Project Summary Cystic fibrosis (CF) is a progressive genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature stop codon mutations including W1282X are among the most severe and there are no curative treatments for patients. Genome editing agents could offer promising therapeutics applicable to all CF patients. Engineered nucleases including CRISPR/Cas9 systems that can catalyze correction of disease-causing mutation(s) have shown promise and entered clinical trials. To mitigate aberrant nuclease activity and reduce off-target effects, prime editing technology combines a catalytically impaired Cas9 endonuclease fused with an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that also encodes the desired edit. As an alternative technology, triplex-forming peptide nucleic acids (PNAs) have no intrinsic nuclease activity and stimulate endogenous DNA repair with low off-target effects when bound adjacent to the target site and co-delivered with donor DNA oligonucleotides. Despite advances in gene editing technology, in vivo delivery remains a primary barrier to clinical translation. The goal of the proposed research is to develop a genome editing-based therapeutic strategy for treating the W1282X nonsense CF mutation as well as high-throughput technologies for identifying effective vehicles for in vivo therapeutic nucleic acid delivery. In Aim 1, PNA- and CRISPR/Cas9 prime editing-based gene editing reagents will be designed to correct the W1282X mutation, encapsulated into poly(amine-co-ester) (PACE) nanoparticles (NPs), and tested in vitro and in vivo. In Aim 2, novel PACE materials will be developed for in vivo delivery of nucleic acid-based therapeutics to the lungs and assessed using high-throughput in vivo platforms to determine the structure-function relationships guiding physiological fate. In Aim 3, physiologically relevant 3D culture models will be developed as high-throughput screening tools to assess delivery and efficacy of CF therapies. Overall, the proposed interdisciplinary research is highly clinically relevant, furthering the translation of promising gene editing/nucleic acid therapeutics for CF and other genetic diseases. Dr. Piotrowski-Daspit received her Ph.D. in Chemical and Biological Engineering and is currently a postdoctoral fellow in the Department of Biomedical Engineering at Yale University. Thus far, she has been developing polymeric NPs for nucleic acid delivery and high-throughput in vivo tools. The career development plan outlines a comprehensive strategy for acquiring the technical, conceptual, and professional skills required to complete the proposed studies and launch an independent research career. The proposed training would enable her to gain significant experience in therapeutic development for CF and integrate her into the CF research community. The training plan, together with her background in biomedical engineering, biomaterials and drug delivery, will place her among a select group of scientists with the skills and breadth of knowledge necessary to effectively pursue interdisciplinary work on nucleic acid delivery and editing of genetic disorders.

Project Narrative The proposed research is directly relevant to public health in that it is aimed at developing a cure for cystic fibrosis and incorporates key steps towards the clinical translation of a therapeutic. Further, the research will develop tools for therapeutic screening and enhancing fundamental knowledge of drug delivery mechanisms. Overall, this work has the potential to contribute to the treatment of devastating genetic diseases, significantly improving the health of patients and reducing the burden of illness.