In Vivo Gene Therapy for Treatment of Osteoarthritis

描述（由申请人提供）：异常骨形成是骨关节炎（OA）关节病理的主要因素，是OA相关临床症状治疗的关键问题。在类风湿性关节炎（RA）研究中，抑制典型Wnt信号通路拮抗剂Dkk-1，在RA转基因小鼠模型中逆转了骨破坏作用，但诱导骨癣或骨刺的形成，骨刺是OA的标志(8)。RA导致骨吸收，OA导致新骨形成。逆转骨性关节炎骨形成病理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。本提案的具体目的是通过引入Wnt转基因（Wnt10b）和Wnt抑制转基因（Dkk-1）来上调和下调Wnt信号传导，从而促进这一评估。目的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Wnt信号。目的2的重点是在OA大鼠模型中操纵Wnt信号。更好地了解Wnt信号在慢性关节炎出生后关节生物学和关节重塑中的作用，将有助于改善OA病理原因的表征，并促进未来对该疾病的更有效治疗。为了实现我们的目标，我们打算通过体外研究来完善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。细胞和组织转导效率将通过报告基因检测进行评估。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Western blotting评估基因表达和蛋白质产生。Wnt/2-catenin信号的保真度将通过抗2-catenin抗体的Western blotting进行评估。为了确定在体内操纵Wnt10b和Dkk-1信号的有效性，我们将使用由前交叉韧带横断（ACLT）联合内侧半月板切除术（MMx）组成的已建立的大鼠OA模型，该模型在软骨降解和骨刺形成方面模拟了人类OA的发病机制。将含有Wnt10b和Dkk-1转基因的载体注射到手术关节和对照关节中，并通过活体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Western blotting来评估基因治疗引起的改变的效果。

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.