Activating cystic fibrosis transmembrance conductance regulator: the therapeutic potential of RNA directed gene activation

 描述（由申请人提供）：囊性纤维化（CF）是一种常染色体隐性遗传疾病，是细胞表面膜上CF跨膜传导调节因子（CFTR）离子转运功能缺陷的结果。遭受这种命运的人可以指望 到30多岁影响大多数CF患者的常见突变是在至少一个CF等位基因中以> 80%的频率发现的F508del突变。该突变体F508del CFTR是CFTR的错误折叠形式，其运输至细胞质顶膜的能力受损，在细胞质顶膜处， 主要起cAMP依赖性Cl通道的作用，尽管效率降低。已经鉴定了参与CFTR的转录调控的长非编码RNA（lncRNA）。初步研究表明，这种lncRNA的抑制可以导致突变CFTR在细胞表面的表达增加。因此，本文提出的研究将检验突变CFTR的可持续表达可以在人细胞中实现的假设，使得在上皮细胞的顶膜处存在生理和治疗有效的CFTR相关cAMP依赖性Cl离子转运功能。我们制定了三个目标来检验我们的假设。在目标1中，我们将开发和对比CFTR的载体和寡核苷酸靶向激活，在目标2中，我们将表征参与CFTR的lncRNA调节的功能组分，并且在目标3中，我们将确定这些lncRNA BG213071和AI805947在表达和阻遏细胞中的基因组靶向和基因表达谱。这些研究代表了CF的新治疗范式的发展，即，靶向lncRNA以影响它们调节的蛋白质编码基因靶点的功能。在这种情况下，将开发和测试几种针对CFTR相关lncRNA的新方法，以增强CFTR的表达和功能。这种通过lncRNA调节CFTR表达的新途径可能导致对发育期间和继发感染应答中参与CFTR表达调节的那些细胞途径的重要见解，以及开发一类新的治疗CF的治疗剂。

 DESCRIPTION (provided by applicant): Cystic Fibrosis (CF) is an autosomal recessive disease that is the result of defective CF transmembrane conductance regulator (CFTR) ion transport function at the cell surface membrane. Individuals suffering this fate can expect to live to their late-thirties. A frequent mutation affecting most CF patients is the F508del mutation found in at least one CF allele at > 80% frequency. This mutant F508del CFTR is a misfolded form of CFTR that is impaired in its ability to traffic to the cytoplasmic apical membrane where it primarily functions as a cAMP-dependent Cl channel, albeit with lowered efficiency. A long non-coding RNA (lncRNA) has been identified that is involved in transcriptional regulation of CFTR. Preliminary studies indicate that inhibition of this lncRNA can result in increased expression of mutant CFTR at the cell surface. Therefore, the studies proposed here will test the hypothesis that sustainable expression of mutant CFTR can be achieved in human cells such that there is physiologically and therapeutically efficacious CFTR-associated cAMP-dependent Cl ion transport function present at the apical membrane of epithelial cells. We have developed three aims to test our hypothesis. In aim 1, we will develop and contrast vector and oligonucleotide targeted activation of CFTR, in aim 2, we will characterize the functional components involved in lncRNA modulation of CFTR and in aim 3 we will determine the genome targeting and gene expression profile of these lncRNAs BG213071 and AI805947 in expressing and repressed cells. These studies represent the development of a new therapeutic paradigm for CF, i.e., targeting lncRNAs to affect the function of the protein-coding gene targets that they regulate. In this context, several novel approaches targeted to CFTR-associated lncRNAs will be developed and tested to enhance CFTR expression and function. This new pathway for regulation of CFTR expression by lncRNA could lead to significant insights into those cellular pathways involved in the modulation of CFTR expression during development and in response to secondary infections as well as to the development of a new class of therapeutic agents to treat CF.