Improved yield of human albumin from transgenic cow milk

描述(申请人提供)：该计划的总体目标是通过在转基因奶牛的乳汁中生产重组人白蛋白(RHA)，为人类治疗提供丰富、经济有效的生物安全来源。人类白蛋白是从捐赠的人类血浆中分离出来的，但血浆分离行业的变化、对成本的担忧以及对血液安全的担忧促使人们努力生产重组人白蛋白，作为人类治疗应用的替代来源。GTC BioTreateutics Inc.(GTC-Bio)已经在转基因奶牛中以2g/L的水平表达了RHA，这一水平足以满足制药辅料市场，但不足以满足全球每年40万公斤的治疗市场。我们已经开发了一种有选择地将RHA产量提高到10g/L的策略，这一水平将使该生产平台在治疗性人体白蛋白市场上具有商业可行性。初步工作表明，编码序列中CpG基序的超甲基化可能限制转基因动物中RHA的表达，而在不改变白蛋白氨基酸序列的情况下移除CpG位点可能显著增加RHA的表达。在这个快速通道第一阶段项目中，我们建议通过以下方式为RHA的高水平生产建立原则证明：1)改造RHA基因以移除CpG位点；2)评估我们的标准测试系统-转基因小鼠中改造的结构；3)优化一种新的高通量筛选方法，该方法将大大减少回收转基因细胞系用于通过体细胞核移植产生转基因动物的工作量和成本；以及4)优化替代选择方案以优先恢复高转基因拷贝数的转基因细胞系。该项目的成功完成将导致快速通道第二阶段项目，以培育和鉴定在牛奶中产生高水平RHA的转基因牛犊。如果该项目成功，这将是RHA的第一次商业规模生产，对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.