The Use of Regional Gene Delivery to Heal Critical Sized Bone Defects

描述(申请人提供)：体外区域基因疗法有可能解决与大量骨缺乏、丰富的疤痕组织和有限的血管供应有关的棘手的骨丢失问题，目前还没有始终如一的满意解决方案。我们成功地用携带BMP-2基因的腺病毒载体(Ad-BMP-2)或慢病毒载体(LV-BMP-2)转导的大鼠和人骨细胞修复了这一策略的潜在临床应用。在最近的一项骨缺损处的修复比较中，Ad-BMP-2与LV-BMP-2治疗获得了质量更好的骨。我们推测，与慢病毒载体相关的更长时间的BMP表达是LV载体获得更好结果的原因，并且这种更长的表达持续时间可能是在患者所见的具有挑战性的生物环境中实现充分的骨修复所必需的。为了将基因疗法应用于临床，它必须是经济有效、安全的，并且必须更好地了解利用转导细胞进行骨修复的生物学，以便成功地管理在人类中看到的广泛变化的骨丢失场景。这项建议的目标是评估一种易于人类使用的“同一天”体外方法的临床潜力；了解转导细胞和宿主细胞在骨形成和修复中的作用；以及评估使用慢病毒载体的体外策略的安全性。在特定目标1中提出的研究将比较“同一天”EV活体策略与标准的两步细胞采集和扩增方法的效果。在特定的目的下，将使用2只在细胞分化特异性启动子控制下表达GFP的转基因小鼠来描述转导细胞和宿主细胞在修复临界大小的骨缺损中的作用。在具体目标3中，将通过生物分布分析和器官毒性评估来评估使用慢病毒载体进行体外基因治疗的安全性。这些建议的目的应该提供关于体外基因治疗用于骨修复的可行性、安全性和生物学方面的有价值的信息。这些数据应该会让我们离实现这一人类用途更近一步。 公共卫生相关性：这项研究提案的最终目标是提高我们对骨修复生物学和区域基因疗法的了解，开发一种新的具有成本效益的治疗策略，并评估这种疗法的安全性，以便使这项技术更接近于人类使用。在许多情况下，我们目前的治疗方案不足以处理难治性骨丢失问题。区域基因疗法有可能提高我们治疗与骨折不愈合、翻修全关节置换术和脊柱融合相关的骨修复难题的能力。

DESCRIPTION (provided by applicant): Ex vivo regional gene therapy has the potential to solve difficult bone loss problems associated with large bone stock deficiency, abundant scar tissue and limited vascular supply for which at the present time there is no consistently satisfactory solution. We have demonstrated proof of concept the potential clinical utility of this strategey by successfully healing critical sized rodent femoral defects with rat and human bone cells transduced with either an adenoviral vector (Ad- BMP-2) or lentiviral vector (LV-BMP-2) containing the cDNA for BMP-2. In a recent comparison of healing of a bone defect with Ad-BMP-2 versus LV-BMP-2 better quality bone was obtained with the LV-BMP-2 treatment. We hypothesized that the more prolonged BMP expression associated with the lentiviral vector was responsible for the better results with the LV vector and that this increased duration of expression may be necessary to achieve adequate bone repair in the challenging biological enviorments seen in patients. In order for gene therapy to be adopted for clinical use it must be cost-effective, safe and there must be a better understanding of the biology of bone repair with transduced cells in order to successfully manage the wide variability bone loss scenarios seen in humans. The goals for this proposal are to assess the clinical potential of a "same day" ex vivo approach that can be easily adapted for human use; understand the role of transduced cells and host cells in bone formation and repair and; evaluate the safety of an ex vivo strategy using a lentiviral vector. The research proposed in Specific Aim 1 will compare the efficacy of a "same day" ev vivo strategy with the standard two step cell harvest and expansion approach. In Specific Aim 2 transgenic mice that express GFP under the control of cell differentiation specific promoters will be used to delineate the role of transduced cells and host cells in the healing of criticial sized bone defects. In Specific Aim 3 the safety of ex vivo gene therapy with a lentiviral vector will be assessed with a biodistribution assay and an evaluation of organ toxicity. These proposed Aims should provide valuable information regarding the feasibility, saftey, and biology of ex vivo gene therapy for bone repair. This data should move us one step closer to implementing this human use. PUBLIC HEALTH RELEVANCE: The ultimate goals of this research proposal are to enhance our knowledge of both the biology of bone repair and regional gene therapy, develop a new cost-effective treatment strategy and assess the safety of this therapy in order to move this technology closer to use in humans. In many cases our present treatment regimens are not adequate to manage difficult bone loss problems. Regional gene therapy has the potential to enhance our ability to treat difficult bone repair problems associated with fracture non-union, revision total joint arthroplasty and spinal fusion.