Manipulation of Sox9-positive osteochondroprogenitor cells towards cell-based gene therapy for genetic bone diseases.

项目摘要 目前，已描述了约500种临床上不同的骨骼发育不良，其中2/3的遗传基础 他们已经被揭露了。然而，患有遗传性骨骼疾病的患者，如成骨不全 (OI)，尽管它的遗传学和病理生理一直很好，但治疗选择有限 描述。 对于遗传性骨骼疾病，用健康细胞取代突变的骨细胞是最直接的方法之一。 治疗策略。基于细胞的治疗已经厌倦了包括OI在内的几种遗传性骨骼疾病，而且它是 研究表明，骨髓(BM)细胞或骨髓来源的间充质干细胞(MSCs)移植 改善OI患者和小鼠的病情。然而，细胞移植治疗还没有得到 积极寻求多种原因，包括系统性骨移植后供体细胞对骨骼的贡献较差 临床和临床前研究中的给药。基于细胞的治疗是安全和有效的，实现 有效的长期植入和使用患者来源的细胞似乎很重要。 为了实现这一目标，我们建议利用稀有的骨基质种群，包括长期的 骨祖细胞与高效的等位基因特异性基因组编辑系统相结合。这个想法是基于 关于罕见的Sox9阳性细胞群包含长期的骨祖细胞的发现 向MSCs致敬。由于这些细胞在体内可以自我更新祖细胞，它们很可能表现出长期植入。在……里面 此外，它们的体积小，将使它们比大型MSCs更适合全身给药。然而， 由于这些细胞是有活力的，因此有必要对它们进行扩增，以便进一步应用。 在R61，我们将开发通过体外扩增Sox9阳性细胞的方法，通过优化 刺激不对称细胞分裂的条件，并通过使用非遗传方法从MSCs重新编程 方法：研究方法。然后，我们评估它们可移植到骨中的能力。 要将自体移植用于治疗，患者来源的Sox9阳性细胞的突变需要 修好了。在R33中，为了演示概念验证，我们将使用具有 I型胶原基因杂合点突变。首先，我们将开发一种高效的、特定于突变的 一种结合新的CRISPR工具、腺苷碱基编辑(ABE)和HIGH的基因组编辑方法 修复突变小鼠Sox9阳性细胞中AGa2突变的腺病毒基因转移系统。 然后我们将评估将基因组编辑的AGa2Sox9阳性细胞移植到 AGa2小鼠。 该项目不仅将通过改变基于细胞的骨病治疗来产生翻译影响，而且 也为研究骨祖细胞生物学提供了独特的研究材料。 好了！

Project Summary Currently, about 500 clinically distinct skeletal dysplasias have been described, and the genetic basis of 2/3 of them has been revealed. However, patients with genetic bone diseases, such as osteogenesis imperfecta (OI), have limited therapeutic options despite the fact that its genetics and pathophysiology have been well described. For genetic bone diseases, replacing mutated bone cells by healthy cells is one of most straightforward therapeutic strategies. Cell-based therapy was tired to several genetic bone diseases including OI, and it was shown that transplantation of bone marrow (BM) cells or BM-derived mesenchymal stromal cells (MSCs) ameliorated the condition of patients and mice with OI. However, cell transplantation therapy has not been actively pursued for multiple reasons, including poor donor-cell contribution to bone after systemic administration in clinical and pre-clinical studies. For cell-based therapy to be safe and effective, achieving efficient long-term engraftment and use of patient-derived cells appear important. To achieve this goal, we propose to utilize a rare bone stromal population that includes long-term osteoprogenitor cells in combination with an efficient allele-specific genome-editing system. This idea is based on the findings that a rare Sox9-positive cell population contains long-term osteoprogenitor cells that give rise to MSCs. Since these cells self-renewing progenitors in vivo, they likely show long-term engraftment. In addition, their small size will make them more suitable than large MSCs for systemic administration. However, since these cells are rate, it is necessary to amplify them for further application. In R61, we will develop methods to amplify Sox9-positive cells by ex vivo expansion by optimizing the condition that stimulates asymmetric cell division and by reprogramming from MSCs using non-genetic methods. We then evaluate their transplantability to bone. To use autografts for treatment, the mutation of patient-derived Sox9-positive cells needs to be repaired. In R33, to demonstrate the proof-of-concept, we will use Aga2 OI model mice that have a heterozyous point mutation in the type I collagen gene. First, we will develop an efficient, mutation-specific genome editing method by combining the new CRISPR tool, adenosine base editor (ABE) and the high capacity adenovirus gene transfer system to repair the Aga2 mutation in Sox9-postive cells from mutant mice. We will then evaluate the therapeutic effect of transplantation with genome-edited Aga2 Sox9-postive cells in Aga2 mice. This project will not only translational impacts by changing the cell-based therapy for bone diseases but also provide unique research materials to investigate osteoprogenitor biology. !