Gene Therapy for Cystic Fibrosis Using Novel Compact Promoters

项目总结/摘要 在美国，超过30，000人患有囊性纤维化（CF），这是一种由以下原因引起的常染色体隐性遗传疾病： CFTR编码的氯离子通道中的突变。至少有1，800种CFTR致病突变已被发现。 尽管取得了显著进展，但最有效的药物选择并不能使约10%的人受益， 患者对所有CF患者有益的治疗方法，尤其是对 目前的治疗方法是迫切需要的。在这里，我们提出了一个概括和突变的发展- 使用能够实现全长CFTR递送的新型紧凑型启动子的不可知基因治疗方法。腺- 相关病毒（AAV）提供了一种安全的治疗基因递送手段;然而，一个主要的障碍限制了 AAV载体的效用：其小的有效载荷容量。除了启动子之外，CFTR基因的大尺寸， 终止子和2个反向末端重复序列对AAV包装呈现显著屏障。目前的战略 为了规避有限的能力，采用小的合成病毒启动子和截短的CFTR基因;两者都是 这些可能具有显著的缺点。截短的CFTR基因比全长CFTR活性低，并且 数亿年的进化保护进一步支持了重要的功能。病毒启动子 具有随时间沉默的倾向，并且由这些启动子驱动的高转基因表达也可以 有毒。我们方法的根本创新是发现和表征了数百种 紧凑的哺乳动物启动子，其使得全长CFTR基因能够在重组质粒中包装和表达。 单个AAV载体。与目前仅限于单一合成病毒特性的方法相比， 启动子，我们证明了一个显着的能力，微调转基因表达水平，利用内源性 发起人。在这个阶段I中，我们提出通过表征紧凑型结构来优化CFTR表达和包装。 在体外和体内的启动子。将优化CFTR编码序列，并测试RNA稳定性、蛋白质 表达和功能处理。最后将优化后的部件组装成AAV进行验证 使用体外和体内模型进行适当包装和CFTR表达。第一阶段的提案使 平行跟踪发展的CF治疗，我们预计，这些实验将提供基础 II期通过IND使能研究推进先导治疗。

PROJECT SUMMARY/ABSTRACT More than 30,000 people in the U.S. suffer from cystic fibrosis (CF), an autosomal recessive disease caused by mutations in the CFTR-encoded chloride channel. At least 1,800 disease-causing mutations in CFTR have been identified, and despite remarkable progress, the most effective pharmaceutical options do not benefit ~10% of patients. Treatment approaches that are beneficial to all CF patients, especially those who do not respond to current therapies, are urgently needed. Here we propose the development of a generalizable and mutation- agnostic gene therapy approach with novel compact promoters that enable full-length CFTR delivery. Adeno- associated viruses (AAV) provide a safe means of therapeutic gene delivery; however, a major obstacle limits an AAV vector’s utility: its small payload capacity. The large size of the CFTR gene, in addition to a promoter, terminator, and 2 inverted terminal repeats, presents a significant barrier to AAV packaging. Current strategies to circumvent the limited capacity employ a small synthetic viral promoter and a truncated CFTR gene; both of these could have significant drawbacks. Truncated CFTR genes are less active than full-length CFTR, and hundreds of millions of years of evolutionary conservation further support important functions. Viral promoters have a propensity to be silenced over time and high transgene expression driven by these promoters can also be toxic. The fundamental innovation of our approach is the discovery and characterization of hundreds of compact mammalian promoters that enable packaging and expression of the full-length CFTR gene within a single AAV vector. In contrast to current approaches that are limited to the properties of a single synthetic viral promoter, we demonstrate a significant capacity to fine-tune transgene expression levels using endogenous promoters. In this Phase I we propose to optimize CFTR expression and packaging by characterizing compact promoters in vitro and in vivo. CFTR coding sequences will be optimized and tested for RNA stability, protein expression, and functional processing. Finally, the optimized components will be assembled into AAV to validate proper packaging and CFTR expression using both in vitro and in vivo models. This Phase I proposal enables parallel track development for a CF therapeutic, and we anticipate that these experiments will provide the basis for a Phase II to advance a lead therapeutic through IND-enabling studies.