Multimeric RNA Aptamers for Glucose-Responsive Insulin Formulations

描述(由申请人提供)： 具有里程碑意义的糖尿病控制和并发症试验(DCCT)和英国前瞻性糖尿病研究(UKPDS)已经临床证明，以较低的糖化血红蛋白(HbA1c)水平衡量，更严格的血糖控制可以显著降低糖尿病并发症的发生率。真正的血糖控制需要提供血糖调节的胰岛素替代治疗。SmartCells正在通过提供SmartInsulinTM来满足这一需求，这是一种新型的、微创的、每天一次的注射，以临床相关的方式对变化的血糖水平进行感知和反应。 与泵和细胞系统不同，SmartInsulin是一种纳米结构材料，由两个生物分子构建块自组装而成：糖基化的胰岛素-聚合物共轭化合物(IPC)和多价葡萄糖结合分子(GBM)。为了取代潜在的免疫原性、基于蛋白质的基底膜，我们开始在NIH SBIR第一阶段拨款(DK072774-01)下，进化出更安全、非免疫原性的合成基底膜，这些基底膜由基于核酸酶的核糖核酸(RNA)适配子构建。该项目的结果是产生了一系列单克隆、单体适配子，它们与基于糖原的IPC结合，并在葡萄糖存在的情况下洗脱。此外，这些序列已经被四聚化，不仅增强了糖原结合，而且还准备好通过糖原IPC的葡萄糖响应性交联来自组装系统。然而，这些适配子的结合亲和力太高，不具有生理相关性。 目前的两年提案寻求对现有池进行突变和进化，以获得与我们的基于蛋白质的GBM相同结合特性的多聚体适配子。我们进一步寻求评估它们在体内形成SmartInsulin产品、控制糖尿病大鼠、帮助将不良注射部位反应降至最低以及在长期给药条件下将不必要的组织堆积降至最低的能力。

DESCRIPTION (provided by applicant): The landmark Diabetes Control and Complications Trial (DCCT) and the UK Prospective Diabetes Study (UKPDS), have clinically proven that tighter glycemic control, as measured by lower glycosylated hemoglobin A1c (HbA1c) levels, significantly reduces the incidence of diabetic complications. True glycemic control requires treatments that provide plasma glucose-regulated insulin replacement. SmartCells is addressing this need by offering SmartInsulinTM, a novel, minimally invasive, once-a-day injection that both senses and responds to altered serum glucose levels in a clinically relevant way. Unlike pump- and cell-based systems, SmartInsulin is a nanostructured material that self-assembles from two biomolecular building blocks: a glycosylated insulin-polymer conjugate (IPC) and a multivalent glucose-binding molecule (GBM). In order to replace potentially immunogenic, protein-based GBMs, we set out to evolve safer, non-immunogenic, synthetic GBMs constructed from nuclease-resistant, ribonucleic acid (RNA)-based aptamers under an NIH SBIR Phase I grant (DK072774-01). The result of that project has yielded a family of monoclonal, monomeric aptamers that bind glycogen-based IPCs and elute in the presence of glucose. Furthermore, the sequences have been tetramerized to not only enhance glycogen binding but also prepare the system to self assemble through glucose-responsive crosslinking of glycogen IPCs. Nevertheless, the binding affinities for these aptamers are too high to be physiologically relevant. The current two-year proposal seeks to mutagenize and evolve the existing pool to obtain multimeric aptamers with the same binding characteristics as our protein-based GBMs. We further seek to evaluate their ability to form SmartInsulin products, control diabetic rats in vivo, help minimize adverse injection site reaction, and minimize unwanted tissue accumulation under chronic dosing conditions.