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

一种新型骨靶向 AAV 介导的基因疗法可促进骨质疏松症的骨形成

• 批准号: 10292759
• 负责人: Jae-Hyuck Shim
• 金额: $ 18.43万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10292759
• 关键词: Adaptor Signaling Protein; Adult; Adverse effects; Affinity Chromatography; Age; Aging; American; Anabolic Agents; Angiogenic Factor; Antibodies; Binding Sites; Biodistribution; Biological; Biomechanics; Bone Density; Bone Resorption; Bone necrosis; Bone structure; Candidate Disease Gene; Capsid; Cell Lineage; Cells; Clinical; Data; Deterioration; Disease; Endothelium; Engineering; Equilibrium; Estrogens; FDA approved; Femoral Fractures; Gene Transduction Agent; Gene Transfer; Health; Histology; Histopathology; In Vitro; Infection; Injections; Intravenous; Jaw; Label; Liver; Mass Spectrum Analysis; Measures; Mediating; MicroRNAs; Modification; Mus; Myocardium; Operative Surgical Procedures; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Osteoporotic; Ovariectomy; PTH gene; Pathogenicity; Pathway interactions; Patients; Phenotype; Postmenopausal Osteoporosis; Proteins; Proteomics; RNA Interference; Recombinant adeno-associated virus (rAAV); Repression; Reverse Transcriptase Polymerase Chain Reaction; Senile Osteoporosis; Serotyping; Serum Calcium Level; Skeletal Muscle; Testing; Therapeutic; Therapeutic Agents; Therapeutic Intervention; Tissues; Tropism; Validation; Viral Vector; WNT Signaling Pathway; aged; base; bone; bone aging; bone loss; bone mass; calcification; fracture risk; gene therapy; immunogenicity; improved; in vivo; innovation; knock-down; long bone; mechanical properties; mouse model; new therapeutic target; novel; optical imaging; osteogenic; osteoporosis with pathological fracture; parathyroid hormone-related protein; response; side effect; skeletal; spine bone structure; targeted treatment; therapeutic gene; therapeutic target; transcriptome; transduction efficiency; transgene expression; vector

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.

项目概要 成人骨量由成骨细胞（OB）形成的骨和骨之间的平衡决定 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 吸收导致骨质疏松症。大多数现有的骨质疏松症治疗方法都是通过抑制 OCs 起作用， 但这些方法不能治愈骨质疏松症，并且受到罕见副作用的限制。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）和抗硬化素抗体的存在可促进 成骨细胞（OB）具有治疗骨质疏松症患者的功能。然而，这些代理也受到担忧的限制 脱靶不良反应和疗效减弱。之前，我们和其他人证明了抑制 强效 OB 抑制剂、硬化蛋白 (SOST) 和接头蛋白 schnurri-3 (SHN3)，可促进骨生长 绝经后和老年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进行基于 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向性和转基因表达的 rAAV9 载体，我们将 开发新的基因疗法，通过单一系统促进骨质疏松症的骨形成 行政。此外，我们将确定潜在的新型成骨和/或血管生成因子 SOST/SHN3 通路可作为骨质疏松症的治疗靶点。目标 1 将检查 骨特异性 rAAV9 介导的 SHN3 或 SOST 沉默是否可以逆转小鼠骨质流失 骨质疏松症模型。为了避免非骨骼组织中潜在的脱靶副作用，rAAV9 的骨- 通过衣壳修饰和组织特异性进一步改善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 rAAV9 介导的 SHN3 或 SOST 沉默的治疗潜力 用于绝经后和老年骨质疏松症。目标 2 将识别新的成骨和/或血管生成因子 受 SHN3/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骨驻留 OB 谱系细胞中携带 amiR-shn3 或 amiR-sost 的 rAAV9 的准确性 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 谱系细胞 将从 AAV 处理的 OVX 小鼠中进行 FACS 分选，并进行转录组分析。作为一个 补充方法，蛋白质组学将在 AAV 转导的 OB 谱系细胞中进行，其中分离自 MetRS;Prx1-cre 小鼠在 OVX 手术后具有 OB 特异性标记的新生蛋白。这些组合 方法将使我们能够共同或差异地识别新的成骨和/或血管生成因子 通过 SHN3 和 SOST 通路调节雌激素缺乏引起的骨质疏松症。 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。