In Vivo Gene Therapy for Treatment of Osteoarthritis

描述（由申请方提供）：骨形成异常是骨关节炎（OA）关节病理学的主要因素，是提出OA相关临床症状治疗方案时的关键问题。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，但可诱导骨赘或骨刺（OA的标志物）的形成（8）。RA导致骨吸收，而OA导致新骨形成。逆转OA的骨形成病理学的一种策略可以是逆转上述策略，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。该提案的具体目的旨在通过引入Wnt转基因（Wnt10b）和Wnt抑制转基因（Dkk-1）上调和下调Wnt信号传导，从而有助于该评估。目的1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。目的2是在OA大鼠模型中体内操纵Wnt信号。对Wnt信号在出生后关节生物学和慢性关节炎关节重塑中的作用的更好理解将导致OA病理学原因的更好表征，并促进未来对这种疾病的更有效治疗。为了实现我们的目标，我们打算使用体外研究来完善几种候选重组腺相关病毒载体（rAAV）血清型对关节炎细胞单层和组织外植体的能力。将使用报告基因试验评估细胞和组织转导效率。然后将细胞和外植体用含有Wnt10b和Dkk-1的载体转导，并使用RT-PCR和Western印迹法评估基因表达和蛋白质产生。Wnt/2-连环蛋白信号传导的保真度将通过使用抗2-连环蛋白抗体的蛋白质印迹来评估。为了确定体内操纵Wnt10b和Dkk-1信号传导的有效性，我们将使用建立的大鼠OA模型，该模型由前交叉韧带横断（ACLT）和内侧韧带切除（MMx）组成，其在软骨降解和骨赘形成方面模拟人类OA的发病机制。将含有Wnt10b和Dkk-1转基因的载体注射到手术和对照关节中，并使用实时成像和确证性组织切片进行数字成像、半定量组织病理学分级和Western印迹法评估基因治疗诱导的变化的疗效。 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岁以后表现出骨关节炎的一些放射学证据，这是功能障碍和独立性降低的主要原因。这项研究可能会导致一种侵入性更小，创伤更小，基于基因治疗的方法来治疗这种使人衰弱的疾病。

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.