Receptor-mediated transport of lysosomal enzymes

描述（由申请人提供）：酶穿过血脑屏障递送以纠正神经元储存是一个主要的未满足需求，因为近90%的溶酶体储存疾病涉及中枢神经系统，并且常规酶替代疗法不能纠正脑中的储存。这项研究的广泛目标是继续研究新发现的溶酶体酶向大脑的转运途径及其与通过酶替代疗法纠正神经元储存的相关性。我们将使用2-葡萄糖醛酸酶和1-艾杜糖醛酸酶作为模型酶，并使用小鼠粘多糖沉积病VII型（MPS VII; Sly病）和MPS I（Hurler病）作为模型溶酶体贮积病。我们寻求利用最近的突破来解决神经元存储的校正，包括：1）发现甘露糖6-磷酸（M6 P）介导的转胞吞作用（在腔膜处的内吞作用，随后快速地在远腔膜处的胞吐作用而不通过溶酶体）可以在成年小鼠脑中被上调，从而允许酶穿过血液的运输。脑屏障与新生儿中所见相当; 2）发现了一种新的化学修饰酶途径，当在血浆中维持高循环水平持续一段时间时，该途径可纠正神经元储存;和3）发现天然酶不能到达的抗性位点可以使用靶向其它细胞表面受体的嵌合酶成功靶向。我们有四个具体目标：1）确定M6 P受体介导的溶酶体酶跨血脑屏障的转胞吞作用的药理学上调的机制和意义。2)确定高碘酸盐（PerT）修饰酶神经元纠正的机制、功效和一般性。3)确定转铁蛋白-艾杜糖醛酸苷酶和转铁蛋白-2-葡萄糖醛酸苷酶嵌合酶在穿过BBB和校正小鼠MPS I和MPS VII小鼠模型中的神经元蓄积方面的功效。我们将使用各种生物化学、细胞生物学、免疫学和分子遗传学方法，并利用我们实验室通过转基因和小鼠基因敲除技术生产的MPS VII新型小鼠模型。我们结合联合收割机组织化学、组织病理学和免疫电镜来测量酶向大脑和其他耐药储存部位的递送。所寻求的答案具有根本意义，并应提供信息，导致新的治疗方法，酶替代溶酶体和其他涉及中枢神经系统的储存疾病。

DESCRIPTION (provided by applicant): Delivery of enzyme across the blood-brain barrier to correct neuronal storage represents a major unmet need since nearly 90% of lysosomal storage diseases involve the central nervous system and conventional enzyme replacement therapy does not correct storage in the brain. The broad goal of this research is to continue studies of newly discovered routes of transport of lysosomal enzymes to brain and their relevance to correcting neuronal storage by enzyme replacement therapy. We will use 2- glucuronidase and 1-iduronidase as model enzymes and murine mucopolysaccharidosis type VII (MPS VII; Sly disease) and MPS I (Hurler disease) as model lysosomal storage diseases. We seek to capitalize on recent breakthroughs addressing correction of neuronal storage, including: 1) The discovery that mannose 6-phosphate (M6P) mediated transcytosis (endocytosis at the luminal membrane followed rapidly by exocytosis at the abluminal membrane without going through the lysosome) can be upregulated pharmacologically in adult mouse brain to allow transport of enzyme across the blood-brain barrier comparable to that seen in the neonate; 2) The discovery of a novel route for chemically modified enzyme to correct neuronal storage when maintained at a high circulating level in plasma over a sustained period; and, 3) The findings that resistant sites not accessible to native enzyme can be targeted successfully using chimeric enzymes that target other cell surface receptors. We have four Specific Aims: 1) Determine the mechanism and significance of pharmacological upregulation of M6P receptor mediated transcytosis of lysosomal enzymes across the blood-brain barrier. 2) Determine the mechanism, efficacy, and generality of neuronal correction by periodate (PerT) modified enzyme. 3) Determine the efficacy of transferrin-iduronidase and transferrin-2-glucuronidase chimeric enzymes in crossing the BBB and correcting neuronal storage in murine MPS I and MPS VII mouse models. We will use a variety of biochemical, cell biological, immunological, and molecular genetic approaches and take advantage of novel mouse models of MPS VII produced in our laboratory by transgenic and mouse knockout technologies. We combine histochemistry, histopathology, and immunoelectron microscopy to measure enzyme delivery to brain and other resistant sites of storage. The answers sought have fundamental significance and should provide information leading to novel therapeutic approaches to enzyme replacement for lysosomal and other storage diseases involving the central nervous system.