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.
