SMART stem cells that autonomously down-modulate TFG-β signaling for Articular Cartilage Repair

SMART 干细胞自主下调 TFG-β 信号传导以修复关节软骨

• 批准号: 10590752
• 负责人: Farshid Guilak
• 金额: $ 15.51万
• 依托单位: STEADMAN PHILIPPON RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10590752
• 关键词: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animal Model; Anti-Inflammatory Agents; Antihypertensive Agents; BMP4; Binding; Biochemical; Biological; Biological Assay; Biological Response Modifier Therapy; Biomechanics; Bone Morphogenetic Proteins; CRISPR/Cas technology; Cartilage; Cartilage injury; Cell Therapy; Cells; Collaborations; Custom; Dedications; Degenerative polyarthritis; Development; Dose; Environment; Feedback; Fibrocartilages; Fibrosis; Gene Expression; Genes; Genome engineering; Human; Hyaline Cartilage; In Vitro; Inferior; Inflammatory; Injury; Interleukin-1; Knock-in; Laboratories; Losartan; Maintenance; Mechanics; Mediating; Methods; Mus; Muscle; Musculoskeletal; Natural regeneration; Nude Rats; Open Reading Frames; Oral Administration; Oryctolagus cuniculus; Paper; Pharmaceutical Preparations; Physiologic Ossification; Pluripotent Stem Cells; Process; Property; Proteoglycan; Regenerative Medicine; Reporting; Reproducibility; Research Personnel; Signal Transduction; Signaling Protein; Site; Structure; System; Systems Development; TGFB1 gene; TNF gene; Technology; Testing; Therapeutic; Time; Tissues; Transforming Growth Factor beta; Transplantation; articular cartilage; bone marrow mesenchymal stem cell; cartilage regeneration; cartilage repair; cytokine; decorin; design; effective therapy; experimental study; genomic locus; healing; improved; improved outcome; inhibitor; injury and repair; innovation; mesenchymal stromal cell; muscle transplantation; novel; novel strategies; pharmacologic; prevent; programs; regenerative therapy; repaired; side effect; spatiotemporal; stem cell therapy; stem cells; therapy outcome; tissue repair; tool

ABSTRACT Articular cartilage is an important hypovascular tissue structure that, once damaged, does not spontaneously regenerate and often leads to osteoarthritis. Considerable efforts have been made to establish therapies that biologically repair damaged articular cartilage, which rely heavily on endogenous or exogenous chondrogenic stem/progenitor cells (CSPCs). One major drawback of current biological therapies is that fibrocartilage tends to be regenerated, which shows inferior biomechanical properties compared with the healthy hyaline articular cartilage. Although a number of therapies have been developed to improve the situation, a reproducible method to regenerates hyaline cartilage that resists endochondral ossification is yet to be developed. Recently, we have demonstrated that oral administration of type 1 angiotensin II receptor antagonist, losartan, regenerates mostly hyaline cartilage after microfracture in rabbits, and concomitantly reduces transforming growth factor-beta 1 (TGF-b1) expression. These results suggest that a proper spatiotemporal suppression of TGF-b1 may be critical to prevent fibrocartilage formation and allow hyaline cartilage regeneration. However, TGF-b is a chondrogenic factor for CSPCs, and involved in the maintenance of articular cartilage. Furthermore, pharmacological anti-TGF- b therapies can cause significant unwanted side effects. Therefore, we hypothesize that effective hyaline cartilage regeneration without overt side effects may be achieved by a cell therapy that also inhibits TGF-b1 signaling locally as needed. Using the CRISPR/Cas9 technology, Dr. Farshid Guilak (mPI) have reported a novel approach that reprograms stem cells (called Stem cells Modified for Autonomous Regenerative Therapy or SMART) to make it possible to deliver anti-inflammatory factor in an auto-regulated, feedback-controlled manner, and demonstrated its utility for musculoskeletal regenerative medicine. In this proposal, we aim to reprogram therapeutic cells to be able to suppress TGF-b1 action locally around the cells by inducing TGF-b inhibitor from them whenever TGF-b1 is present in the environment (i.e., autonomous suppression of fibrotic environment). We consequently propose to test whether such SMART cells may improve cartilage repair when compared to conventional cells. For this purpose, we will use muscle-derived stem cells (MDSCs) and mesenchymal stromal cells (MSCs) to reprogram Decorin (Dcn) as the TGF-b1 inhibitor, and the TGF-b-inducible Smad7 gene as the site to knock-in Dcn (Dcn-KI), using the CRISPR/Cas9 technology. We have already reprogrammed MDSCs, and our preliminary in vitro results indicate that Decorin is induced in a time & dose dependent manner after TGF-b1 exposure, and can suppress the fibrotic cascade. We propose to reprogram MSCs using a similar tactic, and test whether these SMART cells (Dcn-KI MDSCs, Aim1; Dcn-KI MSCs Aim 2) mitigate the effects of TGF- b1 autonomously and induce long-term repair of hyaline articular cartilage, when compared with control unmodified cells. Thus, results of this study will provide a proof-of-concept on the utility of the innovative autoregulatory gene circuit system for development of effective & safe cellular tools for articular cartilage repair.

抽象的 关节软骨是一种重要的缺血管组织结构，一旦受损，不会自发地 再生并经常导致骨关节炎。为了建立治疗方法已经付出了相当大的努力 生物修复受损的关节软骨，严重依赖内源性或外源性软骨形成 干/祖细胞（CSPC）。当前生物疗法的一个主要缺点是纤维软骨往往会 被再生，与健康的透明关节相比，其生物力学特性较差 软骨。尽管已经开发了许多疗法来改善这种情况，但一种可重复的方法 再生抵抗软骨内骨化的透明软骨的方法尚未开发出来。最近，我们有 证明口服 1 型血管紧张素 II 受体拮抗剂氯沙坦可大部分再生 兔子微骨折后的透明软骨，并同时减少转化生长因子-β 1 (TGF-b1) 表达。这些结果表明适当的时空抑制 TGF-b1 可能至关重要 防止纤维软骨形成并允许透明软骨再生。然而，TGF-b 是一种软骨形成物质。 CSPC 的因子，并参与关节软骨的维护。此外，药理学抗TGF- b 疗法可能会导致严重的不良副作用。因此，我们假设有效的透明质 通过同时抑制 TGF-b1 的细胞疗法可以实现无明显副作用的软骨再生 根据需要在本地发出信号。 Farshid Guilak 博士 (mPI) 利用 CRISPR/Cas9 技术报道了一种新的 重新编程干细胞的方法（称为自主再生疗法改良干细胞或 SMART）使以自动调节、反馈控制的方式输送抗炎因子成为可能， 并证明了其在肌肉骨骼再生医学中的实用性。在这个提案中，我们的目标是重新编程 治疗细胞能够通过诱导 TGF-b 抑制剂来抑制细胞周围局部 TGF-b1 的作用 只要环境中存在 TGF-b1（即纤维化环境的自主抑制），它们就会发挥作用。 因此，我们建议测试与传统细胞相比，这种 SMART 细胞是否可以改善软骨修复。 常规细胞。为此，我们将使用肌肉源性干细胞 (MDSC) 和间充质基质 细胞 (MSC) 将核心蛋白聚糖 (Dcn) 重新编程为 TGF-b1 抑制剂，将 TGF-b 诱导型 Smad7 基因重新编程为 使用 CRISPR/Cas9 技术敲入 Dcn (Dcn-KI) 位点。我们已经对 MDSC 进行了重新编程， 我们的初步体外结果表明，核心蛋白聚糖以时间和剂量依赖性方式诱导 TGF-b1暴露，并能抑制纤维化级联反应。我们建议使用类似的策略重新编程 MSC， 并测试这些 SMART 细胞（Dcn-KI MDSC、Aim1；Dcn-KI MSC Aim 2）是否减轻 TGF- 与对照相比，b1 自主并诱导透明关节软骨的长期修复 未修饰的细胞。因此，这项研究的结果将为创新的实用性提供概念验证 自动调节基因回路系统，用于开发用于关节软骨修复的有效且安全的细胞工具。