Modernize animal pharming with emerging gene targeting technologies

利用新兴基因靶向技术实现动物制药现代化

• 批准号: 8712085
• 负责人: JIE XU
• 金额: $ 18.62万
• 依托单位: APPLIED STEMCELL, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8712085
• 关键词: Animals; Antithrombin III; Biological; Bioreactors; Blood; Breeding; Candidate Disease Gene; Cattle; Cell Culture Techniques; Collaborations; Coupled; DNA; Developing Countries; Development; Docking; Drug Approval; Embryo; Embryology; Europe; European; Factor VIIa; Family suidae; Female; Future; Gene Targeting; Genes; Genotype; Goat; Guide RNA; Human; Injection of therapeutic agent; Integrase; Knock-in Mouse; Live Birth; Mammary gland; Mediating; Medicine; Methods; Michigan; Microinjections; Milk; Milk Proteins; Nuclear; Oryctolagus cuniculus; Pharmaceutical Preparations; Phase; Process; Production; Proteins; Recombinant Proteins; Recombinants; Ribonucleases; Scientist; Site; System; Technology; Testing; Transcription Coactivator; Transgenes; Transgenic Animals; Transgenic Organisms; United States Food and Drug Administration; Universities; Vaccines; Work; base; beta-Casein; cost; endonuclease; genome sequencing; improved; interest; nuclear transfer; nuclease; phase 2 study; pregnant; promoter; public health relevance; tool; transgene expression; vector

DESCRIPTION (provided by applicant): Animal pharming is the process of using transgenic animals to produce human drugs, mostly in the mammary glands of female animals. The US Food and Drug Administration (FDA) issued its first approval for ATryn, the recombinant human antithrombin III, produced in the milk of transgenic goats in 2009. Prior to FDA approval, this drug gained the approval by the European Medicines Agency in 2006. Another drug, Ruconest, produced in the milk of rabbits was approved in Europe in 2011. Because of the increasing global demand for human proteins and vaccines, the decreasing volume of donated blood especially in developing countries, and the fact that animal bioreactors can significantly reduce the unit cost per protein by as much as ten folds comparing to the traditional cell culture based method, it is important to continue exploring and improving the animal pharming technologies. There are mainly two methods to produce transgenic pharming animals, namely pronuclear DNA microinjection and nuclear transfer. The first one, pronuclear microinjection, suffers from problems such as random copy numbers and insertion sites of the transgene, unpredictable level of the transgene expression, and generally low transgenic rates. The second one, nuclear transfer, can only be applied to animals that can be cloned efficiently such as cattle, pigs, and goats, but not to other species such as rabbits. Furthermore, nuclear transfer suffers from low rates of healthy live births. Here we propose to modernize, streamline, and possibly standardize the 30-yr old animal pharming technology by integrating the emerging gene targeting technologies, the recently available whole genome sequence information of rabbits, and our expertise in rabbit embryology and transgenics. We propose to use Cas9 based RNA Guided Endonucleases(RGEN) or Transcription Activator-like Effector Nuclease (TALEN) to facilitate the knock in of a transgene at the locus of one major rabbit milk protein gene (i.e. beta-casein). We further propose to introduce "docking" sites (attP) flanking the transgene, enabling modularized insertion of new transgene(s) between the docking sites mediated by the integrase system in a "cassette exchange manner" with high knock- in efficiency. This new system will bring the following improvements to the current pharming technologies: (i) a more reliable expression system that takes the advantage of the promoter of an endogenous major milk protein, resulting in elevated recombinant protein yield up to the level of 10 g/L; (ii) a modularized system that can be used for streamlined production of many other recombinant proteins; and (iii) a standardized production system for different biologicals of interests with known insertion site and copy number of the transgene, and predictable expression level of the product. More importantly this system can be readily adapted for other large animal species such as pigs and cattle.

描述（由申请人提供）：动物制药是利用转基因动物生产人类药物的过程，主要是在雌性动物的乳腺中。2009年，美国食品和药物管理局（FDA）首次批准了用转基因山羊的奶生产的重组人抗凝血酶III （ATryn）。在FDA批准之前，该药于2006年获得了欧洲药品管理局的批准。另一种用兔奶生产的药物Ruconest于2011年在欧洲获得批准。由于全球对人类蛋白质和疫苗的需求不断增加，特别是在发展中国家献血量不断减少，以及动物生物反应器与传统的基于细胞培养的方法相比，可以显著降低每种蛋白质的单位成本高达十倍，因此继续探索和改进动物制药技术非常重要。制备转基因转基因动物的方法主要有两种，即原核DNA显微注射和核移植。第一种方法是原核显微注射，存在转基因拷贝数和插入位点随机、转基因表达水平不可预测、转基因率普遍较低等问题。第二种方法是“核移植”，这种方法只适用于牛、猪、山羊等能够有效克隆的动物，而不适用于兔子等其他物种。此外，核移植的健康活产率很低。在这里，我们建议通过整合新兴的基因靶向技术，最近可用的家兔全基因组序列信息，以及我们在家兔胚胎学和转基因方面的专业知识，使已有30年历史的动物种植技术现代化，简化并可能标准化。我们建议使用基于Cas9的RNA引导内切酶（RGEN）或转录激活因子样效应核酸酶（TALEN）来促进转基因在兔乳蛋白一个主要基因（即β -酪蛋白）位点的敲入。我们进一步建议在转基因两侧引入“对接”位点（attP），使新的转基因能够在整合酶系统介导的对接位点之间以“盒式交换方式”以高敲入效率模块化插入。该新系统将对目前的装药技术带来以下改进：(1)利用内源性主要牛奶蛋白启动子的更可靠的表达系统，使重组蛋白产量提高到10 g/L的水平；（ii）可用于精简生产许多其他重组蛋白的模块化系统；（iii）具有已知转基因插入位点和拷贝数以及可预测产品表达水平的不同感兴趣生物制剂的标准化生产系统。更重要的是，这个系统可以很容易地适应其他大型动物物种，如猪和牛。