AAV-Mediated Gene Therapy for Metabolic Bone Disease

描述(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。无致病性、低免疫原性和稳定表达的独特特性使该载体成功地进行了临床前和临床评估。利用rAAV进行基因治疗的潜在领域之一是代谢性骨缺陷，其特征是单位体积的骨量减少，如骨质疏松，或合成代谢骨重建机制不足，如脊柱融合和骨折。虽然目前可用的激素和药物治疗骨量减少的目的是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治疗将是非常有益的。由于药物和纯化蛋白质的半衰期短，通过合成代谢剂增加骨密度的治疗方法无效。因此，诱导体内持续成骨的新方法应该会改善疾病的病理生理学。 我们早期的研究证实了rAAV转导的间充质干细胞(MSC)在小鼠模型中选择性植入骨、重新填充和表达转基因的长期有效性。有关这一应用的初步研究表明，重组AAV-2(RAAV)高效转导人和小鼠的MSC和成骨细胞，并通过rAAV介导的骨形态发生蛋白-2(BMP-2)的转移使其分化为成骨细胞系。因此，我们推测，在成骨细胞特异性启动子的控制下，用编码BMP-2的rAAV转导培养扩增的MSC自体移植将导致成骨细胞的丰富和骨量的增加。在目前的提案中，我们将评估这一假设：1)确定体外培养的MSC的植入，并通过异位表达骨归巢信号来优化骨归巢的富集性；2)确定BMP-2在体内的骨特异性表达及其成骨意义；3)确定AAV介导的基因治疗在体内骨量减少小鼠模型中的效果。这些研究的成功结果可能为未来开发针对人类骨质疏松症和其他骨质疏松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.