Peptide Transduction-based Therapies for CF

描述：(申请人提供)：囊性纤维化(CF)是一种遗传性疾病，由编码CFTR的基因突变引起，导致寿命显著缩短以及相关的病理改变。不幸的是，目前可用的治疗方法都不能阻止疾病的发展。主要的CFTR突变，deltaF508，导致突变蛋白保留在内质网(ER)中，随后被泛素-蛋白酶体途径降解。导致deltaF508内质网滞留的机制包括与细胞质伴侣蛋白如HSC/Hsp70和Hdj2的关联。我们研究了含有苯丙氨酸缺失的CFTRdelta508的NBD1区域的过度表达是否会竞争与伴侣蛋白的结合，导致突变的全长蛋白运输到细胞膜。我们已经证明，表达deltaF508核苷酸结合结构域1(DeltaF508 NBD1)和NBD1+R结构域(deltaF508NBD1-RD)的腺病毒载体可以恢复感染的人CFTR deltaF508突变气道细胞的氯离子通道活性。然而，由于腺病毒转导人呼吸道的效率存在问题，我们已经检测了传递带有12个氨基酸的蛋白转导结构域(PTD5)的NBD1-RD结构域的能力。在原代培养的人DeltaF508细胞中，用对照PTD5-EGFP多肽处理后，未观察到明显的cAMP刺激卤化物外流，而经PTD5-deltaF508 NBD1-RD融合处理后，SPQ分析和膜片钳显示cAMP依赖的阴离子通透性恢复。此外，PTD5-deltaF508 NBD1-RD融合处理减少了与全长deltaF508蛋白相关的Hdj2的数量。这些结果表明，蛋白质介导的deltaF508片段的转导可以竞争结合Hdj2，导致内质网释放并将突变的CFTR蛋白运输到细胞表面。因此，这一第一阶段提案的目标是建立一种快速、可重复和定量的检测方法，该方法基于电压敏感染料来检测CFTR活性，可用于相关的第二阶段应用，以绘制竞争与deltaF508结合所需的CFTR中的最小区域。此外，该提案的第二个目标是开发纯化可溶性PTD-NBD融合蛋白的方法，以便在II期研究中进行分析。拟议的I期和II期研究的成功完成将导致I期临床试验，以测试这种基于PTD的新治疗方法治疗CF的有效性。

DESCRIPTION: (provided by applicant): Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CFTR that results in a significantly shorter lifespan as well as associated pathologies. Unfortunately, none of the currently available treatments are able to halt the progression of the disease. The predominant CFTR mutation, deltaF508, results in a mutant protein that is retained in the endoplasmic reticulum (ER) and subsequently degraded by the ubiquitin-proteosome pathway. The mechanisms responsible for ER retention of deltaF508 include associations with cytoplasmic chaperone proteins such as Hsc/Hsp70 and Hdj2. We have examined whether over-expression of NBD1 region of CFTRdelta508 containing the phenylalanine deletion could compete for binding to the chaperone proteins, resulting in trafficking of the mutant full-length protein to the cell membrane. We have demonstrated that adenoviral vectors expressing the deltaF508 nucleotide binding domain 1 (deltaF508 NBD1) as well as the NBD1 plus R domain (deltaF508NBD1-RD) could restore Cl- channel activity restoration in infected human CFTR deltaF508 mutant airway cells. However, due to problems with the efficiency of adenoviral transduction of human airway, we have examined the ability to deliver the NBD1-RD domain with a 12 amino acid protein transduction domain (PTD5). In primary human deltaF508 airway cells treated with a control PTD5-eGFP peptide, no significant cAMP stimulated halide efflux was observed, whereas treatment with the PTD5-deltaF508 NBD1-RD fusion restored cAMP-dependent anion permeability as measured by SPQ analysis and patch-clamp. Furthermore, treatment with the PTD5-deltaF508 NBD1-RD fusion reduced the amount of Hdj2 associated with full-length deltaF508 protein. These results suggest that protein-mediated transduction of a fragment of deltaF508 can compete for binding to Hdj2, resulting in release from the ER and trafficking of the mutant CFTR protein to the cell surface. Thus the goal of this Phase I proposal is to establish a rapid, reproducible and quantitative assay an assay based on voltage-sensitive dyes to detect CFTR activity that could be used in the associated Phase II application to map the minimal region in CFTR required to compete for binding to deltaF508. In addition, a second goal of the proposal is to develop methods for purifying soluble PTD-NBD fusion proteins for analysis in the phase II study. The successful completion of the proposed Phase I and Phase II studies should lead to a phase I clinical trial to test the efficacy of this novel PTD-based therapeutic approach to treat CF.