Autonomous RUNX2 suppression in genetically modified stem cell-derived chondrocytes as a disease modifying therapeutic for PTOA

转基因干细胞衍生软骨细胞中的自主 RUNX2 抑制作为 PTOA 的疾病修饰疗法

• 批准号: 10574780
• 负责人: Rhima Coleman
• 金额: $ 19.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-03 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10574780
• 关键词: Address; Affect; Anti-Inflammatory Agents; Arthralgia; Biological; Biomedical Engineering; Bone remodeling; Cartilage; Cell Differentiation process; Cell Separation; Cell Therapy; Cells; Chondrocytes; Clinical; Data; Deposition; Development; Disease; Doctor of Philosophy; Environment; Equilibrium; Exhibits; Extracellular Matrix; Female; Fluorescent Probes; Functional disorder; Genes; Genetic; Genetic Engineering; Growth Factor; Histologic; Hyperalgesia; Hypertrophy; Image; Immune; Immune response; In Vitro; Inflammation; Inflammatory; Injections; Injury; Interleukin-4; Joints; Knee; Knee joint; Label; Luciferases; Macrophage; Matrix Metalloproteinases; Measures; Mediating; Mesenchymal; Methods; Modality; Modeling; Monitor; Mus; Musculoskeletal; Orthopedic Surgery; Outcome; Pain; Pathologic; Pathology; Pathway interactions; Peptide Hydrolases; Phenotype; Process; Production; Property; Protein Analysis; Randomized; Research; Research Personnel; Severities; Signal Transduction; Site-Directed Mutagenesis; Stromal Cells; Synovial Fluid; Synovitis; Testing; Therapeutic; Tissues; Traumatic Arthropathy; Undifferentiated; adult stem cell; anterior cruciate ligament rupture; arthropathies; articular cartilage; autocrine; cartilage degradation; cartilage regeneration; cell type; chondroprotection; clinically relevant; cytokine; disability; effective therapy; efficacious treatment; engineered stem cells; experimental study; gain of function; genetically modified cells; immunoregulation; improved; in vitro Model; in vivo; in vivo imaging; inflammatory marker; inflammatory milieu; joint inflammation; joint injury; male; mid-career faculty; mouse model; next generation; novel; paracrine; prevent; professor; regenerative; response; runx proteins; stem; stem cells; structural imaging; tissue repair; transcription factor; transcriptome sequencing; treatment effect

ABSTRACT INVESTIGATORS: Rhima Coleman, PhD (PI) is an Associate Professor of Biomedical Engineering focused on genetic engineering of stem cells for the treatment of joint pathology. Tristan Maerz, PhD (Co-PI) is an Assistant Professor of Orthopaedic Surgery with expertise in PTOA pathophysiology and synovial inflammation. RESEARCH CONTEXT: Persistent intra-articular inflammation is a recognized driver of joint pathology during post-traumatic osteoarthritis (PTOA) progression. Mesenchymal stem/multipotent stromal cells (MSCs) possess potent anti-inflammatory properties, and MSC-derived chondrocytes (MdChs) have shown promise as a regenerative intra-articular treatment. Successful implementation of MdChs as a cellular therapy is currently hindered by rapid hypertrophic maturation of MdChs driven by the transcription factor RUNX2, which is exacerbated by cytokines known to be enriched in the PTOA joint. We have genetically engineered MdChs to autonomously suppress RUNX2 (sRX2-MdChs), and, therefore, chondrocyte hypertrophy, which supports cartilage formation, even under inflammation. Critically, our preliminary data also indicate that the secretome of these genetically modified cells markedly reduce the local pro-inflammatory environment by inducing repolarization of pro-inflammatory macrophages (M1) into the anti-inflammatory (M2) phenotype. OBJECTIVE: To test the disease-modifying and anti-inflammatory efficacy of genetically engineered RUNX2- suppressing MdChs as a cellular treatment in a murine model of PTOA. SPECIFIC AIMS: 1). Demonstrate that a single sRX2-MdCh injection blocks pathological inflammation and PTOA progression following joint injury. 2). Characterize the mechanisms that mediate the autocrine and paracrine effects of the sRX2-MdChs secretome on cartilage formation and the anti-inflammatory cellular crosstalk between sRX2-MdChs and macrophages in vitro. RESEARCH PLAN: In Aim 1, we will employ a noninvasive murine joint injury model of PTOA to test the anti- inflammatory and PTOA-mitigating effects of a single sRX2-MdCh injection following injury. Male and female mice will be randomized to 1) sRX2-MdChs; 2) Scramble-MdChs; 3) Unmodified/WT MSCs; or 4) Vehicle. We will use a multifaceted set of live, in vivo and ex vivo outcomes to assess joint pain, intra-articular inflammation and protease activity, bone remodeling, and articular cartilage damage to assess PTOA severity. We will track intra-articular retention of injected cells via live fluorescent monitoring of luciferase activity and a near-infrared tag. In Aim 2, we will test the hypothesis that the observed autocrine and paracrine effects of the sRX2-MdCh secretome are mediated by IL-4 and assess the mechanism by which IL-4 effects MdCh cartilage formation and M1-to-M2 macrophage polarization.

抽象的 调查员：Rhima Coleman 博士（PI）是生物医学工程副教授，专注于 用于治疗关节病理的干细胞基因工程。 Tristan Maerz 博士（Co-PI）是助理 骨外科教授，拥有 PTOA 病理生理学和滑膜炎症方面的专业知识。 研究背景：持续性关节内炎症是关节病理学公认的驱动因素 创伤后骨关节炎（PTOA）进展。间充质干细胞/多能基质细胞（MSC）具有 有效的抗炎特性，MSC 衍生的软骨细胞 (MdChs) 已显示出作为 再生性关节内治疗。 MdChs 作为一种细胞疗法目前已成功实施 受转录因子 RUNX2 驱动的 MdChs 快速肥大成熟的阻碍，该转录因子 已知在 PTOA 关节中富集的细胞因子会加剧这种情况。我们对 MdChs 进行了基因改造，以 自主抑制 RUNX2 (sRX2-MdChs)，从而抑制软骨细胞肥大，这支持 软骨形成，即使在炎症下也是如此。至关重要的是，我们的初步数据还表明， 这些转基因细胞通过诱导细胞显着减少局部促炎环境 促炎巨噬细胞 (M1) 复极化为抗炎 (M2) 表型。 目的：测试基因工程 RUNX2- 的疾病缓解和抗炎功效 抑制 MdChs 作为 PTOA 小鼠模型的细胞治疗方法。 具体目标： 1).证明单次 sRX2-MdCh 注射可阻断病理性炎症并 关节损伤后 PTOA 进展。 2）。表征介导自分泌和 sRX2-MdChs 分泌组对软骨形成和抗炎细胞的旁分泌作用 体外 sRX2-MdChs 和巨噬细胞之间的串扰。 研究计划：在目标1中，我们将采用PTOA的非侵入性小鼠关节损伤模型来测试其抗- 损伤后单次 sRX2-MdCh 注射的炎症和 PTOA 缓解作用。男女 小鼠将被随机分配到 1) sRX2-MdChs； 2) Scramble-MdChs； 3) 未修饰/WT MSC；或 4) 车辆。我们 将使用多方面的活体、体内和离体结果来评估关节疼痛、关节内炎症 以及蛋白酶活性、骨重塑和关节软骨损伤，以评估 PTOA 严重程度。我们将追踪 通过实时荧光监测荧光素酶活性和近红外将注射细胞保留在关节内 标签。在目标 2 中，我们将测试以下假设：观察到的 sRX2-MdCh 的自分泌和旁分泌效应 分泌组由 IL-4 介导，并评估 IL-4 影响 MdCh 软骨形成的机制和 M1 至 M2 巨噬细胞极化。