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低血管组织结构，一旦受损，不会自发发生 会再生，通常会导致骨关节炎。已经做出了相当大的努力来建立治疗方法， 生物修复严重依赖内源性或外源性软骨生成的受损关节软骨 干细胞/祖细胞(CSPC)。目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，其生物力学性能较差 软骨。尽管已经开发了许多治疗方法来改善这种情况，但一种可重复使用的方法 为了再生透明软骨，抵抗软骨内骨化的能力还有待开发。最近，我们有 证实口服1型血管紧张素II受体拮抗剂氯沙坦最能再生。 兔微骨折后软骨透明，并伴随转化生长因子-β1的减少 (转化生长因子-β1)表达。这些结果表明，适当的时空抑制转化生长因子-β1可能是至关重要的。 防止纤维软骨形成，让透明软骨再生。然而，转化生长因子-b是一种软骨性的 CSPC的因子，并参与关节软骨的维护。此外，药理抗转化生长因子-1 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蛋白 没有明显副作用的软骨再生可以通过抑制转化生长因子-β1的细胞疗法来实现。 根据需要在本地发送信号。利用CRISPR/Cas9技术，Farshid Guilak博士(MPI)报告了一种新的 对干细胞(称为干细胞)重新编程以进行自主再生治疗的方法 SMART)使得能够以自动调节、反馈控制的方式传递抗炎因子， 并展示了其在肌肉骨骼再生医学中的应用。在这项提案中，我们的目标是重新编程 治疗性细胞能够通过诱导转化生长因子-β抑制物在细胞周围局部抑制转化生长因子-β1的活动 只要环境中存在转化生长因子-β1(即，自主抑制纤维化环境)，它们就会被激活。 因此，我们建议测试这种智能细胞是否可以改善软骨修复 常规细胞。为此，我们将使用肌肉来源的干细胞(MDSCs)和间充质基质 细胞(MSCs)重新编程核心蛋白(DCN)作为转化生长因子-β1抑制因子，并将转化生长因子-β诱导的Smad7基因作为 站点敲入DCN(DCN-KI)，使用CRISPR/Cas9技术。我们已经对MDSC进行了重新编程， 我们的初步体外结果表明，核心蛋白聚糖是以时间和剂量依赖的方式在 TGF-β1暴露，并可抑制纤维化级联反应。我们建议使用类似的策略对MSCs进行重新编程， 并测试这些智能细胞(DCN-KI MDSCs，Aim1；DCN-KI MSCs Aim 2)是否可以缓解转化生长因子-1的影响 B1与对照组相比，自主和诱导透明关节软骨的长期修复 未修改的单元格。因此，这项研究的结果将为创新的实用性提供概念验证 用于开发安全有效的关节软骨修复细胞工具的自动调节基因回路系统。

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.