In Vivo Gene Therapy for Treatment of Osteoarthritis

异常骨形成是骨关节炎(OA)关节病理中的一个主要因素，在提出与OA相关的临床症状的治疗方案时是一个关键问题。在类风湿性关节炎(RA)的研究中，抑制典型的Wnt信号通路的拮抗剂DKK-1逆转了RA转基因小鼠模型的骨破坏效应，但诱导了骨赘或骨刺的形成，骨刺是OA的标志(8)。类风湿性关节炎导致骨吸收，骨性关节炎导致新骨形成。逆转骨性关节炎骨形成病理的一个策略可能是逆转上述策略，即上调Wnt拮抗剂DKK-1的表达。 我们的长期目标是利用一种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。这项建议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，从而有助于这一评估。目的1着重于确定最有效的基因转移策略，以有效地调控关节组织中的Wnt信号。目的2致力于在大鼠骨性关节炎模型中操纵Wnt信号。更好地理解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，将有助于更好地表征OA的病理原因，并有助于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提炼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。细胞和组织的转导效率将使用报告基因分析进行评估。然后用含有Wnt10b和DKK-1的载体转导细胞和外植体，并用RT-PCR和Western blotting检测基因表达和蛋白质产量。Wnt/2-catenin信号的保真度将通过抗2-catenin抗体的Western blotting进行评估。 为了确定在体内调控Wnt10b和DKK-1信号的有效性，我们将使用一种已建立的大鼠OA模型，该模型包括前交叉韧带横断(ACLT)和内侧半月板切除(MMX)，该模型在软骨降解和骨赘形成方面模仿人类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，并将使用数字成像、半定量组织病理学分级和Western blotting的实时成像和确证组织切片来评估基因治疗诱导的变化的有效性。

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.