Gene Therapy for Cystic Fibrosis Using Novel Compact Promoters

项目摘要/摘要 美国有超过3万人患有囊性纤维化，这是一种常染色体隐性遗传病，由 Cftr编码的氯离子通道突变。至少1800个致病基因突变已被证实 尽管取得了显著进展，但最有效的药物选择并未使约10%的人受益 病人。对所有慢性萎缩性胃炎患者有益的治疗方法，特别是对 目前的治疗方法是迫切需要的。在这里，我们建议开发一种可泛化和突变的- 不可知的基因治疗方法与新的紧凑启动子，使全长CFTR交付。腺体- 相关病毒(AAV)提供了一种安全的治疗基因传递方式；然而，一个主要障碍限制了 AAV载体的效用：它的有效载荷能力很小。除了启动子外，cftr基因的大小， 终止子和2个倒置的末端重复序列是AAV包装的一个重要障碍。当前战略 为了绕过有限的能力，使用一个小的合成病毒启动子和一个截短的cftr基因； 这些可能会有很大的缺点。截短的cftr基因比全长cftr基因活性低，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 随着时间的推移有被沉默的倾向，由这些启动子驱动的高转基因表达也可以 是有毒的。我们方法的根本创新是发现并表征了数百个 紧凑的哺乳动物启动子，使全长cftr基因在 单一AAV载体。与局限于单个合成病毒特性的当前方法形成对比 启动子，我们展示了显著的能力微调转基因表达水平使用内源 推动者。在这一阶段，我们建议通过描述紧凑结构来优化cftr的表达和包装 体外和体内的启动子。CFTR编码序列将被优化并测试RNA稳定性、蛋白质 表达和功能加工。最后，将优化后的组件组装成AAV进行验证 使用体外和体内模型，正确包装和表达CFTR。此第一阶段提案可实现 CF治疗的平行轨道开发，我们预计这些实验将提供基础 对于第二阶段，通过启用IND的研究推进铅治疗。

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.