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

描述（由申请方提供）：溶酶体贮积症（LSD）是一种遗传性疾病，个体罕见，但总体上会造成严重的医疗和人类负担。酶替代疗法，预计了几十年，成为现实戈谢病在20世纪90年代，MPS I和法布里病在2003年，目前正在临床试验中的其他几个LSD。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地表现在中枢神经系统中。由于脑微血管系统产生的血脑屏障（BBB），所施用的酶不会进入CNS。然而，存在用于运输大脑所需的蛋白质的生理系统。我们建议利用转铁蛋白受体（TfR）的转胞吞作用来运送治疗酶穿过BBB。与TfR的结合将由选择用于结合该受体的适体介导。适体是从大的随机文库中选择的单链核酸分子，用于结合任何靶分子，很像单克隆抗体。在初步研究中，我们选择了在体外结合到小鼠TfR的胞外结构域的RNA适体。我们发现，这样的适体可以结合在饱和的方式培养的淋巴瘤细胞和生物素化的适体-链霉亲和素缀合物可以内吞培养的L细胞。具体目标1是产生对核糖核酸酶具有抗性的修饰的RNA适体，用于随后的体内研究，并开发将适体缀合至模型蛋白的一般程序。具体目标2是定量测量缀合物与TfR的结合，将体外测量的性质与细胞培养物中的结合和内吞作用相关联。具体目标3是建立用于研究适体和蛋白-适体缀合物的转胞吞作用的BBB的细胞培养模型，以选择最有希望的候选物用于体内研究。具体目标4是测试适体修饰的溶酶体酶进入CNS并纠正LSD小鼠模型中的病理的能力。选择用于研究的疾病/酶组合按优先顺序为MPS I/α-L-艾杜糖醛酸酶、MPS III B/α-N-乙酰氨基葡萄糖苷酶、MPS VII/β-葡萄糖醛酸酶和桑德霍夫病/β-氨基己糖苷酶。如果成功，这项研究将提供一种将治疗分子引入大脑和治疗累及CNS的溶酶体贮积症的一般方法，并可能对治疗其他神经系统疾病产生影响。

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.