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

项目摘要 目前，已经描述了大约500种临床上不同的骨骼发育不良，并且2/3的骨骼发育不良的遗传基础是由基因引起的。 他们被揭露了。然而，患有遗传性骨病的患者，如成骨不全症， (OI)尽管其遗传学和病理生理学已经很好地研究， 介绍了 对于遗传性骨病，用健康细胞替换突变的骨细胞是最简单的方法之一。 治疗策略以细胞为基础的治疗已经厌倦了包括OI在内的几种遗传性骨病， 显示移植骨髓（BM）细胞或BM衍生的间充质基质细胞（MSC）， 改善OI患者和小鼠的病情。然而，细胞移植疗法还没有被广泛应用。 由于多种原因，包括全身性移植后供体细胞对骨的贡献差， 在临床和临床前研究中，为了使基于细胞的治疗安全有效， 有效的长期移植和使用患者来源的细胞显得很重要。 为了实现这一目标，我们建议利用一种罕见的骨基质人口，包括长期 骨祖细胞与有效的等位基因特异性基因组编辑系统的组合。这个想法是基于 一种罕见的Sox 9阳性细胞群含有长期的骨祖细胞， MSC。由于这些细胞在体内自我更新祖细胞，它们可能显示出长期植入。在 此外，它们的小尺寸将使它们比大MSC更适合于全身施用。然而，在这方面， 由于这些细胞是经过评级，因此有必要将它们放大以用于进一步应用。 在R61中，我们将开发通过体外扩增扩增Sox 9阳性细胞的方法， 刺激不对称细胞分裂的条件，并通过使用非遗传的方法从MSC重编程， 方法.然后我们评估它们的骨移植性。 为了使用自体移植物进行治疗，需要对患者来源的Sox 9阳性细胞的突变进行检测。 修复.在R33中，为了证明概念验证，我们将使用Aga 2 OI模型小鼠，其具有 I型胶原基因中的杂合子点突变。首先，我们将开发一种高效的、突变特异性的 通过结合新的CRISPR工具，腺苷碱基编辑器（ABE）和高 容量腺病毒基因转移系统修复突变小鼠Sox 9阳性细胞中的Aga 2突变。 然后，我们将评估基因组编辑的Aga 2 Sox 9阳性细胞移植在乳腺癌中的治疗效果。 Ag 2小鼠。 该项目不仅将通过改变基于细胞的骨疾病治疗产生转化影响， 也为研究骨祖细胞生物学提供了独特的研究材料。 !

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. !