NIS PROTEINS WITH ALTERED ANION SPECIFICITY

描述（由申请人提供）：NIS是钠碘同向转运体，介导甲状腺中碘的摄取和浓度，为诊断甲状腺疾病提供基础。 放射性成像和放射性碘治疗，这是在临床和兽医实践中常规使用。当NIS基因被引入非甲状腺细胞时，它们可以像甲状腺细胞一样，使用碘化物/高锝酸盐放射成像检测和/或使用碘化物放射疗法破坏。 Imanis Life Sciences正在开发NIS报告基因技术，用于准确、灵敏、高分辨率地监测活体受试者的基因调控和细胞命运。与依赖于光学成像的荧光素酶和GFP报告基因不同，NIS可用于大型动物和人类以及深层小鼠组织中的定量表达监测，因为与光子不同，伽马射线通过其穿过的组织的衰减最小。NIS的其他优点是临床批准的NIS特异性放射性同位素的现成可用性，缺乏免疫原性（NIS是一种自身蛋白质）和对靶组织无害。 来自循环放射性示踪剂和来自天然表达NIS的组织（如甲状腺、胃和唾液腺）的背景信号可显著限制NIS报告基因技术的灵敏度、分辨率和实用性。 重要的是，NIS介导的NIS特异性放射性示踪剂阴离子的摄取可以被高氯酸盐有效地抑制，高氯酸盐可以安全地施用给人类受试者用于此目的。因此，本申请的目标是分离高氯酸盐抗性NIS突变体，其即使在高氯酸盐存在下也能有效转运碘化物或高锝酸盐离子。SPECT和PET成像研究中的背景信号将通过非放射性高氯酸盐阴离子与NIS放射性示踪剂的共同给药来降低。如果高氯酸盐抗性NIS报告蛋白是离子选择性的，并且对高氯酸盐抑制具有抗性，则示踪剂摄取将发生在表达突变NIS蛋白的遗传修饰的细胞/组织中，但在天然表达NIS的器官中被阻断。对于第一阶段的申请，我们有以下具体目标。具体目标1：筛选表达NIS变异体文库的细胞，以寻找转运碘或高锝酸盐的能力相对耐受高氯酸盐抑制的变异体。具体目的2：构建并验证人NIS中的底物和共转运离子结合位点，并合理设计突变，以允许在高氯酸盐存在下转运放射性碘或高锝酸盐。目标1或2的里程碑将触发提交第二阶段SBIR资助申请，这将是分离出一种NIS变体，该变体在高氯酸盐存在下比野生型（WT）NIS转运至少多40%的碘化物。这个目标是非常可行的，因为我们已经有了第一代NIS变体，它选择性地摄取NIS底物。

DESCRIPTION (provided by applicant): NIS, the sodium iodide symporter, mediates the uptake and concentration of iodide in the thyroid gland, providing the basis for diagnostic thyroid radioimaging and radioiodine therapy, which are used routinely in clinical and veterinary practice. When the NIS gene is introduced into non-thyroid cells, they can, like thyroid cells, be detected using iodide/pertechnetate radioimaging and/or destroyed using iodide radiotherapy. Imanis Life Sciences is developing NIS reporter gene technology for accurate, sensitive, high resolution monitoring of gene regulation and cell fate in living subjects. Unlike luciferase and GFP reporter genes, which rely on optical imaging, NIS can be used for quantitative expression monitoring in large animals and humans as well as in deep-seated mouse tissues because, unlike photons, gamma rays are minimally attenuated by the tissues through which they pass. Additional advantages of NIS are the ready availability of clinically approved NIS-specific radioisotopes, lack of immunogenicity (NIS is a self-protein) and harmlessness to targeted tissues. Background signals from circulating radiotracer and from tissues naturally expressing NIS, such as thyroid, stomach and salivary glands, can significantly limit the sensitivity, resolution and utility of the NIS reporter gene technology. Importantly, NIS-mediated uptake of NIS specific radiotracer anions can be potently inhibited by perchlorate, which can be safely administered to human subjects for this purpose. The goal of the current application is therefore to isolate perchlorate resistant NIS mutants that efficiently transport iodide or pertechnatate ion even in the presence of perchlorate. Background signal in SPECT and PET imaging studies will then be reduced by coadministration of nonradioactive perchlorate anions with a NIS radiotracer. If the perchlorate-resistant NIS reporter protein is ion selective, and resistant to perchlorate inhibition, tracer uptake will occur in genetically modified cells/tissue expressing th mutated NIS protein, but be blocked in organs naturally expressing NIS. For the phase I application we have the following specific aims. SPECIFIC AIM 1: Screen cells expressing a library of NIS variants for a variant whose ability to transport iodide or pertechnetate is relativly resistant to perchlorate inhibition SPECIFIC AIM 2: To construct and validate substrate and co-transported ions binding sites in human NIS and rationally design mutations that will allow radioiodide or pertechnetate transport in the presence of perchlorate. The milestone for aim 1 or 2 that will trigger submission of phase II SBIR grant application will be the isolation of a NIS variant that transports at least 40% more iodide than wild-type (WT) NIS in the presence of perchlorate. The aim is highly feasible as we have already a first generation NIS variant that selectively uptakes NIS substrates.