用mSclhFcPD治疗超过6周的时间导致小梁体积骨矿物质密度(vBMD)和骨体积分数(BV/TV)的适度但显著的降低,20%和15%，分别。
■施用重组mSclhFcPD部分纠正了SOST-/-小鼠的高骨量表型，提示在SOST-/-硬化病小鼠模型中,以提高其半衰期为目标的硬化素能够负向调节骨形成。
■这些发现支持外源性硬化素能减少骨量的概念，然而，适度的疗效表明，硬骨素替代可能不是缓解硬骨病中骨过度形成的最佳策略。因此，应该探索替代方法。
