Laser-guided gene therapy for cartilage defects

描述（由申请人提供）：肌肉骨骼研究的最新进展极大地受益于分子生物学的进步，揭示了特定基因在骨关节炎（OA）中的作用。在这种以基因为中心的模式中，OA的发病机制是由于软骨损伤后缺乏适当的基因表达。因此，诱导所需基因的表达至关重要。不幸的是，缺乏一个安全有效的基因传递系统，禁止这种方法的基因治疗关节软骨修复。LAGeT，LLC开发了一种专有技术，旨在以位点特异性方式安全有效地递送和激活目的基因。这种技术被称为光激活基因转导方法（LAGT），其工作原理是用激光的长波紫外线（UVA）照射靶组织，从而诱导宿主的DNA修复酶，促进重组腺相关病毒（rAAV）第二链的合成。激光照射的细胞的后续rAAV感染导致有效的转导，而未照射的旁观者细胞保持未转导。生长/分化因子5（GDF 5）是最有前途的基因治疗之一，因为它负责发育过程中的软骨形成。先前已经表明，GDF 5可以在缺损内的关节软骨细胞中被激活，启动适当的修复反应。因此，我们建议使用GDF 5验证LAGT技术。LAGT技术和支持性体外和体内数据引起了骨科专业公司的极大兴趣，并正在寻找其基因的输送系统（即DePuy、Medtronic、Genzyme、斯特赖克）。已经形成了一个共识，即在LAGT技术被许可或收购之前，LAGeT LLC必须进行体内实验，以证明LAGT治疗关节软骨缺损的商业潜力。因此，我们提出了这些在兔关节软骨缺损模型中的临床前实验：1）确定对体内关节软骨细胞无害的UVA的最大剂量; 2）确定用于关节软骨的激光激活基因转导的最佳UVA剂量范围，其由体内最大rAAV-eGFP转导和最小软骨细胞凋亡定义;和3）在兔模型中验证激光激活的GDF 5基因治疗关节软骨缺损。公共卫生相关性：用于组织修复和再生的有效基因治疗需要将位点特异性基因递送到受损组织的边缘。为此，LAGeT LLC正在开发一种专有技术，旨在实现激光引导基因递送。在这里，我们将评估这种技术在与骨关节炎相关的软骨模型中的疗效。

DESCRIPTION (provided by applicant): Recent progress in musculoskeletal research has greatly benefited from the advances in molecular biology uncovering the role of specific genes in osteoarthritis (OA). In this gene- centered paradigm, OA pathogenesis results from the lack of appropriate gene expression follow cartilage injury. Therefore inducing the expression of desirable genes is critical. Unfortunately, the absence of a safe and effective gene delivery system has prohibited this approach of gene therapy for articular cartilage repair. LAGeT, LLC has developed a proprietary technology that aims for safe and efficient delivery and activation of a gene of interest in a site- specific manner. This technology, termed light-activated gene transduction method (LAGT), works by irradiating the target tissue with long wave ultraviolet light (UVA) from a laser, thus inducing the host's DNA repair enzymes needed to promote recombinant adeno-associated virus (rAAV) second-strand synthesis. Subsequent rAAV infection of laser-irradiated cells leads to efficient transduction, while non-irradiated bystander cells remain untransduced. Growth/differentiation factor 5 (GDF5) is one of the most promising for gene therapy because it is responsible for chondrogenesis during development. It has been shown previously that GDF5 could be activated in articular chondrocytes within the defect, initiating appropriate repair response. We therefore propose to use GDF5 for validation of LAGT technology. The LAGT technology and supporting in vitro and in vivo data have generated serious interest from the companies specializing in the orthopedics and searching for a delivery system for their genes (i.e. DePuy, Medtronic, Genzyme, Stryker). A consensus opinion has been formed that before LAGT technology is to be licensed or acquired, LAGeT LLC must perform in vivo experiments to demonstrate the commercial potential of LAGT for articular cartilage defects. Therefore, we propose these pre-clinical experiments in a rabbit articular cartilage defects model to: 1) determine the maximum dose of UVA that is not harmful to articular chondrocytes in vivo; 2) determine the optimal UVA dose range for laser-activated gene transduction of articular cartilage as defined by the maximal rAAV-eGFP transduction and minimal chondrocyte apoptosis in vivo; and 3) validate laser-activated GDF5 gene therapy of articular cartilage defects in a rabbit model. PUBLIC HEALTH RELEVANCE: Effective gene therapy for tissue repair and regeneration requires site-specific gene delivery to the edge of the damaged tissue. To this end, LAGeT LLC is developing a proprietary technology designed to achieve laser-guided gene delivery. Here we will evaluate the efficacy of this technology in a cartilage model that is relevant to osteoarthritis.