CRISPR-Cas9 Genome Edited MSCs to Target OA Cartilage Regeneration

项目摘要 膝关节骨关节炎患者的软骨缺损通常由纤维软骨填充。关节炎症 使得软骨缺损成为难治性疾病。骨髓间充质干细胞（MSCs），一种有前途的细胞 软骨缺损的治疗来源，表现出位点依赖性分化偏好：骨髓- 衍生的MSC（BMSC）表现出软骨形成分化，但以软骨内骨化结束;脂肪- 来源的MSC（ADSC）是容易获得的，但具有有限的软骨形成潜力， 纤维软骨而不是关节软骨;滑膜来源的MSC（SDSC）是组织特异性干细胞， 细胞用于软骨形成分化，但可及性有限。此外，更大数量的MSC是 组织工程和再生所需的;然而，MSC在体外培养过程中容易衰老， 体内扩增导致其增殖和分化潜能的丧失。由于MSC和 纤维软骨广泛表达I型胶原（COL 1A 1），在这个提议中，我们假设， 在COL 1A 1基因座处整合OStrio（OCT 4和SOX三联体）允许MSC再生和关节再生。 通过负反馈回路抑制纤维软骨分化。为了实现这一假设，我们 计划使用尖端的CRISPR-Cas9基因组编辑技术，将OStrio精确整合到 C 0 L1 A1基因座的MSC（Aim 1），随后微调的增殖和软骨形成分化的MSC（Aim 1）， 通过WNT信号表达OStrio的MSC（目标2）;最后，将在 通过皮下植入（具有足够的血管供应）来自 在免疫缺陷小鼠模型中表达人OStrio的MSC用于未来的临床转化（目的3.1） 和关节内植入来自兔OStrio表达MSC的未成熟组织构建体， 兔骨软骨缺损模型（Aim 3.2）。我们的期望是表达OStrio的MSC具有上级 在体外研究中的增殖和软骨形成分化能力以及在软骨中的上级能力 在动物中的表面重塑和对炎症和氧化以及血管生成的恶化的抗性 问题研究我们的目标是设计“理想的”MSC，它可以被大量扩增， 向关节软骨分化，具有强大的抵抗恶劣骨关节炎环境的能力， 防止肥大软骨和纤维软骨形成。为了将这一发现转化为临床环境， 我们将使用尖端的CRISPR-Cas9基因组编辑技术，通过靶向的方式引入这些因子， 整合，而不是使用慢病毒载体，以提高这种新疗法的安全性。这个项目 通过安全地提供大的， 大量的患者特异性功能性软骨祖细胞，可以在恶劣的骨关节炎环境中存活。

Project Summary Cartilage defects in patients with knee osteoarthritis are often filled with fibrocartilage. Inflammation in joints renders cartilage defects a refractory disease. Mesenchymal stromal/stem cells (MSCs), a promising cell source for the treatment of cartilage defects, exhibit a site-dependent differentiation preference: bone marrow- derived MSCs (BMSCs) exhibit chondrogenic differentiation but end with endochondral ossification; adipose- derived MSCs (ADSCs) are readily available but have limited chondrogenic potential and tend toward fibrocartilage instead of articular cartilage; and synovium-derived MSCs (SDSCs) are a tissue-specific stem cell for chondrogenic differentiation but have limited accessibility. Moreover, a greater number of MSCs is needed for tissue engineering and regeneration; however, MSCs are prone to become senescent during ex vivo expansion leading to a loss of their proliferation and differentiation potentials. Given that MSCs and fibrocartilage have extensive expression of collagen I (COL1A1), in this proposal, we hypothesize that integration of OStrio (both OCT4 and SOX trio) at the COL1A1 locus allows for MSC rejuvenation and articular differentiation while suppressing fibrocartilage by a negative feedback loop. To achieve this hypothesis, we plan to use cutting-edge CRISPR-Cas9 Genome Editing technology for precise integration of OStrio at the COL1A1 locus of MSCs (Aim 1), followed by fine-tuning of proliferation and chondrogenic differentiation of OStrio-expressing MSCs via WNT signals (Aim 2); lastly, genetically engineered MSCs will be assessed in vivo through subcutaneous implantation (with a sufficient vascular supply) of premature tissue constructs from human OStrio-expressing MSCs in an immunodeficient mouse model for future clinical translation (Aim 3.1) and intra-articular implantation of premature tissue constructs from rabbit OStrio-expressing MSCs in an osteochondral defect rabbit model (Aim 3.2). Our expectation is that OStrio-expressing MSCs have superior proliferation and chondrogenic differentiation capacity in an in vitro study as well as superior ability in cartilage resurfacing and resistance to deterioration from inflammation and oxidation as well as angiogenesis in animal studies. Our objective is to engineer “ideal” MSCs which can be prodigiously expanded and specifically differentiated toward articular cartilage with a robust capacity to resist the harsh osteoarthritic environment by preventing hypertrophic cartilage and fibrocartilage formation. To translate this finding into the clinical setting, we will use cutting-edge CRISPR-Cas9 Genome Editing technology to introduce these factors by targeted integration instead of using lentiviral vectors to improve the safety profile of this new therapy. This project has the potential to bypass a critical bottleneck in articular cartilage regeneration by safely providing large quantities of patient-specific functional chondroprogenitors that can survive a harsh osteoarthritic environment.