Oral FGF21 delivery to limit its action to the liver

内分泌作用蛋白成纤维细胞生长因子21 （FGF21）是一种重要的代谢调节因子，在啮齿动物模型和非人灵长类动物中被证明可以减少肥胖和相关的代谢紊乱。FGF21类似物的首次临床研究证实了人体体重、循环脂质和胰岛素的降低，并指出肝脏是最重要的靶点，特别是在患有脂质紊乱和非酒精性脂肪性肝病的患者中。肝脏也是循环FGF21的主要来源，但对FGF21对肝脏的直接影响知之甚少。FGF21的生物学非常复杂，由于其在多个靶器官中具有多种代谢功能，并且对其确切的细胞靶点和细胞内机制仍存在争议，因此仅部分了解。此外，从药理学上讲，血浆浓度升高会导致不良副作用，如骨量减少。我们假设通过口服给药系统将FGF21靶向递送到肝脏将导致代谢改善，同时避免不良反应。此外，口服可以方便地每天使用。该项目的主要目标是(i)澄清FGF21作用的肝脏特异性作用，以及（ii）在表达FGF21的可食用植物的帮助下建立FGF21的口服给药。目的(i)将研究重组FGF21对野生型和FGF21消融小鼠肝脏和原代肝细胞的影响。利用无偏倚的转录组学和代谢组学方法，将在体外和体内鉴定和验证肝细胞中FGF21的新代谢靶点。这也将有助于建立一个稳健可靠的读出系统来分析口服FGF21对肝脏的影响。对于目标（ii），将建立一个植物表达系统。可食用植物提供了一个很好的系统，因为植物细胞保护细胞内化合物在胃中不被降解，并在植物物质被分解时将它们释放到肠道中（在植物生物胶囊中）。FGF21的肠道释放将导致肝脏通过门静脉大量暴露于FGF21，但由于FGF21的半衰期短，易于快速蛋白水解，因此循环水平较低。为了提高植物细胞的保护作用，FGF21会附着在一个油结合结构域上。转铁蛋白作为第二融合体从肠道进入血液。它会通过内源性蛋白酶在血清中被分离。使用瞬时产生的纯化FGF21变体，将在模拟胃肠道和Ussing室系统的体外系统中分析其有效性。最好的变种将用于稳定转化食用油丰富的烟草。将通过FGF21消融小鼠和肥胖小鼠的喂养研究来研究它们的生物利用度和代谢效应。

The endocrine acting protein fibroblast growth factor 21 (FGF21) is an important metabolic regulator shown to reduce obesity and associated metabolic disorders in rodent models and non-human primates. The first clinical studies with FGF21 analogues confirmed the lowering of body weight, circulating lipids and insulin in humans and pointed to the liver as the most important target, particularly in patients suffering from lipid disorders and non-alcoholic fatty liver disease. Liver is also the main source of circulating FGF21 but little is known about direct hepatic effects of FGF21. The biology of FGF21 is quite complex and only partially understood due to its diverse metabolic functions in multiple target organs and ongoing controversies about the exact cellular targets and intracellular mechanisms. Also, pharmacologically increased plasma concentrations can lead to adverse side effects such as the reduction of bone mass.We hypothesize that the targeted delivery of FGF21 to the liver through an oral administration system will lead to metabolic improvements while avoiding adverse effects. In addition, oral delivery would allow an easy daily application. The main objectives of this project are (i) clarification of the hepatic specific effects of FGF21 action, and (ii) establishing the oral administration of FGF21 with the help of edible plants that express FGF21.For objective (i) the effects of recombinant FGF21 on liver and primary hepatocytes of wildtype and FGF21 ablated mice will be investigated. Using an unbiased transcriptomic and metabolomic approach, novel metabolic targets of FGF21 in hepatocytes will be identified and verified in vitro and in vivo. This will also serve to establish a robust and reliable read out system for analyzing hepatic effects of orally applied FGF21. For objective (ii) a plant expression system will be established. Edible plants provide an excellent system since the plant cells protect intracellular compounds from degradation in the stomach and release them in the intestine when the plant material is decomposed (in planta bioencapsulation). The intestinal release of FGF21 will lead to a high exposure of the liver to FGF21 through the portal vein, but low circulating levels due to the short half-life of FGF21 which is subject to rapid proteolysis. To improve the protective effect of the plant cell, FGF21 will be attached to an oilbinding domain. For transcytosis from the intestine into the blood transferrin is used as second fusion partner. It will be cleaved off in the serum via an endogenous protease. Using transiently produced, purified FGF21 variants, the effectiveness will be analysed in in vitro systems of a simulated gastrointestinal tract and the Ussing chamber system. The best variant will be used to stably transform edible oil-rich tobacco. Their bioavailability and metabolic effects will be investigated by feeding studies using FGF21 ablated and obese mice.