Regional Gene Therapy to Enhance Bone Repair

项目摘要 这项建议的目标是评估人类骨髓细胞的骨修复潜力。 用含有BMP-2基因的慢病毒载体进行转导。有很多种 没有始终如一的令人满意的解决方案的骨修复困难场景包括： 骨折不愈合，急性骨折伴广泛骨丢失，翻修全关节置换术和 脊柱假关节脊柱假关节。传统上，自体骨移植一直是黄金标准，但 这种骨骼的供应有限，人们对相关的发病率感到担忧 有了嫁接收获。重组骨形态发生蛋白(BMP)获FDA批准使用 在脊柱融合和新鲜胫骨骨折的治疗中。然而，BMP的成功与否参差不齐 并与包括软组织水肿和异位在内的副作用有关 骨化。骨科医生一直在寻找替代组织工程的方法 加强骨骼修复的策略。我们的计划是开发区域性基因疗法，使用 转导骨髓细胞作为综合组织工程策略的一个方面 加强骨修复。 已经在动物模型中证明了用BMP进行基因治疗可以产生骨髓 通过慢病毒基因转移产生的细胞有可能治疗中型或大型骨骼 缺陷或当宿主的生物环境受到严重损害时。在本建议书中 我们计划测试我们的假设，即人类骨髓细胞被慢病毒载体转导 过表达BMP-2有可能促进人类的骨修复。人骨 将从接受全髋关节手术的患者的髓内管中获取骨髓细胞 关节成形术。细胞将被慢病毒载体(LV-TSTA-IC9/BMP-2)转导，该载体可 含有BMP-2的cDNA和一个可诱导的自杀基因(ICaspase 9)，然后植入 在一种临界大小的裸鼠股骨缺损区模型中。我们提出以下目标： 完成的目标包括：1)比较骨后局部基因治疗的疗效 用新鲜分离或培养的扩增的人骨髓细胞修复；2)建立 可放大用于人类的转导细胞的“细胞剂量”；以及3)确定 骨形态发生蛋白转导的人细胞的局部和系统生物分布及器官评价 毒性、载体遗传毒性和异位骨化。从这项提案中获得的数据 将提供有关将这一区域基因治疗策略调整为 人类的治疗方案。

Project Summary The goal of this proposal is to assess the bone repair potential of human bone marrow cells transduced with a lentiviral vector containing the cDNA for BMP-2. There are a number of difficult bone repair scenarios for which there is no consistently satisfactory solution including: fracture nonunion, acute fractures with extensive bone loss, revision total joint arthroplasty and pseudarthrosis of the spine. Traditionally, autologous bone graft has been the gold standard but there is a limited supply of this bone and there are concerns regarding the morbidity associated with graft harvest. Recombinant bone morphogenetic proteins (BMP) are FDA approved for use in spinal fusion and treatment of fresh tibial fractures. However, BMPs have had mixed success in humans and are associated with side effects including soft tissue edema and heterotopic ossification. Orthopaedic surgeons have been searching for alternative tissue engineering strategies to enhance bone repair. Our plan is to develop regional gene therapy using transduced bone marrow cells as one aspect of a comprehensive tissue engineering strategy to enhance bone repair. It has been demonstrated in animal models that gene therapy with BMP producing bone marrow cells created via lentiviral gene transfer has the potential to treat either medium or large bone defects or when the biological environment of the host is severely compromised. In this proposal we plan to test our hypothesis that human bone marrow cells transduced with a lentiviral vector to overexpress BMP-2 have the potential to enhance bone repair in humans. Human bone marrow cells will be harvested from the intramedullary canals of patients undergoing total hip arthroplasty. The cells will be transduced with a lentiviral vector (LV-TSTA-iC9/BMP-2) that contains the cDNA for BMP-2 and an inducible suicide gene (iCaspase 9) and then implanted in a critical sized femoral defect model in nude rats. We propose the following aims to accomplish the goal including: 1) compare the efficacy of regional gene therapy after bone repair with freshly isolated or cultured expanded human bone marrow cells; 2) establish a “cellular dose” of transduced cells that can be scaled up for use in humans; and 3) determine the local and systemic biodistribution of BMP transduced human cells and assess for organ toxicity, vector genotoxicity and heterotopic ossification. The data obtained from this proposal will provide critical information with respect to adapting this regional gene therapy strategy as a treatment regimen in humans.