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

操纵 Sox9 阳性骨软骨祖细胞进行遗传性骨疾病的细胞基因治疗。

• 批准号: 9906987
• 负责人: TATSUYA KOBAYASHI
• 金额: $ 40.33万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-17 至 2022-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906987
• 关键词: Address; Adenosine; Adenoviruses; Alleles; Allografting; Autologous Transplantation; Biology; Bone Diseases; Bone Marrow; Bone Marrow Cell Transplantation; Bone Marrow Cells; Cell Size; Cell Therapy; Cell Transplantation; Cell Transplants; Cell division; Cells; Cellular biology; Chondrocytes; Clinical; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen Gene; Collagen Type I; Consensus; Data; Disease; Engraftment; Ethics; Functional disorder; Gene Transfer; Genes; Genetic; Goals; In Vitro; Life; Mesenchymal; Methods; Minor; Mus; Mutant Strains Mice; Mutate; Mutation; Osteoblasts; Osteogenesis Imperfecta; Patients; Peripheral; Phase; Point Mutation; Population; Research; Stem cells; Stromal Cells; System; Systemic Therapy; Technology; Testing; Therapeutic; Therapeutic Effect; Transplantation; base; bone; bone cell; gene therapy; genome editing; improved; in vivo; mesenchymal stromal cell; mouse model; non-genetic; novel; osteoprogenitor cell; preclinical study; progenitor; repaired; self renewing cell; self-renewal; skeletal dysplasia; tool; translational impact; treatment risk

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. !

项目概要 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，患有遗传性骨病的患者，例如成骨不全症 (OI)，尽管其遗传学和病理生理学已取得良好进展，但治疗选择有限 描述的。 对于遗传性骨病，用健康细胞替换突变的骨细胞是最直接的方法之一 治疗策略。细胞疗法对包括成骨不全症在内的多种遗传性骨病感到厌倦， 研究表明，骨髓（BM）细胞或BM衍生的间充质基质细胞（MSC）的移植 改善成骨不全症患者和小鼠的状况。然而，细胞移植疗法尚未得到广泛应用。 出于多种原因积极追求，包括系统性移植后供体细胞对骨的贡献较差 临床和临床前研究中的给药。为了使基于细胞的治疗安全有效，实现 患者来源的细胞的有效长期植入和使用显得很重要。 为了实现这一目标，我们建议利用稀有的骨基质群体，其中包括长期 骨祖细胞与有效的等位基因特异性基因组编辑系统相结合。这个想法是基于 研究结果表明，一种罕见的 Sox9 阳性细胞群含有长期骨祖细胞，可产生 间充质干细胞。由于这些细胞在体内是自我更新的祖细胞，因此它们可能表现出长期的移植能力。在 此外，它们的小尺寸使它们比大的 MSC 更适合全身给药。然而， 由于这些细胞是有速率的，因此有必要对其进行放大以供进一步应用。 在 R61 中，我们将开发通过优化离体扩增来扩增 Sox9 阳性细胞的方法 刺激不对称细胞分裂的条件，并通过使用非遗传性从 MSC 重新编程 方法。然后我们评估它们向骨骼移植的能力。 为了使用自体移植物进行治疗，需要对源自患者的 Sox9 阳性细胞进行突变 修复了。在 R33 中，为了演示概念验证，我们将使用 Aga2 OI 模型小鼠，该小鼠具有 I 型胶原基因杂合点突变。首先，我们将开发一种高效的、针对突变的 结合新的CRISPR工具、腺苷碱基编辑器（ABE）和高通量测序技术的基因组编辑方法 腺病毒基因转移系统修复突变小鼠 Sox9 阳性细胞中的 Aga2 突变。 然后我们将评估基因组编辑的 Aga2 Sox9 阳性细胞移植的治疗效果 Aga2 小鼠。 该项目不仅会通过改变骨疾病的细胞疗法产生转化影响，而且还会产生转化影响。 还提供独特的研究材料来研究骨祖细胞生物学。 ！