Aptamer-directed crossing of BBB therapy of MPS 111B

描述（由申请人提供）： MPS III B（Sanfilippo综合征III B）是一种由NAGLU基因突变和溶酶体酶α-N-乙酰氨基葡萄糖苷酶缺失引起的破坏性疾病。临床特征包括严重的智力迟钝、行为问题和死亡，通常发生在青春期。没有有效的治疗方法。酶替代疗法，一种越来越多的溶酶体贮积病的治疗方法，不被认为是圣菲利波综合征的一种选择，因为血脑屏障（BBB）阻止治疗酶到达大脑。然而，必需蛋白质如转铁蛋白通常通过受体介导的转胞吞作用通过毛细血管内皮细胞转运穿过BBB。本申请的目的是开发一种新的策略，以在MPS III B小鼠模型中将α-N-乙酰氨基葡糖苷酶运送穿过BB B。该策略将利用与转铁蛋白受体结合的适体。适体是单链核酸，其可以从大的随机化文库中选择以结合任何期望的靶标;在这方面，它们类似于单克隆抗体。具体目标1是分离和表征与小鼠转移受体的胞外结构域结合的RNA适体。通过掺入2 ′-氟嘧啶，使适体对RNA酶降解具有相对抗性，并测定其与转铁蛋白受体的结合亲和力。具体目标2是将选定的适体缀合至蛋白质-首先缀合至作为模型蛋白的eGFP，然后缀合至重组人α N-乙酰葡糖胺糖苷酶。具体目的3是通过小鼠细胞系和从MPS III B小鼠脑分离的培养的神经元测试蛋白适体缀合物的转铁蛋白受体介导的内吞作用。具体目标4是在体内测试适体-酶缀合物，以确定它们是否可以将α-N-乙酰氨基葡糖苷酶运送到脑实质中以及该酶是否在神经细胞中起作用。将在目标3的内吞试验中发现有用的α-N-乙酰氨基葡糖苷酶-适体缀合物给予MPS III B小鼠，并对脑进行生化和形态学检查。如果这种策略显示出有希望的结果，它可以很容易地适用于其他神经退行性溶酶体贮积病的酶递送，以及常见疾病如阿尔茨海默氏症和帕金森氏症的药物递送。

DESCRIPTION (provided by applicant): MPS III B (Sanfilippo syndrome III B) is a devastating disease caused by mutation of the NAGLU gene and absence of the lysosomal enzyme alpha-N-acetylglucosaminidase. The clinical features include profound mental retardation, behavioral problems and death, usually in adolescence. There is no effective treatment. Enzyme replacement, a therapy becoming available for a growing number of lysosomal storage diseases, is not considered an option for the Sanfilippo syndrome because the blood brain barrier (BBB) prevents therapeutic enzyme from reaching the brain. However, essential proteins such as transferrin are normally transported across the BBB by receptor-mediated transcytosis through capillary endothelial cells. The goal of this application is to develop a novel strategy to ferry alpha-N-acetylglucosaminidase across the BBB in a mouse model of MPS III B. This strategy will make use of aptamers that bind to the transferrin receptor. Aptamers are single stranded nucleic acids that can be selected from large randomized libraries to bind to any desired target; in this respect, they resemble monoclonal antibodies. Specific Aim 1 is to isolate and characterize RNA aptamers that bind to the extracellular domain of the mouse transferring receptor. The aptamers will be made relatively resistant to RNase degradation by incorporation of 2'fluoropydmidines, and their binding affinities to the transferrin receptor will be determined. Specific Aim 2 is to conjugate selected aptamers to proteins - first to eGFP as a model protein, then to recombinant human alpha N-acetylglucosaminidase. Specific Aim 3 is to test the protein aptamer conjugates for transferrin receptor-mediated endocytosis by a mouse cell line and by cultured neurons isolated from brain of MPS III B mice. Specific Aim 4 is to test aptamer-enzyme conjugates in vivo, in order to determine whether they can ferry alpha-N-acetylglucosaminidase into the brain parenchyma and whether the enzyme will be functional in neural cells. The alpha-N-acetylglucosaminidase-aptamer conjugates found useful in the endocytosis test of Aim 3 will be administered to MPS III B mice and the brains subjected to biochemical and morphological examination. Should this strategy show promising results, it could easily be adapted for enzyme delivery for other neurodegenerative lysosomal storage diseases, as well as for drug delivery in common diseases such as Alzheimer's and Parkinson's.