CRISPR-Cas9 Genome Edited MSCs to Target OA Cartilage Regeneration

项目摘要 膝关节骨性关节炎患者的软骨缺损处通常充满纤维软骨。关节炎症 使软骨缺陷成为一种难治性疾病。间充质基质/干细胞--一种很有前途的细胞 治疗软骨缺陷的来源，表现出位置依赖的分化偏好：骨髓- 来源的骨髓间充质干细胞(BMSCs)表现为软骨分化，但以软骨内成骨结束；脂肪- 来源的骨髓间充质干细胞(ADSCs)很容易获得，但软骨形成潜力有限，倾向于 纤维软骨而不是关节软骨；滑膜来源的间充质干细胞是一种组织特异性干细胞。 细胞具有向软骨细胞分化的能力，但可及性有限。此外，更多的MSCs是 组织工程和再生所需；然而，骨髓间充质干细胞在运动中容易衰老 体内扩增导致其增殖和分化潜能丧失。鉴于MSCs和 纤维软骨广泛表达I型胶原(COL1A1)，在本方案中，我们假设 OStrio(包括OCT4和SOX Trio)在COL1A1基因座的整合允许MSC年轻化和关节 分化的同时通过负反馈环路抑制纤维软骨。为了实现这一假设，我们 计划使用尖端的CRISPR-Cas9基因组编辑技术在 骨髓间充质干细胞的COL1A1基因座(Aim 1)，随后对其增殖和成软骨分化的微调 OStrio通过WNT信号表达MSCs(Aim 2)；最后，基因工程MSCs将在 通过皮下植入(有足够的血管供应)构建的早期组织 在免疫缺陷小鼠模型中表达人OStrio的骨髓间充质干细胞，用于未来的临床翻译(目标3.1) 兔表达OStrio的MSCs构建的早期组织结构在兔关节腔内的移植 骨软骨缺损兔模型(AIM 3.2)。我们的期望是表达OStrio的MSCs具有更好的 体外研究中的增殖和成软骨分化能力以及优异的软骨分化能力 动物的表面修复和对炎症、氧化以及血管生成恶化的抵抗 学习。我们的目标是设计出“理想的”骨髓间充质干细胞，这种干细胞可以大量扩增，并且 分化成关节软骨，具有强大的抵抗恶劣骨关节环境的能力 防止软骨肥大和纤维软骨的形成。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，有针对性地介绍这些因素 整合，而不是使用慢病毒载体，以改善这一新疗法的安全性。这个项目有 通过安全地为关节软骨再生提供大量的 大量患者特定的功能性软骨前体细胞，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.