AAV-mediated gene therapy for metabolic bone disease

描述（由申请人提供）：腺相关病毒（AAV）载体是长期治疗代谢缺陷的理想载体。该载体具有无致病性、低免疫原性和稳定表达等特点，已成功进行了临床前和临床评价。使用rAAV的基因治疗的一个潜在的、尚未探索的领域是代谢性骨缺陷，其特征在于如骨质疏松症中的每单位体积的骨质量减少或如脊柱融合和骨折中的合成代谢性骨重建机制不足。尽管目前可用的用于骨质减少的激素和药物疗法旨在防止破骨细胞进一步破坏骨，但通过增加成骨事件来增加骨量的疗法将是非常有益的。通过合成代谢剂增加骨密度的治疗由于无效的递送方法和药物和纯化蛋白质的短半衰期而受到限制。因此，新的方法来诱导持续的体内成骨应改善疾病的病理生理。 我们早期的研究建立了rAAV转导的间充质干细胞（MSC）在小鼠模型中选择性移植到骨、重新增殖和表达转基因的长期功效。与该应用相关的初步研究表明，重组AAV-2（rAAV）以高效率转导人和鼠MSC和骨祖细胞，并且rAAV介导的骨形态发生蛋白-2（BMP-2）的转移导致它们分化成骨细胞谱系。因此，我们假设自体移植培养扩增的MSC，在骨祖细胞特异性启动子的控制下用编码BMP-2的rAAV转导，将导致成骨细胞富集和骨量增加。在目前的建议中，我们将评估这一假设：1）确定离体培养的MSC的植入，并通过骨归巢信号的异位表达优化归巢到骨的富集，2）确定BMP-2的骨特异性表达及其在体内的成骨意义，3）确定AAV介导的基因治疗在骨质疏松小鼠模型中的体内作用。这些研究的成功结果可能为将来开发用于人类骨质疏松症和其他骨质减少性疾病的基因治疗方法奠定基础。

DESCRIPTION (provided by applicant): Adeno-associated virus (AAV) vectors are ideal for the long-term treatment of metabolic defects. Unique features including non-pathogenicity, low-immunogenicity and stable expression have positively resulted in successful preclinical and clinical evaluation of this vector. One of the potential, yet, unexplored areas of gene therapy using rAAV is metabolic bone defects characterized by a reduction in the mass of bone per unit volume as in osteoporosis or insufficient mechanisms for anabolic bone remodeling as in spinal fusion and fracture. Although, currently available hormonal and drug therapies for osteopenia aim to prevent further bone destruction by osteoclasts, therapies directed towards increasing bone mass by increasing the event of osteogenesis will be greatly beneficial. Treatments to increase bone density by anabolic agents are limited due to ineffective delivery methods and a short half-life of the drugs and purified proteins. Thus, novel methods to induce sustained in vivo osteogenesis should improve the pathophysiology of the disease. Our earlier studies established long-term efficacy of rAAV-transduced mesenchymal stem cells (MSC) to selectively engraft to bone, repopulate and express a transgene in a mouse model. Preliminary studies pertaining to this application indicated that recombinant AAV-2 (rAAV) transduces human and murine MSC and osteoprogenitors in high-efficiency and that rAAV-mediated transfer of bone morphogenetic protein-2 (BMP-2) leads to their differentiation into osteoblast lineage. Thus, we hypothesize that autologous transplantation of culture-expanded MSC, transduced with rAAV encoding BMP-2 under the control of osteoprogenitor-specific promoters, will result in osteoblast enrichment and increased bone mass. In the current proposal, we will evaluate this hypothesis to: 1) Determine the engraftment of ex vivo cultured MSC and optimize enrichment of homing to bone by ectopic expression of a bone homing signal, 2) Determine bone-specific expression of BMP-2 and its osteogenic significance in vivo and 3) Determine the effects of AAV-mediated gene therapy in osteopenic mice models in vivo. A successful outcome of these studies may form the basis for future development of gene therapy approaches for osteoporosis and other osteopenic diseases in humans.