TRANS-SPLICING TO REPAIR CYSTIC FIBROSIS MRNA

本研究的最终目标是开发一种新的通用机制，在前信使RNA水平上对任何人类或真核生物基因进行重编程。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]），用于生产治疗囊性纤维化的实用基因疗法。在第一阶段，我们生产了一系列治疗前RNA分子（PTMs），能够与突变的囊性纤维化跨膜调节因子（CFTR）前mRNA启动SMaRT反应，修复mRNA并在培养细胞中表达全长CFTR蛋白。2期的目标是在最具临床相关性的囊性纤维化模型中找到一种最佳的PTM，该PTM可以修复突变的CFTR mRNA，适当表达正常的CFTR蛋白，并恢复氯通道功能。这一PTM治疗候选药物将进入三期临床前测试，如果结果保证，最终将进行人体临床试验。许多其他遗传疾病也可能适用于SMaRT疗法。反式剪接PTMs可以通过赋予细胞内特异性、减小传递基因的大小和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。建议的商业应用：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的进展。仅在美国，就有临床需求来服务和治疗这个大约有30,000名患者的市场。此外，有效剪接体介导的RNA反式剪接技术的发展将在许多基因转移应用中发挥作用，包括治疗其他遗传疾病、剪接病毒感染（HIV、EBV、乳头状瘤等）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.