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）已在临床上证明，更严格的血糖控制（通过较低的糖化血红蛋白A1 c（HbA 1c）水平测量）可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浆葡萄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，SmartInsulinTM是一种新型的微创的，每天一次的注射，它可以以临床相关的方式感知和响应血清葡萄糖水平的变化。 与基于泵和细胞的系统不同，SmartInsulin是一种纳米结构材料，由两种生物分子构建块自组装而成：糖基化胰岛素-聚合物缀合物（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GBM，我们着手发展更安全，非免疫原性，合成GBM构建的核酸酶抗性，核糖核酸（RNA）为基础的适配体根据NIH SBIR第一阶段的资助（DK 072774 -01）。该项目的结果产生了一个单克隆单体适体家族，其在葡萄糖存在下结合基于糖原的IPC和APC。此外，序列已被四聚化以不仅增强糖原结合，而且使系统准备通过糖原IPC的葡萄糖响应性交联进行自组装。然而，这些适体的结合亲和力太高而不具有生理学相关性。 目前为期两年的提案旨在诱变和进化现有的池，以获得与我们的蛋白质为基础的GBM相同的结合特性的多聚体适体。我们进一步寻求评估其形成SmartInsulin产品的能力，在体内控制糖尿病大鼠，帮助最大限度地减少注射部位不良反应，并在慢性给药条件下最大限度地减少不必要的组织蓄积。

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.