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

描述（由申请人提供）：在健康人体中，红细胞产生的速率由缺氧调节的促红细胞生成素（EPO）表达控制。患有各种慢性疾病的患者通常会出现贫血，这种贫血可以通过EPO替代疗法来缓解。目前，外源性EPO作为经常施用的重组蛋白提供。一个有吸引力的替代方案是将EPO基因递送到身体的异位部位，为慢性疾病的长期管理提供内部激素来源。为了将基于EPO的基因治疗过渡到临床，需要调节表达水平的工具来维持红细胞计数在正常范围内。大多数目前可用的药物依赖性基因调控系统依赖于对人体来说是外来的蛋白质因子。为了避免由这种外源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质的系统，用于药物依赖性调节外源EPO基因递送到患者肢体肌肉中的表达。该系统将基于将药物敏感核糖开关插入EPO mRNA中，当患者服用药物配体时，该核糖开关可以打开表达。该I期项目的主要目标是：（1）使用随机化的ssRNA库来选择经历配体依赖性构象变化的适体，以及（2）将它们插入EPO表达盒中，以评估它们在体外瞬时转染培养细胞中调节EPO表达的能力。我们的第一阶段的努力应该提供的概念证明，所选择的核糖开关可以作为一个功能，药物依赖性调节剂的表达水平的情况下，全长EPO mRNA，在活细胞。第二阶段的工作将在体内测试最有希望的调节EPO表达构建体，首先在小鼠中，然后在非人灵长类动物中进行研究。总体目标是通过给予或停用诱导剂药物稳定维持所需的红细胞生成水平，如患者血液学参数的变化所示。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。药物敏感核糖开关也可以掺入编码其他治疗性蛋白质如胰岛素和其他激素、生长因子或治疗性抗体的表达盒中。公共卫生相关性：重组人EPO蛋白目前是医疗保险最高的药物费用。基因治疗是一种有前途的，更经济的长期管理慢性贫血的替代方案。然而，由于EPO的缺乏和过度表达都可能导致严重的健康问题，因此开发安全的基因治疗的基石是找到一种可靠的工具来调节表达。我们提出的非免疫原性调节系统可以通过服用已经被FDA批准用于人类患者长期使用的药物来控制EPO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.