Modernize animal pharming with emerging gene targeting technologies

描述(申请人提供)：动物制药是使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。美国食品和药物管理局(FDA)于2009年首次批准了ATryn，这是一种从转基因山羊奶中生产的重组人抗凝血酶III。在FDA批准之前，该药于2006年获得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日益增加，献血量的减少，特别是在发展中国家，动物生物反应器可以显著降低单位蛋白质成本，与传统的基于细胞培养的方法相比，单位成本可降低多达10倍，因此，继续探索和改进动物制药技术具有重要意义。转基因药用动物的制备主要有两种方法，即原核DNA显微注射和核移植。第一种是原核显微注射，存在着转基因拷贝数和插入位置随机、转基因表达水平不可预测、转化率普遍较低等问题。第二种是核移植，只适用于牛、猪、山羊等可以高效克隆的动物，而不适用于兔子等其他物种。此外，核移植还存在健康活产率低的问题。在这里，我们建议通过整合新兴的基因打靶技术、最近可用的兔全基因组序列信息以及我们在兔胚胎学和转基因方面的专业知识，来现代化、简化并可能标准化已有30年历史的动物配药技术。我们建议使用基于Cas9的RNA引导的内切酶(RGEN)或转录激活因子样效应子核酸酶(TALEN)来促进一个主要的兔乳蛋白基因(即β-酪蛋白)上的转基因敲入。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(S)能够以一种高效的“盒式交换方式”在整合酶系统介导的对接位点之间进行模块化插入。这一新系统将为目前的制药技术带来以下改进：(I)利用内源主要乳蛋白启动子的更可靠的表达系统，使重组蛋白产量提高到10g/L的水平；(Ii)模块化系统，可用于许多其他重组蛋白的简化生产；以及(Iii)针对不同生物的标准化生产系统，已知转基因插入位点和拷贝数，并可预测产品的表达水平。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。

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.