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; 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低血管组织结构，一旦损坏，就不会自发地 再生并经常导致骨关节炎。已经做出了巨大的努力来建立疗法 生物修复受损的关节软骨，严重依赖内源性或外源性软骨 茎/祖细胞（CSPC）。当前生物疗法的一个主要缺点是纤维球纤维属倾向于 被再生，与健康的透明关节相比，它显示出劣质的生物力学特性 软骨。尽管已经开发了许多疗法来改善这种情况，但一种可再现的方法 为了再生透明的透明软骨，尚未开发前软骨软骨。最近，我们有 证明口服1型1型血管紧张素II受体拮抗剂Losartan重新生成 兔子微骨折后透明软骨，并随同降低了转化生长因子-beta 1 （TGF-B1）表达。这些结果表明，适当的TGF-B1的时空抑制可能至关重要 为了防止纤维球形成并允许透明软骨再生。但是，TGF-B是软骨 CSPC的因素，并参与关节软骨的维护。此外，药理学抗TGF- B疗法会引起明显的不良副作用。因此，我们假设有效的透明碱 软骨再生无明显的副作用可以通过抑制TGF-B1的细胞疗法来实现 根据需要在本地发出信号。 Farshid Guilak博士（MPI）使用CRISPR/CAS9技术报告了一本小说 重编程干细胞的方法（称为为自动再生疗法修饰的干细胞或 智能）可以以自动调节，反馈控制的方式提供抗炎因素 并证明了其对肌肉骨骼再生医学的实用性。在此提案中，我们的目标是重新编程 治疗细胞能够通过诱导TGF-B抑制剂从细胞周围局部抑制TGF-B1作用 每当TGF-B1存在于环境中（即对纤维化环境的自主抑制）时，它们。 因此，我们建议测试此类智能细胞是否可以改善软骨维修 常规细胞。为此，我们将使用肌肉衍生的干细胞（MDSC）和间质基质 细胞（MSC）重新编程Decramin（DCN）作为TGF-B1抑制剂，而TGF-B诱导的SMAD7基因为 使用CRISPR/CAS9技术的网站敲入DCN（DCN-KI）。我们已经重新编程了MDSC， 我们的初步体外结果表明，在 TGF-B1暴露，可以抑制纤维化的级联反应。我们建议使用类似策略重新编程MSC， 并测试这些智能细胞（DCN-KI MDSC，AIM1; DCN-KI MSC AIM 2）减轻TGF-的影响 与对照相比，B1自主并诱导透明关节软骨的长期修复 未改性的细胞。因此，这项研究的结果将提供有关创新效用的概念证明 开发有效且安全的细胞修复工具的自动调节基因电路系统。