Muscle Stem Cells Reprogrammed Through Genome Engineering for Autonomously Regulated Anti-Fibrotic Therapy

我们前期的研究表明，转化生长因子β 1（TGF-β1）在骨骼肌中起着关键作用 损伤后纤维化抑制TGF-β1的抗纤维化药物可以减少肌肉纤维化， 肌肉再生和修复。此外，我们还证明了肌肉移植- 骨髓间充质干细胞（MDSCs）可以促进损伤后肌肉的再生，但注射后的MDSCs的分化能力明显低于注射前。 细胞转化为纤维化细胞限制了对肌肉修复的有益作用。事实上，我们观察到， TGF-β1对损伤肌肉微环境的影响，不仅诱导了TGF-β1自分泌表达， TGF-β1还促进MDSC分化为肌成纤维细胞，这有助于MDSC的发育。 纤维化我们最近报道，联合氯沙坦（抗纤维化剂）和MDSC移植， 通过阻止MDSC的再生，显著提高了骨骼肌中MDSC的再生潜力， 分化为纤维化。虽然全身使用氯沙坦是安全的，FDA也批准了，但它可能导致 广泛阻断TGF-β1，这可能是不希望的。此外，药物抗纤维化疗法 通常有效减少纤维化，但使用高，不受管制的剂量，并有显着的副作用 方面的影响.最近，使用CRISPR/Cas9基因组编辑系统，我们的联合首席研究员（Guilak博士） 创造了干细胞，可以拮抗IL-1α或TNF-α介导的炎症，在一个自动调节，反馈， 肌肉骨骼再生医学应用的控制方式。他们证明了 定制设计对促炎细胞因子免疫的干细胞的能力的概念， 最佳组织修复的细胞来源。因此，我们提出，这种新的基因组工程系统也可以 可用于拮抗TGF-β1介导的纤维化，并进一步促进损伤后的肌肉愈合。我们建议 靶向TGF-β可溶性受体II型（TSRTII），以拮抗TGF-β1介导的纤维化的应用。 我们建议在MDSC中开发一种自调节基因回路，这样TGF-β1基因将被 通过核酸酶介导的TSRTII（一种TGF-β1拮抗剂）整合， TGF-β1信号通路。内源性TGF-β1基因座的转基因表达 MDSC将提供快速反馈控制以响应于TGF-β1产生TSRTII。我们将首先测试 基因编辑的MDSC显示出在体外减轻TGF-β1纤维化作用的能力，通过检查 MDSC向肌成纤维细胞的分化以及TSRTII和TGF-β1相关基因的表达 通路接下来，我们建议确定对照MDSC的肌肉再生和修复是否显示出 显著的纤维化事件响应于TGF-β1，而用基因组编辑的MDSC的肌肉修复将是 保护免受体内损伤后的纤维化。

Our previous studies indicated that transforming growth factor (TGF)-β1 plays a key role in skeletal muscle fibrosis after injury. Antifibrotic agents that inactivate TGF-β1 can reduce muscle fibrosis and significantly improve muscle regeneration and repair. Furthermore, we have also demonstrated that the transplantation of muscle- derived stem cell (MDSCs) could improve muscle regeneration after injury, but the differentiation of the injected cells into fibrotic cells limits the beneficial effect on muscle repair. In fact, we have observed that MDSCs under the influence of TGF-β1 from the injured muscle microenvironment, not only induce an autocrine expression of TGF-β1 but also promote the MDSCs’ differentiation into myofibroblasts that contribute to the development of fibrosis. We have recently reported that combining losartan (anti-fibrotic agent) with MDSC transplantation significantly improved the regenerative potential of MDSCs in skeletal muscle by preventing the MDSC’s differentiation into fibrosis. Although the systemic use of losartan is safe and FDA-approved, it likely leads to widespread blockade of TGF-β1 which might not be desirable. In addition, pharmacological anti-fibrotic therapies are often effective at diminishing fibrosis, but are used at high, unregulated doses and have significant side effects. More recently, using the CRISPR/Cas9 genome editing system, our co-Principal Investigator (Dr. Guilak) created stem cells that can antagonize IL-1α or TNF-α-mediated inflammation in an auto-regulated, feedback- controlled manner for musculoskeletal regenerative medicine applications. They have demonstrated proof-of- concept of the ability to custom-design stem cells that are immune to pro-inflammatory cytokines as a potential cell source for optimal tissue repair. We therefore propose that this novel genome engineering system can also be used to antagonize TGF-β1-mediated fibrosis and further improve muscle healing after injury. We propose to target the TGF-β soluble receptor type II (TSRTII), to antagonize TGF-β1-mediated fibrosis in the application. We are proposing to develop an autoregulatory gene circuit in MDSCs, such that the TGF-β1 gene will be reprogrammed by nuclease-mediated integration of the TSRTII, a TGF-β1 antagonist, immediately downstream of the TGF-β1 signaling pathway. Transgene expression from the endogenous TGF-β1 locus in engineered MDSCs will provide rapid feedback-control to produce TSRTII in response to TGF-β1. We will first test whether the gene-edited MDSCs show the ability to mitigate the fibrotic effects of TGF-β1 in vitro, by examining the differentiation of MDSCs into myofibroblasts and the expression of TSRTII and genes involved in the TGF-β1 pathway. Next, we propose to determine whether muscle regeneration & repair with control MDSCs show significant fibrotic events in response to TGF-β1, whereas muscle repair with genome-edited MDSCs will be protected from fibrosis after injury in vivo.