Production of recombinant human glucocerebrosidase in Escherichia coli

描述（由申请方提供）：戈谢病是人类最常见的溶酶体贮积病，导致脂肪葡糖脑苷脂在脾、肝、肺、骨髓和脑中有害蓄积。戈谢病患者表现出遗传性葡萄糖脑苷脂酶（GCase）缺乏，但有效的酶替代疗法可用于大多数戈谢病患者。重组GCase目前每年产生超过10亿美元的收入，但GCase的生产成本很高。由此产生的费用使许多病人无法负担治疗费用，而保健机构也不愿承担终身治疗费用。目前，糖蛋白GCase在中国人卵巢（CHO）细胞中表达，并在体外进一步加工，以暴露人类患者生物摄取所需的末端甘露糖残基。除了昂贵之外，哺乳动物细胞培养物还容易受到病毒污染。事实上，病毒污染导致了GCase的严重短缺，这阻碍了收入，并危及依赖定期静脉注射该药物的患者。这为替代的工业规模GCase表达平台打开了市场，这些平台被充分表征，不易受病毒污染，并且不需要复杂的体外化学修饰。Glycobia专门从事糖工程细菌作为治疗性糖蛋白立体特异性生物合成的平台。这些研究的具体假设是，糖工程大肠杆菌可用于生产活性重组GCase，而不需要在体外化学修饰。E.与酵母、CHO、植物或所有其他真核细胞不同，不存在导致不受控制的糖型的天然糖基化途径。我们预计，一个E。大肠杆菌表达平台将能够以可控、快速和成本有效的方式生产活性GCase。本研究的目的是通过在大肠杆菌中克隆和表达甘露寡糖合成的遗传机制来产生GCase。coli（Aim 1）中表达活性GCase。coli（Aim 2）。这种细菌表达平台代表了一种变革性的解决方案，以应对向患者提供具有成本效益的GCase酶替代疗法这一悬而未决的生物医学挑战。 公共卫生相关性：葡萄糖脑苷脂酶替代疗法已经彻底改变了戈谢病的临床治疗，但是生产平台的效率低下导致医疗保健消费者的成本过高。重组葡糖脑苷脂酶在哺乳动物细胞培养物中表达，使得该方法昂贵，易受病毒污染，并且经受不可控糖型的进一步体外加工。拟议的研究重点是在大肠杆菌发酵中生产活性葡萄糖脑苷脂酶，而不需要哺乳动物细胞培养或体外化学修饰。

DESCRIPTION (provided by applicant): Gaucher's disease is the most common lysosomal storage disease in humans resulting in the harmful accumulation of fatty glucocerebroside in the spleen, liver, lungs, bone marrow, and brain. Gaucher patients exhibit a hereditary deficiency of glucocerebrosidase (GCase), but effective enzyme replacement therapy is available for most patients with Gaucher's disease. Recombinant GCase currently generates over $1 billion in annual revenue, but production of GCase is expensive. The resulting cost leaves many patients unable to afford treatment and health carriers reluctant to underwrite lifelong treatment. Currently, the glycoprotein GCase is expressed in Chinese Hamster ovary (CHO) cells and further processed in vitro to expose requisite terminal mannose residues for biological uptake in human patients. In addition to being expensive, mammalian cell culture is susceptible to viral contamination. In fact, viral contamination has resulted in a severe shortage of GCase that has set back revenues and endangered patients who depend on regular intravenous administration of the drug. This has opened the market for alternative industrial scale GCase expression platforms that are well-characterized, not susceptible to viral contamination, and do not require intricate in vitro chemical modification. Glycobia specializes in glycoengineering bacteria as a platform for the stereospecific biosynthesis of therapeutic glycoproteins. The specific hypothesis of these proposed studies is that glycoengineered Escherichia coli can be used to produce active recombinant GCase without the need for in vitro chemical modification. The advantage of E. coli as a host for GCase expression is that - unlike yeast, CHO, plant or all other eukaryote cells - there are no native glycosylation pathways to result in uncontrolled glycoforms. We anticipate that an E. coli expression platform will be capable of producing active GCase in a controlled, rapid, and cost-effective manner. The objective of this proposal is to generate GCase by cloning and expressing the genetic machinery for mannose oligosaccharide synthesis in E. coli (Aim 1) and expressing active GCase in glycoengineered E. coli (Aim 2). This bacterial expression platform represents a transformative solution to the unanswered biomedical challenge of delivering a cost-effective GCase enzyme replacement therapy to patients. PUBLIC HEALTH RELEVANCE: Glucocerebrosidase enzyme replacement therapy has revolutionized the clinical treatment of Gaucher's disease, but inefficiencies in the production platform have resulted in prohibitive costs to the healthcare consumer. Recombinant glucocerebrosidase is expressed in mammalian cell culture making the process expensive, susceptible to viral contamination, and subject to further in vitro processing of uncontrollable glycoforms. The proposed studies focus on producing active glucocerebrosidase in Escherichia coli fermentation without the need for mammalian cell culture or in vitro chemical modification.