Supporting data for "Regulation of osteoblast function with osteoinductive aptamers"

Bone mass is maintained constant by the coupled action of osteoclasts and osteoblasts through bone remodeling cycles. Patients with low bone mass disorders such as osteoporosis and osteogenesis imperfecta are prone to bone fractures and in severe cases anti-resorptive therapy is ineffective. With limited choices of existing anabolic agents, it is beneficial to develop alternative improved therapeutic approaches. Targeting functional proteins regulating differentiation and activities of osteoblasts with therapeutic antibodies showed promising outcomes in promoting bone growth. Oligonucleotide aptamers are “chemical antibodies” with advantages over conventional antibodies such as specificity and ease of production. Aptamers inhibiting negative regulators of bone formation such as sclerostin (SOST) and WW-containing protein 1 (WWP1) have been generated and functionally tested in vitro. To extend this for in vivo and translational studies, we aim to assess the functions of these aptamers with stabilization modifications and to evaluate their pharmacokinetics and bioavailability. Serum stability of aptamers for SOST and WWP1 were enhanced with inverted thymidine modification at the 3’ end, namely SOST-dT and WWP1-dT, and were tested to verify that the modification did not affect aptamers’ functionality. Metatarsal and femur bones were cultured ex vivo in osteogenic medium supplemented with aptamer SOST-dT. Bone morphometric analysis revealed no significant difference in cortical bone mineral density with and without aptamer SOST-dT treatment, immunohistochemistry examination suggested reduced sclerostin protein expression in an ex vivo setting. Osteoblastic cells incubated with aptamer WWP1-dT in culture medium seemed to have increased RUNX2 protein expression yet one-way ANOVA analysis suggested that it was not statistically significant. Before we can conclude the effect of these aptamers, we elect to evaluate the efficacy of aptamer SOST-dT and WWPT-dT directly in mouse model. Aptamer extraction and quantification assay with SYBR Green qPCR were developed and optimized for monitoring bioavailability and pharmacokinetics in tissues. Aptamer retrieval from serum, bone marrow, liver and kidney have been successfully optimized with various incubation conditions, tissue volume, tissue homogenization methods and effect of DNA carrier tested out. To investigate for PCR interference by inhibiting factors presence in tissues, correction factors and detection range were deduced. These assessments provided key information on optimal injection frequency and dosage for evaluating the efficacy of aptamers in promoting bone formation in mouse models, demonstrating their therapeutic potentials in treating low bone mass disorders.骨量通过破骨细胞（osteoclasts）与成骨细胞（osteoblasts）的协同作用，经由骨重塑（bone remodeling）循环维持恒定。患有骨质疏松症（osteoporosis）、成骨不全症（osteogenesis imperfecta）等骨量减少性疾病的患者易发生骨折，且重症患者对抗吸收治疗应答不佳。现有促骨合成制剂（anabolic agents）的选择十分有限，因此开发更优化的新型治疗方案具有重要价值。以调控成骨细胞分化与活性的功能蛋白为靶点的治疗性抗体（therapeutic antibodies），在促进骨生长方面已展现出良好的应用前景。寡核苷酸适配子（oligonucleotide aptamers）被称为“化学抗体”，相较于传统抗体，其具备特异性强、制备简便等优势。针对骨形成负调控因子（如硬化蛋白sclerostin, SOST以及含WW结构域蛋白1（WW-containing protein 1, WWP1)）的适配子已被成功制备，并完成了体外（in vitro）功能验证。为将该研究拓展至体内（in vivo）实验与转化医学领域，本研究拟对这些适配子进行稳定化修饰，评估其功能，并检测其药代动力学（pharmacokinetics）与生物利用度（bioavailability）。通过在3'端引入反向胸腺嘧啶修饰（inverted thymidine modification），靶向SOST与WWP1的适配子的血清稳定性得到提升，分别命名为SOST-dT与WWP1-dT；后续实验验证了该修饰不会影响适配子的功能活性。将跖骨与股骨置于添加了SOST-dT适配子的成骨培养基中进行离体（ex vivo）培养。骨形态计量学分析（bone morphometric analysis）显示，经SOST-dT适配子处理与否，皮质骨骨密度（cortical bone mineral density）无显著差异；免疫组织化学（immunohistochemistry）检测则表明，离体培养环境中硬化蛋白的蛋白表达水平有所下调。在培养基中加入WWP1-dT适配子培养的成骨细胞，其RUNX2蛋白表达水平看似有所升高，但单因素方差分析（one-way ANOVA）结果显示该差异无统计学意义。在最终明确这些适配子的作用效果前，我们决定直接在小鼠模型中评估SOST-dT与WWPT-dT适配子的疗效。我们开发并优化了基于SYBR Green实时定量聚合酶链反应（SYBR Green qPCR）的适配子提取与定量检测方法，用于检测组织内适配子的生物利用度与药代动力学特征。通过优化多种孵育条件、组织样本体积、组织匀浆方法，并验证了DNA载体的影响，我们已成功建立了从血清、骨髓、肝脏与肾脏中回收适配子的方法。为探究组织中存在的抑制因子对PCR反应的干扰情况，我们推导得出了校正因子与检测范围。上述实验评估为确定小鼠模型中适配子促进骨形成的最佳注射频率与给药剂量提供了关键依据，同时证实了这些适配子在治疗骨量减少性疾病中的治疗潜力。