The Chloroplast Expression System as a platform for orally bioavailable muscle therapeutics

摘要 靶向的两个主要途径增强胰岛素样生长因子I（IGF-I）的作用，并抑制胰岛素样生长因子I（IGF-I）的作用。 Myostatin/Gdf 8及其相关家族成员的抗生长途径。已经进行了几次临床试验 和/或正在研究蛋白质/抗体和基因治疗方法。虽然这些方法可能 提供这些途径的有效调节，并可能保留功能性肌肉质量，迄今为止没有研究 开发了一种口服生物可利用的化合物，可以促进肌肉生长。因此，我们断言， 递送促生长化合物的挑战是缺乏口服生物利用度，因为目前所有的选择都依赖于 肌肉内或静脉内/动脉注射。在本申请中，我们建议利用 叶绿体生物囊化生产肌肉生长抑制素前肽和IGF-I的生物有效性和稳定性， 相对于植物中的总可溶性蛋白质，能够高产量地生产蛋白质。合作团队将 联合收割机在叶绿体表达方面的专业知识与骨骼肌生理学方面的专业知识相结合， 生长促进剂的递送策略可以增加小鼠的功能性肌肉质量。 在目标1中，将与IgG的Fc结构域融合的鼠肌肉生长抑制素前肽在烟草中表达， benthamiana中进行，并评估肌生长抑制素抑制的基于细胞的活性和体内功效。同样，在Aim 2，将制备类似的Pro-IGF-I-Fc构建体，并且也以相同的方式进行测试。完成时 这个项目，这一战略的全面评估将完成，并可能至少有1名候选人将 确定进入生菜生产，这是一个临床相关的平台， 治疗性蛋白质的无标记生产。最终，叶绿体表达系统可能是 作为一种潜在的战略来表达和交付既定的因素以及新发现的因素， 可以促进肌肉生长的蛋白质。

ABSTRACT Two of the major pathways targeted enhance the actions of insulin-like growth factor I (IGF-I), and inhibit the anti-growth pathways of Myostatin/Gdf8 and its related family members. Several clinical trials have occurred and/or are underway examining protein/antibody and gene therapy approaches. While these approaches may provide potent regulation of these pathways, and potentially preserve functional muscle mass, no study to date has developed an orally bioavailable compound that can promote muscle growth. Thus, we assert that a major challenge for delivery of pro-growth compounds is the lack of oral bioavailability, as all current options rely on intramuscular or intravenous/arterial injections. In this application, we propose to capitalize on the oral biovailability and stability of chloroplast bioencapsulation to produce myostatin propeptide and IGF-I, which enable high production of protein relative to the total soluble protein in plants. The collaborative team will combine expertise in chloroplast expression with expertise in skeletal muscle physiology to determine if a novel delivery strategy for growth promoting agents can increase functional muscle mass in mice. In Aim 1, the murine myostatin propeptide fused to the Fc domain of IgG will be expressed in Nicotiana benthamiana and evaluated for cell-based activity and in vivo efficacy of myostatin inhibition. Likewise, in Aim 2, a similar construct of Pro-IGF-I-Fc will be made, and also tested in the same manner. At the completion of this project, a thorough evaluation of this strategy will be complete, and potentially at least 1 candidate will be identified to move forward into production in lettuce, which is a platform that is clinically relevant and affords marker free production of therapeutic proteins. Ultimately, the chloroplast expression system may be established as a potential strategy to express and deliver established factors as well as newly discovered proteins that can promote muscle growth.