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

项目摘要 成人骨量是由成骨细胞（OB）和骨形成之间的平衡决定的。 破骨细胞（OC）的吸收，以及这种平衡的紊乱有利于破骨细胞（OC）介导的 再吸收导致骨质疏松症。大多数现有的骨质疏松症治疗剂通过抑制OC起作用， 但这些药物不能治愈骨质疏松症，并且受到罕见副作用的限制。当前合成代谢药物，甲状旁腺 激素（PTH）、甲状旁腺相关蛋白（PTHrP）和抗硬化蛋白抗体存在用于促进 成骨细胞（OB）的功能，以治疗骨质疏松症患者。不过，这些代理商也受到关注的限制 治疗脱靶副作用和疗效下降以前，我们和其他人证明， 有效的OB抑制剂，硬化素（SOST）和衔接蛋白schnurri-3（SHN 3），促进骨 在绝经后和老年骨质疏松症的小鼠模型中形成。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）。使用我们的工程rAAV 9载体与骨特异性向性和转基因表达，我们将 开发新的基因疗法，促进骨质疏松症的骨形成， 局此外，我们还将鉴定出受以下因素调节的潜在的新型成骨和/或血管生成因子： SOST/SHN 3通路可能作为骨质疏松症的治疗靶点。目标1将检查 骨特异性rAAV 9介导的SHN 3或SOST沉默是否可以逆转小鼠的骨丢失 骨质疏松症的模型。为了避免在非骨骼组织中的潜在脱靶不良反应，rAAV 9的骨- 通过衣壳修饰和组织特异性修饰，进一步提高了特异性向性和转基因表达 miRNA介导的rAAV表达抑制。使用两种骨质疏松症小鼠模型（卵巢切除 (OVX)和老年小鼠），我们将确定rAAV 9介导的SHN 3或SOST沉默的治疗潜力。 绝经后和老年性骨质疏松症。目标2将鉴定新的成骨和/或血管生成因子 由SHN 3/SOST通路调节，促进骨质疏松症中的骨形成。体内沉默 将通过以下方法检查携带amiR-shn 3或amiR-sost的rAAV 9在骨驻留的OB谱系细胞中的准确性 全转录组数据的散点图分析。一旦验证，wt、shn 3或sost缺陷型OB谱系细胞 将从AAV处理的OVX小鼠中进行FACS分选，并进行转录组谱分析。作为 作为补充方法，蛋白质组学将在从分离的AAV转导的OB谱系细胞中进行， MetRS; Prx 1-cre小鼠，具有OVX手术后新生蛋白质的OB特异性标记。这些组合 方法将使我们能够识别新的成骨和/或血管生成因子共同或差异 通过SHN 3和SOST途径调节雌激素缺乏诱导的骨质疏松症。 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.