Developing a stable cell line expressing recombinant sclerostin

1摘要 2颞下颌关节(TMJ)是一个复杂的关节系统，对牙齿的咬合至关重要， 3咀嚼、呼吸和言语。TMJ由一个肌肉网络组成， 4个韧带和1个纤维软骨盘和一个髁突。颞下颌关节紊乱病(TMD) 5是涉及TMJ和相关的一组复杂的、不同种类的病理 6肌肉学。退行性的TMD，包括TMJ骨关节炎(OA)，会使人虚弱， 7影响生活质量。TMD困扰着1000多万美国人，每年造成 根据NIDCR，80~40亿美元。目前的TMJ OA治疗通常是双重的，涉及到 9疼痛管理或侵入性手术，如高失败率的全关节置换术。 再生医学承诺提供因疾病而失去的娱乐功能组织。 因此，利用常驻干细胞的再生能力来修复TMJ可能 12代表了一种基于干细胞的治疗TMJ OA的微创方法。由NIH赞助 13小企业赠款，我们进一步开发了StemGel®，这是一种缓释制剂，通过 14用透明质酸(HA)包埋硬脂素。每月高效注射StemGel® 15抑制兔和小型猪TMJ骨关节炎的进展和软骨再生 16个型号。 17硬化素是StemGel®的有效成分，是一种糖基化蛋白质，含有213个氨基酸。 18需要哺乳动物的表达系统来忠实地产生翻译后的 19修改。细胞系发育是治疗性蛋白质的关键转折点 20制造业。细胞底物的性质和与细胞底物有关的事件 21可以影响蛋白质的质量和安全，进而对蛋白质的各个方面进行有效的质量控制 22处理细胞底物。这项计划的目标是发展主细胞库 23(MCB)/稳定表达硬化素的细胞系易于转移到合同中 24大规模生产GMP的开发和制造组织(CDMO) 25级蛋白质，用于药代动力学和安全性测试。

1 Abstract 2 The temporomandibular joint (TMJ) is a complex joint system critical for dental occlusion, 3 mastication, respiration and speech. The TMJ is comprised of a network of muscles, 4 ligaments, and a fibrocartilaginous disc and condyle. Temporomandibular disorders (TMDs) 5 are a complex, heterogeneous group of pathologies involving the TMJ and associated 6 musculature. Degenerative types of TMDs, including TMJ osteoarthritis (OA), are debilitating, 7 compromise the quality of life. TMDs afflict over 10 million Americans at an annual cost of 8 ~$4 billion, per the NIDCR. Current TMJ OA treatments are typically two-fold, involving either 9 pain management or invasive surgeries, such as total joint replacements with high failure rates. 10 Regenerative medicine promises the recreation functional tissue that is lost from diseases. 11 Thus exploiting the regenerative capabilities of resident stem cells to repair TMJ may 12 represent a minimally invasive stem cell based treatment for TMJ OA. Sponsored by NIH 13 small business grant, we further developed StemGel®, a sustained-release formulation by 14 encapsulating sclerostin in hyaluronic acid (HA). Monthly injection of StemGel® efficiently 15 inhibits TMJ OA progression and regenerates cartilage in both rabbit and minipig TMJ OA 16 models. 17 The active ingredient of StemGel®, sclerostin is a glycosylated protein with 213 amino acids. 18 A mammalian expressing system is required to faithfully produce the post-translational 19 modification. Cell line development is a critical inflection point for therapeutic protein 20 manufacturing. The properties of the cell substrate and the events linked to cell substrate 21 can affect protein quality and safety, and further, the effective quality control on all aspects of 22 handling the cell substrate. The goal of this proposal is to develop Master Cell Bank 23 (MCB)/stable cell line expressing sclerostin to be readily transferred to the Contract 24 Development and Manufacturing Organization (CDMO) for massive production of the GMP 25 grade protein for pharmacokinetics and safety tests.