MODULATION OF CELLULAR CLEARANCE TO TREAT HUMAN DISEASE

描述（由申请人提供）：本项目的目标是生成基于细胞清除调节的全新治疗策略的原理验证数据。这一策略将在溶酶体贮积病（LSD）上进行测试，这是一组超过50种遗传性疾病，具有进行性，多系统表型，主要影响儿童。目前可用于LSD的疗法具有重大局限性。我们已经发现了控制溶酶体和自噬体的生物发生和功能的转录因子TFEB（Sardiello等人Science 2009; Settembre等人Science，2011），并且我们已经表明TFEB过表达促进LSD的细胞清除（Medina等人Dev. Cell，2011）。最近，我们证明了mTOR抑制剂促进TFEB核转位和活性（Settembre等，EMBO J.，2012年）。在这个项目中，我们将：1）在多发性硫酸酯酶缺乏症（MSD）的小鼠模型中测试诱导型TFEB过表达的治疗潜力。该小鼠模型的表型的高严重性和广谱性将使我们能够测试我们的治疗方法对多种组织的影响; 2）测试AAV介导的TFEB基因递送在庞贝氏症（PD）（一种主要涉及肌肉和心脏的病症）的鼠模型中的治疗功效。这将使我们能够比较基于TFEB的方法与更传统的酶替代和基因替代疗法的疗效。我们已经通过在来源于永生化成肌细胞的培养的肌管中和通过肌内注射AAV-TFEB在PD小鼠模型中的体内病毒介导的TFEB过表达获得了非常令人鼓舞的初步数据; 3）鉴定促进TFEB活性的化合物。我们已经开发了一种基于TFEB核易位的高含量筛选方法。通过高含量药物筛选鉴定的阳性命中将进行一组体外二级测定，并将有希望的化合物施用于MSD和PD的小鼠模型。如果成功，拟议的办法将是处理最不发达国家问题的一个范式转变。与针对单一疾病实体的现有疗法相反，我们基于细胞清除调节的方法可能对许多LSD和常见的迟发性神经退行性疾病的治疗产生影响。

DESCRIPTION (provided by applicant): The goal of this project is to generate proof-of-principle data on a completely novel therapeutic strategy that is based on the modulation of cellular clearance. This strategy will be tested on lysosomal storage diseases (LSDs), a group of over 50 inherited diseases with a progressive, multisystemic phenotype that mostly affects children. Currently available therapies for LSDs have major limitations. We have discovered a transcription factor, TFEB, that controls the biogenesis and function of lysosomes and autophagosomes (Sardiello et al. Science 2009; Settembre et al. Science, 2011) and we have shown that TFEB overexpression promotes cellular clearance of LSDs (Medina et al. Dev. Cell, 2011). Recently, we demonstrated that mTOR inhibitors promote TFEB nuclear translocation and activity (Settembre et al. EMBO J., 2012). In this project, we will: 1) test the therapeutic potential of inducible TFEB overexpression in a mouse model of Multiple Sulfatase Deficiency (MSD). The high severity and broad spectrum of the phenotype of this mouse model will enable us to test the effects of our therapeutic approach on multiple tissues; 2) test the therapeutic efficacy of AAV- mediated gene delivery of TFEB in a murine model of Pompe disease (PD), a disorder that primarily involves muscles and heart. This will allow us to compare the efficacy of a TFEB-based approach with more traditional enzyme replacement and gene replacement therapies. We have already obtained very encouraging preliminary data by viral-mediated TFEB overexpression in cultured myotubes derived from immortalized myoblasts and in vivo in a PD mouse model by intramuscular injection of AAV-TFEB; 3) identify chemical compounds that promote TFEB activity. We have developed a high-content screening assay based on TFEB nuclear translocation. Positive hits identified by high-content drug screening will be subject to a panel of in vitro secondary assays and promising compounds will be administered to mouse models of MSD and PD. If successful, the proposed approach will represent a paradigm shift in the treatment of LSDs. Contrary to existing therapies, which are directed towards single disease entities, our approach based on the modulation of cellular clearance, may have an impact on the therapy of many LSDs and of common, late onset neurodegenerative diseases.