SPLICEOSOME TRANS-SPLICING TO REPAIR CF MRNA

该提案的最终目标是开发一种新的通用机制，以在前信使RNA水平上重新编程任何靶基因。具体而言，该提案将开发用于治疗囊性纤维化的基因疗法的技术。在I期，我们将产生一系列治疗前RNA分子（PTM），其能够促进剪接体介导的RNA与突变型囊性纤维化跨膜调节因子（CFTR）前mRNA的反式剪接反应，修复来自CF患者的培养细胞中的突变型mRNA。近期目标是：1）开发一种或多种可以修复突变CFTR mRNA的PTM;和2）表达校正的CFTR蛋白并恢复培养细胞中的氯离子通道功能。目前，我们有一个PTM能够修复CFTR外显子10，其中包括最常见的突变，Δ F508。根据本提案，将生产该PTM、短PTM和靶向不同CFTR区域的PTM的修改并进行功能测试。其他遗传性疾病也可能适合于反式剪接RNA修复。反式剪接PTMs可以通过赋予细胞内特异性、减小递送基因的大小和获得内源性靶基因的表达模式来改善基因治疗。拟议的商业应用：能够通过反式剪接修复突变CFTR的RNA分子的开发可能导致囊性纤维化的治疗，可以减缓或停止疾病进展。临床上需要服务和治疗美国30，000名受影响个体的市场。此外，有效剪接体介导的RNA反式剪接技术的开发将在许多基因转移应用中具有实用性，包括治疗其他遗传疾病、剪接病毒（HIV、EV、乳头状瘤等）感染和癌症。

The ultimate objective of this proposal is to develop a new, general mechanism to reprogram any targeted gene at the pre-messenger RNA level. Specifically, this proposal will develop the technology to produce a gene therapy for the treatment of Cystic Fibrosis. In Phase I, we will produce a series of pre-therapeutic RNA molecules (PTMs) that are capable of promoting spliceosome mediated RNA trans-splicing reactions with mutant Cystic Fibrosis Transmembrane Regulator (CFTR) pre- mRNA, repairing mutant mRNA in cultured cells from CF patients. The immediate goals are: 1) to develop one or more PTMs that can repair mutant CFTR mRNA; and 2) express corrected CFTR protein and restore chloride channel function in cultured cells. Currently, we have one PTM that is capable of repairing CFTR exon 10, which includes the most prevalent mutation, delta F508. Modifications of this PTM, shorted PTMs, and PTMs targeting different CFTR regions will be produced and functionally tested under this proposal. Other genetic diseases may also be amenable to trans-splicing RNA repair. Trans-splicing PTMs could improve gene therapies by conferring intra-cellular specificity, decreasing the size of the delivered gene, and acquiring the expression pattern of the endogenous target gene. PROPOSED COMMERCIAL APPLICATIONS: Development of RNA molecules capable of repairing mutant CFTR by trans-splicing could lead to a therapy for Cystic Fibrosis that could slow or halt disease progression. There is a clinical need to serve and treat this market of 30,000 affected individuals in the U.S. Additionally, the development of effective spliceosome mediated RNA trans-splicing technology would be of utility in many gene transfer applications including the treatment of other genetic diseases, infection by splicing viruses (HIV, EV, papilloma, etc), and cancer.