Aptamer-directed crossing blood barrier enzyme therapy

描述（由申请人提供）：溶酶体贮积症（lsd）是一种遗传性疾病，个别罕见，但总的来说是严重的医疗和人类负担。期待了几十年的酶替代疗法在20世纪90年代成为戈谢病的现实，在2003年成为MPS I和Fabry病的现实，目前正在其他几种lsd的临床试验中。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。由于脑微血管形成的血脑屏障（BBB），给药酶不能进入中枢神经系统。然而，存在着运送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）的胞吞作用将治疗酶运送过血脑屏障。与TfR的结合将通过选择与该受体结合的适体介导。适配体是从大型随机文库中选择的单链核酸分子，可以与任何目标分子结合，就像单克隆抗体一样。在初步研究中，我们选择了体外结合小鼠TfR细胞外结构域的RNA适体。我们发现这种适体可以饱和结合到培养的淋巴瘤细胞上，并且生物素化的适体-链亲和素缀合物可以被培养的L细胞内吞。具体目标1是为随后的体内研究生成抗核糖核酸酶的修饰RNA适体，并开发将适体偶联到模型蛋白的一般程序。特异性目的2是定量测量缀合物与TfR的结合，将体外测量的特性与细胞培养中的结合和内吞作用相关联。具体目的3是建立血脑屏障细胞培养模型，研究适体和蛋白-适体偶联物的胞吞作用，以选择最有希望的候选物进行体内研究。特异性目的4是检测适配体修饰的溶酶体酶进入中枢神经系统并纠正LSD小鼠模型病理的能力。选择用于研究的疾病/酶组合依次为：MPS I/ α - l -伊杜糖醛酸酶、MPS III B/ α - n -乙酰氨基葡萄糖苷酶、MPS VII/ β -葡萄糖苷酶和Sandhoff病/ β -己糖氨基酶。如果成功，这项研究将为将治疗分子引入大脑和治疗伴有中枢神经系统的溶酶体贮积症提供一种通用方法，并可能对治疗其他神经系统疾病有启示。

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.