A novel bone-targeting AAV-mediated gene therapy to promote bone formation in osteoporosis

项目总结 成人骨量由成骨细胞(OBS)的骨形成和骨之间的平衡决定。 破骨细胞(OCs)的吸收，以及这种平衡的紊乱有利于破骨细胞(OC)介导 吸收会导致骨质疏松症。现有的大多数治疗骨质疏松症的药物都是通过抑制OCS发挥作用的， 但这些药物不能治愈骨质疏松症，而且副作用很少。目前的合成代谢药物甲状旁腺 激素(PTH)、甲状旁腺激素相关蛋白(PTHrP)和抗硬化素抗体存在促进 成骨细胞(OB)具有治疗骨质疏松症的功能。然而，这些代理人也受到担忧的限制。 用于治疗偏离目标的不良反应和疗效减弱。之前，我们和其他人证明了抑制 有效的OB抑制因子，硬化素(Sost)和适配蛋白SchNurri-3(SHN3)，促进骨骼 绝经后和老年性骨质疏松症小鼠模型的形成。一种创新的治疗方法 骨质疏松症是使用重组腺相关病毒的基于RNAi的骨合成代谢基因治疗 (RAAV)。使用我们设计的具有骨特异性定向和转基因表达的rAAV9载体，我们将 开发新的基因疗法，促进骨质疏松症患者的骨形成 行政管理。此外，我们将确定潜在的新的成骨和/或血管生成因子，由 可能成为骨质疏松治疗靶点的SOST/SHN3通路。Aim 1将检查 骨特异性rAAV9介导的SHN3或SOST沉默是否能逆转小鼠的骨丢失 骨质疏松症模型。为了避免非骨骼组织潜在的靶外不良反应，rAAV9‘S骨- 衣壳修饰和组织特异性修饰进一步改善了特异性趋向性和转基因表达 MiRNA介导的rAAV表达抑制。使用两种骨质疏松症小鼠模型(去卵巢 (OVX)和老龄小鼠)，我们将确定rAAV9介导的SHN3或SOST沉默的治疗潜力 绝经后和老年性骨质疏松症。目标2将确定新的成骨和/或血管生成因子 受促进骨质疏松骨形成的SHN3/SOST途径调节。体内沉默 携带Amir-shn3或Amir-Sost的rAAV9在骨驻留的OB谱系细胞中的准确性将通过 整个转录组数据的散点图分析。一旦验证，wt、shn3或sost缺陷的OB系细胞 将从AAV治疗的OVX小鼠中分离出FACS，并进行转录组分析。作为一名 补充方法，蛋白质组学将在AAV转导的OB系细胞中进行，分离自 去卵巢手术后新生蛋白OB特异性标记的pRX1-cre小鼠。这些组合 方法将使我们能够共同或不同地识别新的成骨和/或血管生成因子 在雌激素缺乏引起的骨质疏松中受SHN3和SOST信号通路的调节。 成功完成这些目标将提供概念验证演示并确定 潜在的新型调节剂，可用作骨质疏松症的治疗靶点。

PROJECT SUMMARY Adult bone mass is determined by the balance between bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs), and disturbances in this equilibrium to favor osteoclast (OC)-mediated resorption leads to osteoporosis. The majority of existing therapeutics for osteoporosis act by inhibiting OCs, but these can not cure osteoporosis and are limited by rare side effects. Current anabolic agents, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and anti-sclerostin antibody exist for promoting osteoblast (OB) function to treat patients with osteoporosis. However, these agents are also limited by concern for off-target adverse effects and waning efficacy. Previously, we and others demonstrated that inhibition of potent OB suppressors, sclerostin (SOST) and the adaptor protein schnurri-3 (SHN3), promotes bone formation in mouse models of postmenopausal and senile osteoporosis. One innovative approach to treat osteoporosis is RNAi-based bone anabolic gene therapy using recombinant adeno-associated virus (rAAV). Using our engineered rAAV9 vector with bone-specific tropism and transgene expression, we will develop novel gene therapeutics that promote bone formation in osteoporosis with a single systemic administration. Additionally, we will identify potential novel osteogenic and/or angiogenic factors regulated by the SOST/SHN3 pathway that could be useful as therapeutic targets for osteoporosis. Aim 1 will examine whether bone-specific rAAV9-mediated silencing of SHN3 or SOST can reverse bone loss in mouse models of osteoporosis. To avoid potential off-target adverse effects in non-skeletal tissues, rAAV9’s bone- specific tropism and transgene expression were further improved by capsid modification and tissue-specific miRNA-mediated repression of rAAV expression. Using two mouse models of osteoporosis (ovariectomized (OVX) and aged mice), we will determine therapeutic potentials of rAAV9-mediated silencing of SHN3 or SOST in postmenopausal and senile osteoporosis. Aim 2 will identify novel osteogenic and/or angiogenic factors regulated by the SHN3/SOST pathway that promote bone formation in osteoporosis. In vivo silencing accuracy of rAAV9 carrying amiR-shn3 or amiR-sost in bone-residing OB lineage cells will be examined by scatterplot analysis of whole transcriptome data. Once validated, wt, shn3- or sost-deficient OB-lineage cells will be FACS-sorted from AAV-treated, OVX-mice and subjected for transcriptome profiling. As a complimentary approach, proteomics will be performed in AAV-transduced OB-lineage cells isolated from MetRS;Prx1-cre mice with OB-specific labeling of nascent proteins after OVX-surgery. These combinatory approaches will allow us to identify novel osteogenic and/or angiogenic factors commonly or differentially regulated by the pathways of SHN3 and SOST in response to estrogen deficiency-induced osteoporosis. Successful completion of these aims will provide proof-of-concept demonstration and identify potential novel regulators that could be useful as therapeutic targets for osteoporosis.