Improved yield of human albumin from transgenic cow milk

描述（由申请方提供）：本项目的总体目标是通过在转基因奶牛乳汁中生产重组人血白蛋白（rhA），为人用治疗用途提供丰富、经济、生物安全的人血白蛋白来源。人血白蛋白是从捐献的人血浆中分离出来的，但血浆分离行业的变化、对成本的担忧和对血液安全性的焦虑促使人们努力生产重组人血白蛋白作为人治疗应用的替代来源。GTC生物治疗公司（GTC-Bio）在转基因奶牛中表达了2 g/L水平的rhA，这足以满足药用辅料市场的需求，但不足以满足每年400，000 kg的全球治疗市场。我们已经开发了一种策略，选择性地将rhA产量提高到10 g/L，这一水平将使该生产平台在治疗性人白蛋白市场上具有商业可行性。初步研究表明，编码序列中CpG基序的超甲基化可能会限制转基因动物中rhA的表达，而去除CpG位点而不改变白蛋白氨基酸序列可能会显着增加rhA的表达。在这个快速通道I期项目中，我们建议通过以下方法建立高水平生产rhA的原理证明：1）重塑rhA基因以去除CpG位点; 2）在转基因小鼠中评估重塑的构建体，我们的标准测试系统; 3）优化了一种新型的高-本发明涉及一种通量筛选方法，其将显著减少回收用于产生转基因动物的转基因细胞系的工作量和成本通过体细胞核转移;和4）优化替代选择方案以优先回收具有高转基因拷贝数的转染细胞系。该项目的成功完成将导致一个快速通道第二阶段项目，以生产和表征转基因小牛产生高水平的rhA在他们的牛奶。如果该计划成功，这将是第一次以商业规模生产rhA，对于治疗应用具有成本效益。

DESCRIPTION (provided by applicant): The overall goal of this program is to provide an abundant, cost-effective, biosecure source of human albumin for human therapeutic use by producing recombinant human albumin (rhA) in the milk of transgenic cows. Human albumin is isolated from donated human plasma, but shifts in the plasma fractionation industry, concerns about cost and anxiety about blood safety have motivated efforts to produce recombinant human albumin as an alternative source for human therapeutic applications. GTC Biotherapeutics Inc. (GTC-Bio) has expressed rhA in transgenic cows at levels of 2 g/L, which is adequate for the pharmaceutical excipient market but not the 400,000 kg annual worldwide therapeutic market. We have developed a strategy for selectively boosting rhA production to 10 g/L, a level that would make this production platform commercially feasible for the therapeutic human albumin market. Preliminary work suggests that hypermethylation of CpG motifs in the coding sequence may limit rhA expression in transgenic animals, and that removing CpG sites without altering the albumin amino acid sequence may significantly increase rhA expression. In this Fast-Track Phase I project, we propose to establish proof of principle for high-level production of rhA by: 1) remodeling the rhA gene to remove CpG sites; 2) evaluating the remodeled construct in transgenic mice, our standard test system; 3) optimizing a novel high-throughput screening method that will substantially reduce the effort and cost of recovering transgenic cell lines for use in generating transgenic animals by somatic cell nuclear transfer; and 4) optimizing an alternative selection scheme to preferentially recover transfected cell lines with high transgene copy numbers. Successful completion of this project will lead to a Fast-Track Phase II project to produce and characterize transgenic calves producing high levels of rhA in their milk. If this program is successful, it will be the first production of rhA at a commercial scale that is cost-effective for therapeutic applications.