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

摘要 关节软骨是一种重要的少血管组织结构，一旦受损， 再生并经常导致骨关节炎。已经做出了相当大的努力来建立治疗， 生物修复受损的关节软骨，这在很大程度上依赖于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倾向于 与健康的透明关节相比， 软骨虽然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 但是，抵抗软骨内骨化的再生透明软骨还有待开发。最近我们 表明口服1型血管紧张素II受体拮抗剂，氯沙坦， 透明软骨微骨折后，在兔，并伴随着减少转化生长因子β 1 （TGF-β 1）表达。这些结果表明，适当的时空抑制TGF-β 1可能是至关重要的 以防止纤维软骨形成并允许透明软骨再生。然而，TGF-β是一种软骨形成因子， CSPC的因子，并参与关节软骨的维持。此外，药理学抗TGF-β B疗法可引起显著的不良副作用。因此，我们假设有效的透明质酸 软骨再生没有明显的副作用，可以通过细胞疗法，也抑制TGF-β 1 根据需要在本地发送信号。使用CRISPR/Cas9技术，Farshid Guilak博士（mPI）报告了一种新的 一种重新编程干细胞的方法（称为自主再生疗法的干细胞修饰或 SMART）以使其能够以自动调节、反馈控制的方式递送抗炎因子， 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 治疗性细胞能够通过诱导TGF-β抑制剂从细胞周围局部抑制TGF-β 1作用， 当环境中存在TGF-β 1时（即，纤维化环境的自主抑制）。 因此，我们建议测试这种SMART细胞是否可以改善软骨修复， 传统细胞。为此，我们将使用肌源性干细胞（MDSC）和间充质基质细胞（MSC）。 细胞（MSC）重编程Decorin（Dcn）作为TGF-β 1抑制剂，TGF-β诱导的Smad 7基因作为TGF-β 1抑制剂。 位点敲入Dcn（Dcn-KI），使用CRISPR/Cas9技术。我们已经重新编程了MDSC， 我们的初步体外实验结果表明，核心蛋白聚糖是以时间和剂量依赖性的方式诱导后， TGF-β 1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编程MSC， 并测试这些SMART细胞（Dcn-KI MDSC，Aim 1; Dcn-KI MSC Aim 2）是否减轻TGF-β 1的作用。 与对照组相比，b1自主诱导透明关节软骨的长期修复 未经修饰的细胞因此，这项研究的结果将提供一个概念验证的效用的创新 自动调节基因电路系统，用于开发有效和安全的关节软骨修复细胞工具。

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.