Gene Therapy for Cystic Fibrosis Using Novel Compact Promoters

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.