Protein-free regulation of erythropoietin expression by a drug-sensing riboswitch

描述(申请人提供)：在健康的人体中，红细胞的产生速率受低氧调节的促红细胞生成素(EPO)的表达控制。患有各种慢性病的患者往往会患上贫血，这种贫血可以通过EPO替代疗法来缓解。目前，外源性EPO是作为一种经常使用的重组蛋白提供的。一种有吸引力的替代方案是将EPO基因输送到人体的异位部位，为长期治疗慢性病提供内部激素来源。为了将基于EPO的基因疗法过渡到临床，需要调节表达水平的工具，以将红细胞计数保持在正常范围。目前可用的大多数药物依赖的基因调控系统依赖于对人体来说是外来的蛋白质因子。为了避免由这些外来蛋白引发的免疫排斥反应的风险，我们计划创建一个无蛋白系统，用于药物依赖地调节外源EPO基因进入患者四肢肌肉的表达。该系统将基于在EPO mRNA中插入药物敏感核糖开关，当患者服用药物配体时，该开关可以启动表达。这个第一阶段项目的主要目标是：(1)使用随机的单链RNA池来筛选发生配体依赖构象变化的适体，以及(2)将它们插入EPO表达盒中，以评估它们在体外、在瞬时转染的培养细胞中调节EPO表达的能力。我们的第一阶段工作应该提供概念证据，证明所选的核糖开关可以在活细胞中作为全长EPO mRNA背景下的表达水平的功能性、药物依赖性调节因子。第二阶段的工作将在体内测试最有希望的受调控的EPO表达结构(S)，首先在小鼠身上，然后在非人类灵长类动物上进行研究。总体目标是根据患者血液学参数的变化，通过给予或停用诱导剂药物，稳定地维持所需的红细胞生成水平。这种基因疗法将为慢性贫血患者提供一种安全、长期的治疗选择。药物敏感核糖开关还可以被整合到编码其他治疗性蛋白的表达框中，例如胰岛素和其他激素、生长因子或治疗性抗体。公共卫生相关性：重组人促红细胞生成素蛋白目前代表着联邦医疗保险最高的药品费用。对于慢性贫血的长期治疗，基因疗法是一种有前途的、更经济的替代方案。然而，由于EPO的缺乏和过度表达都会导致严重的健康问题，因此发展安全的基于基因的治疗的基石是找到可靠的工具来调节表达。我们建议的非免疫原性调节系统可以通过服用一种已经被FDA批准在人类患者身上长期使用的药物来控制EPO的表达。

DESCRIPTION (provided by applicant): In the healthy human body the rate of red blood cell production is controlled by the hypoxia-regulated expression of erythropoietin (EPO). Patients suffering from various chronic diseases often develop anemia that can be alleviated by EPO replacement therapy. Currently, exogenous EPO is provided as a frequently administered recombinant protein. An attractive alternative would be to deliver the EPO gene into ectopic sites of the body to provide an internal hormone source for the long-term management of a chronic disease. For the transition of an EPO-based gene therapy to the clinic, tools to regulate expression levels would be required to maintain red blood cell counts in the normal range. Most currently available drug-dependent gene regulatory systems rely on protein factors that are foreign to the human body. To avoid the risk of immune rejection triggered by such foreign proteins, we plan to create a protein-free system for the drug-dependent regulation of expression from exogenous EPO genes delivered into the patient's limb muscles. The system will be based on inserting a drug-sensing riboswitch into the EPO mRNA that can turn expression on when the patient is taking the drug-ligand. The major goals of this Phase I project are: (1) to use a randomized pool of ssRNAs to select for aptamers that undergo ligand-dependent conformational changes, and (2) to insert them into EPO expression cassettes in order to assess their ability to regulate EPO expression in vitro, in transiently transfected cultured cells. Our Phase I efforts should provide proof of concept that the selected riboswitch can serve as a functional, drug-dependent regulator of expression levels in the context of the full-length EPO mRNA, in live cells. Phase II efforts will test the most promising regulated EPO expression construct(s) in vivo, initially in mice, followed by studies in non-human primates. The overall goal is the stable maintenance of a desired level of erythropoiesis by administering or withdrawing the inducer drug, as indicated by changes in the patient's hematological parameters. This gene therapy would provide a safe, long-term treatment option for patients with chronic anemia. The drug-sensing riboswitch can also be incorporated into expression cassettes encoding other therapeutic proteins, such as insulin and other hormones, growth factors or therapeutic antibodies. PUBLIC HEALTH RELEVANCE: Recombinant human EPO protein currently represents Medicare's highest drug expense. Gene therapy is a promising, more economical alternative for the long-term management of chronic anemia. However, since not only the lack, but also the overexpression of EPO can cause serious health problems, the cornerstone of developing safe gene-based therapy is finding a reliable tool for regulating expression. Our proposed non- immunogenic regulatory system could control EPO expression by taking a drug that is already FDA-approved for long-term use in human patients.