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.

摘要 导师：Rhima科尔曼，博士（PI）是生物医学工程的副教授，专注于 用于治疗关节病变的干细胞基因工程。Tristan Maerz，PhD（Co-PI）是一名助理 骨科教授，擅长PTOA病理生理学和滑膜炎症。 研究背景：持续性关节内炎症是公认的关节病理学驱动因素， 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 关节内再生治疗MdChs作为细胞疗法的成功实施目前是 受到转录因子RUNX 2驱动的MdChs快速肥大成熟的阻碍， 由已知在PTOA关节中富集的细胞因子加剧。我们通过基因工程改造了MdCh， 自主抑制RUNX 2（sRX 2-MdChs），因此，软骨细胞肥大，支持 软骨形成，即使在炎症下。重要的是，我们的初步数据还表明， 这些基因修饰的细胞通过诱导炎症反应， 促炎巨噬细胞（M1）复极化为抗炎（M2）表型。 目的：测试基因工程RUNX 2 - 1的疾病改善和抗炎功效。 抑制MdChs作为PTOA鼠模型中的细胞治疗。 具体目标：1）。证明单次sRX 2-MdCh注射可阻断病理性炎症， 关节损伤后PTOA进展。2）。表征介导自分泌和 sRX 2-MdChs分泌体对软骨形成和抗炎细胞的旁分泌作用 sRX 2-MdChs和巨噬细胞之间的体外串扰。 研究目的：在目的1中，我们将采用非侵入性小鼠PTOA关节损伤模型来测试抗- 图10显示了损伤后单次sRX 2-MdCh注射的炎症和PTOA减轻作用。男女 将小鼠随机分配至1）sRX 2-MdChs; 2）Scramble-MdChs; 3）未修饰的/WT MSC;或4）媒介物。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 和蛋白酶活性、骨重建和关节软骨损伤来评估PTOA严重程度。我们将跟踪 通过荧光素酶活性的活体荧光监测和近红外光谱分析， 标签在目的2中，我们将检验所观察到的sRX 2-MdCh的自分泌和旁分泌作用 分泌组由IL-4介导，并评估IL-4影响MdCh软骨形成的机制， M1至M2巨噬细胞极化。