In Vivo Gene Therapy for Treatment of Osteoarthritis

治疗骨关节炎的体内基因疗法

• 批准号: 7913900
• 负责人: JEFFREY B. MASON
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7913900
• 关键词: Adult; Age; Anterior Cruciate Ligament; Antibodies; Arthritis; Biological Assay; Biology; Bone Resorption; Cartilage; Cells; Chronic; Clinical Treatment; Degenerative polyarthritis; Disease; Disease Progression; Effectiveness; Excision; Exostoses; Future; Gene Expression; Gene Transfer; Goals; Human; Image; In Vitro; Joints; Lead; Life; Medial meniscus structure; Modeling; Operative Surgical Procedures; Osteoarthrosis Deformans; Osteogenesis; Pathogenesis; Pathology; Production; Proteins; Rattus; Recombinants; Reporter Genes; Research; Reverse Transcriptase Polymerase Chain Reaction; Rheumatoid Arthritis; Role; Serotyping; Signal Pathway; Signal Transduction; Symptoms; Tissues; Transgenes; Transgenic Mice; Up-Regulation; Western Blotting; adeno-associated viral vector; bone; cellular transduction; design; digital imaging; effective therapy; functional disability; gene therapy; improved; in vivo; monolayer; mouse model; postnatal; public health relevance; transduction efficiency; vector

DESCRIPTION (provided by applicant): Aberrant bone formation, a primary factor in osteoarthritis (OA) joint pathology, is a key issue when proposing treatments for the clinical symptoms associated with OA. In rheumatoid arthritis (RA) research, inhibition of Dkk-1, an antagonist of the canonical Wnt signaling pathway, reversed the bone destructive effect in a transgenic mouse model of RA, but induced the formation of osteophytes, or bone spurs, a marker of OA (8). Whereas RA leads to bone resorption, OA leads to formation of new bone. One strategy to reverse the bone- forming pathology of OA may be to reverse the strategy mentioned above, an up-regulation of the Wnt- antagonist Dkk-1. Our long-term goal is to utilize a gene therapy-based approach to assess the regulatory role of Wnt signaling in the pathogenesis of OA. The specific aims of this proposal are designed to contribute to this assessment by up- and down-regulating Wnt signaling through introduction of a Wnt transgene (Wnt10b) and a Wnt-inhibiting transgene (Dkk-1). Aim 1 is focused on determining the most efficient gene transfer strategy for effective regulatory control of Wnt signaling in articular tissues. Aim 2 is focused on manipulating Wnt signaling in vivo in a rat model of OA. An improved understanding of the role of Wnt signaling in postnatal joint biology and joint remodeling in chronic arthritis will lead to improved characterization of the causes of OA pathologies and facilitate more effective treatments for this disease in the future. To achieve our goals, we intend to use an in vitro study to refine the ability of several candidate recombinant adeno-associated viral vector (rAAV) serotypes to transduce cell monolayers and tissue explants from articular joints. Cell and tissue transduction efficiency will be assessed using reporter gene assays. Cells and explants will then be transduced with Wnt10b and Dkk-1-containing vectors and gene expression and protein production will be assessed using RT-PCR and Western blotting. Fidelity of Wnt/2-catenin signaling will be assessed by Western blotting with anti-2-catenin antibodies. To determine the effectiveness of manipulating Wnt10b and Dkk-1 signaling in vivo, we will use an established rat OA model consisting of anterior cruciate ligament transection (ACLT) in combination with resection of the medial menisci (MMx), which mimics the pathogenesis of human OA in terms of cartilage degradation and osteophyte formation. Vectors containing Wnt10b and Dkk-1 transgenes will be injected into surgical and control joints and the efficacy of gene therapy-induced changes will be assessed using live-imaging and confirmatory tissue sections for digital imaging, semi-quantitative histopathological grading and Western blotting. PUBLIC HEALTH RELEVANCE: The goal of this proposed research is to identify mechanisms that regulate disease progression in osteoarthritic joints. Approximately 80% of adults show some radiographic evidence of osteoarthritis, a major contributor to functional impairment and reduced independence, after the age of age of 65. This study may lead to a less invasive and less traumatic, gene therapy-based approach to treatment of this debilitating disease.

描述（由申请人提供）：骨形成异常是骨关节炎（OA）关节病理学的主要因素，在提出治疗与 OA 相关的临床症状时是一个关键问题。在类风湿性关节炎 (RA) 研究中，抑制 Dkk-1（经典 Wnt 信号通路的拮抗剂）可逆转 RA 转基因小鼠模型中的骨破坏作用，但会诱导骨赘或骨刺（OA 标志物）的形成 (8)。 RA 导致骨吸收，而 OA 则导致新骨形成。逆转 OA 骨形成病理学的一种策略可能是逆转上述策略，即上调 Wnt 拮抗剂 Dkk-1。我们的长期目标是利用基于基因治疗的方法来评估 Wnt 信号在 OA 发病机制中的调节作用。该提案的具体目标旨在通过引入 Wnt 转基因 (Wnt10b) 和 Wnt 抑制转基因 (Dkk-1) 来上调和下调 Wn​​t 信号传导，从而为这一评估做出贡献。目标 1 的重点是确定最有效的基因转移策略，以有效调控关节组织中的 Wnt 信号传导。目标 2 重点是在 OA 大鼠模型中体内操纵 Wnt 信号传导。更好地了解 Wnt 信号在产后关节生物学和慢性关节炎关节重塑中的作用，将有助于更好地了解 OA 病理原因，并促进未来对该疾病进行更有效的治疗。为了实现我们的目标，我们打算利用体外研究来改进几种候选重组腺相关病毒载体（rAAV）血清型转导关节细胞单层和组织外植体的能力。将使用报告基因测定来评估细胞和组织转导效率。然后用含有 Wnt10b 和 Dkk-1 的载体转导细胞和外植体，并使用 RT-PCR 和蛋白质印迹评估基因表达和蛋白质生产。 Wnt/2-连环蛋白信号传导的保真度将通过使用抗 2-连环蛋白抗体进行蛋白质印迹来评估。为了确定体内操纵 Wnt10b 和 Dkk-1 信号传导的有效性，我们将使用已建立的大鼠 OA 模型，该模型由前交叉韧带横断 (ACLT) 结合内侧半月板 (MMx) 切除组成，该模型在软骨退化和骨赘形成方面模拟人类 OA 的发病机制。含有 Wnt10b 和 Dkk-1 转基因的载体将被注射到手术关节和对照关节中，并使用实时成像和验证性组织切片进行数字成像、半定量组织病理学分级和蛋白质印迹来评估基因治疗引起的变化的功效。 公共卫生相关性：这项拟议研究的目标是确定调节骨关节炎关节疾病进展的机制。大约 80% 的成年人在 65 岁之后表现出一些骨关节炎的影像学证据，骨关节炎是导致功能障碍和独立性降低的主要原因。这项研究可能会带来一种侵入性较小、创伤较小的基于基因治疗的方法来治疗这种使人衰弱的疾病。