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毁灭性疾病。临床特征包括严重的智力低下、行为问题和死亡，通常发生在青春期。目前还没有有效的治疗方法。酶替代疗法正在成为一种治疗越来越多的溶酶体储存疾病的方法，但并不被认为是Sanfilippo综合征的一种选择，因为血脑屏障(BBB)阻止了治疗性酶到达大脑。然而，转铁蛋白等必需蛋白通常通过受体介导的细胞转运通过毛细血管内皮细胞跨血脑屏障运输。这项应用的目标是开发一种新的策略来在MPS III B小鼠模型中通过血脑屏障运送α-N-乙酰氨基葡萄糖苷酶。该策略将利用与转铁蛋白受体结合的适体。适配子是单链核酸，可以从大型随机文库中选择与任何所需的靶点结合；在这方面，它们类似于单抗。具体目标1是分离和鉴定与小鼠转移受体的胞外结构域结合的RNA适配子。通过掺入2‘-氟代嘧啶，适体将相对抵抗核糖核酸酶的降解，并将确定它们与转铁蛋白受体的结合亲和力。具体目标2是将选定的适配子与蛋白质偶联-首先与作为模型蛋白的EGFP偶联，然后与重组人α-N-乙酰氨基葡萄糖苷酶偶联。具体目标3是通过小鼠细胞系和从MPS III B小鼠脑中分离的培养神经元来测试转铁蛋白受体介导的内吞作用的蛋白质适配子结合物。具体目标4是在体内测试适体-酶结合物，以确定它们是否能将α-N-乙酰氨基葡萄糖苷酶输送到脑实质，以及这种酶是否会在神经细胞中发挥作用。在AIM 3的吞噬试验中发现有用的α-N-乙酰氨基葡萄糖苷酶-适配子结合物将用于MPS III B小鼠以及接受生化和形态检查的大脑。如果这一策略显示出有希望的结果，它很容易被用于治疗其他神经退行性溶酶体储存疾病的酶制剂，以及用于阿尔茨海默氏症和帕金森氏症等常见疾病的药物输送。

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.