TRANS-SPLICING TO REPAIR CYSTIC FIBROSIS MRNA

这项提案的最终目标是开发一种新的通用机制，在前信使RNA水平上重编程任何人类或真核基因。具体而言，该提案将开发我们的专利技术，剪接体介导的RNA反式剪接（SMaRT[TM]），以生产用于治疗囊性纤维化的实用基因疗法。在第1阶段，我们生产了一系列治疗前RNA分子（PTM），这些分子能够启动与突变型囊性纤维化跨膜调节因子（CFTR）前mRNA的SMaRT反应，修复mRNA并在培养的细胞中表达全长CFTR蛋白。第2阶段的目标是确定一种最佳PTM，该PTM可以修复突变型CFTR mRNA，适当表达正常CFTR蛋白，并在最具临床相关性的囊性纤维化模型中恢复氯离子通道功能。这种领先的PTM治疗候选药物将进入第三阶段的临床前测试，如果结果需要，最终将进入人体临床试验。许多其他遗传性疾病也可能适用于SMaRT[TM]疗法。 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以及获得内源性靶基因的调控表达来改善基因治疗的许多方面。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。仅在美国，就有临床需求来服务和治疗大约30，000名受影响的个体。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、EBV、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 human or eukaryotic gene at the pre- messenger RNA level. Specifically, this proposal will develop our patented technology, spliceosome mediated RNA trans-splicing (SMaRT[TM]), towards the production of a practical gene therapy for the treatment of Cystic Fibrosis. In Phase 1, we produced a series of pretherapeutic RNA molecules (PTMs) that are capable of initiating SMaRT reactions with mutant Cystic Fibrosis Transmembrane Regulator (CFTR) pre-mRNA, repairing the mRNA and expressing full length CFTR protein in cultured cells. The goal in Phase 2 is to identify an optimal PTM that can repair mutant CFTR mRNA, appropriately express normal CFTR protein, and restore chloride channel function in the most clinically relevant models of Cystic Fibrosis. This lead PTM therapeutic candidate will enter full preclinical testing in Phase 3 and ultimately, human clinical trials if results warrant. Many other genetic diseases may also be amenable to SMaRT[TM] therapeutics. Trans-splicing PTMs could improve many aspects of gene therapies by conferring intra-cellular specificity, decreasing the size of the delivered gene, and acquiring the regulated expression of the endogenous target gene. PROPOSED COMMERCIAL APPLICATIONS: Development of RNA molecules capable of repairing mutant CFTR by trans-splicing could lead to a therapeutic for Cystic Fibrosis that could slow or halt disease progression. There is a clinical need to serve and treat this market of approximately 30,000 affected individuals in the U.S. alone. 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, infections by splicing viruses ( HIV, EBV, papilloma, etc) and cancer.