Aptamer-directed crossing of the blood barrier for enzyme therapy of LSDs

描述(申请人提供)：溶酶体储存障碍(LSD)是一种遗传性疾病，个别罕见，但总体上会造成严重的医疗和人类负担。预计了几十年的酶替代疗法在20世纪90年代成为治疗高谢病的现实，2003年成为治疗MPS I和Fabry病的现实，目前正在对其他几种LSD进行临床试验。然而，大多数LSD患者无法从这些进步中受益，因为他们的疾病全部或部分表现在中枢神经系统。由于脑微血管系统产生的血脑屏障(BBB)，给药的酶不会进入中枢神经系统。然而，存在着运输大脑所需蛋白质的生理系统。我们建议利用转铁蛋白受体(TFR)的跨细胞作用来运送治疗酶穿过血脑屏障。与TFR的结合将通过选择与该受体结合的适体来介导。适配子是从大的随机文库中挑选出来的单链核酸分子，可以与任何目标分子结合，很像单抗。在初步研究中，我们选择了在体外与小鼠TFR胞外区域结合的RNA适配子。我们发现，这种适配子可以饱和的方式与培养的淋巴瘤细胞结合，生物素化的适配子-链霉亲和素结合物可以被培养的L细胞内吞。具体目标1是产生抗核糖核酸酶的修饰RNA适配子，用于体内后续研究，并开发将适配子连接到模型蛋白质的一般程序。具体目标2是定量测量结合物与转铁蛋白受体的结合，将体外测量的性质与细胞培养中的结合和内吞作用相关联。具体目的3是建立血脑屏障的细胞培养模型，用于研究适配子和蛋白质-适配子的跨细胞作用，以筛选出最有希望的体内研究候选者。具体目的4是测试适体修饰的溶酶体酶进入中枢神经系统的能力，并纠正LSD小鼠模型的病理。疾病/酶组合依次为MPS I/α-L-艾杜糖苷酶、MPS III B/α-N-乙酰氨基葡萄糖苷酶、MPS VII/β-葡萄糖醛酸酶和桑德霍夫病/β-己糖苷酶。如果成功，这项研究将为将治疗性分子引入大脑和治疗涉及中枢神经系统的溶酶体储存障碍提供一般方法，并可能对其他神经系统疾病的治疗具有借鉴意义。

DESCRIPTION (provided by applicant): Lysosomal storage disorders (LSDs) are genetic diseases, individually rare, but in the aggregate presenting a serious medical and human burden. Enzyme replacement therapy, anticipated for decades, became a reality for Gaucher disease in the 1990s, for MPS I and Fabry disease in 2003, and is currently in clinical trial for several other LSDs. And yet most LSD patients are unable to benefit from these advances because their disease manifests itself, in whole or in part, in the central nervous system. The administered enzyme does not enter the CNS because of the blood brain barrier (BBB) created by the brain microvasculature. However, there exist physiological systems for transporting proteins needed by the brain. We propose to utilize the transcytosis of the transferrin receptor (TfR) to ferry therapeutic enzymes across the BBB. Binding to the TfR will be mediated by aptamers selected for binding to that receptor. Aptamers are single-stranded nucleic acid molecules that are selected from large random libraries for binding to any target molecule, much like monoclonal antibodies. In preliminary studies, we selected RNA aptamers that bind in vitro to the extracellular domain of mouse TfR. We found that such aptamers could bind in saturable fashion to cultured lymphoma cells and that a biotinylated aptamer-streptavidin conjugate could be endocytosed by cultured L cells. Specific Aim 1 is to generate modified RNA aptamers resistant to ribonuclease for subsequent studies in vivo, and to develop general procedures to conjugate aptamers to model proteins. Specific Aim 2 is to measure quantitatively the binding of the conjugates to TfR, correlating properties measured in vitro with binding and endocytosis in cell culture. Specific Aim 3 is to set up a cell culture model of the BBB for the study of transcytosis of aptamers and protein-aptamer conjugates, in order to select the most promising candidates for in vivo studies. Specific Aim 4 is to test the ability of aptamer-modified lysosomal enzymes to enter the CNS and correct the pathology in mouse models of LSD. Disease/enzymes combinations selected for study are, in order of priority, MPS I/ alpha-L-iduronidase, MPS III B/ Alpha-N-acetylglucosaminidase, MPS VII/ beta-glucuronidase and Sandhoff disease/beta-hexosaminidase. If successful, this study will provide a general approach for introducing therapeutic molecules into the brain and treating lysosomal storage disorders with CNS involvement, and may have implications for treating other neurologic diseases.