Project Summary/Abstract Recent advances make the possibility of gene editing for lung diseases such as cystic fibrosis (CF) a promising therapeutic approach. Several designer nucleases can now be engineered to introduce site-specific double strand breaks in DNA, allowing therapeutic modifications at specific genomic loci. A major barrier to progress is the lack of efficient methods to deliver gene editing reagents to airway epithelia in vivo. We will use novel amphiphilic peptides to deliver CRISPR nuclease proteins and guide RNAs (termed ribonucleoproteins, RNPs). The overall goal of this proposal is to advance gene editing as a therapeutic approach for diseases of the respiratory tract that impact the function of airway epithelial cells using a novel peptide-based delivery technology. Our preliminary studies show that these peptides can rapidly and efficiently deliver RNPs. We therefore hypothesize that therapeutically relevant levels of gene editing can be achieved in airway epithelia. In Phase 1 we use RNPs and amphipathic peptide delivery to perform gene editing in cultured human airway epithelial cells and the airways of transgenic mice. Our aims include: 1) Completing an in vitro to screen to discover new amphiphilic peptides with improved RNP delivery efficiency. 2) Quantifying the genome editing efficiency in cultured human airway epithelia and mouse airways following amphiphilic peptide mediated delivery of RNPs. This includes the cell types transduced and the efficiency of editing. We will also assess editing and persistence of progenitor cells types. 3) Investigate the safety and toxicity of amphiphilic peptides in human airway epithelia and mouse airways, including biodistribution, immune response, and pulmonary histopathology. Phase 2 will use RNPs and peptide delivery to perform gene editing in airways of newborn transgenic pigs. These studies build on our extensive experience working with a CF pig model. The aims include: 1) Design and implement peptide and protein scale up and for peptide-mediated delivery of RNPs to pig airways. 2) Quantify the cell targeting and editing efficiency of RNPs following in vivo RNP delivery using amphiphilic peptides. We will identify the long lived and progenitor cell types transduced in the large and small airways and investigate their persistence over time. 3) Investigate the safety and toxicity of candidate peptides and RNPs in the pig lung. This includes studies of pulmonary inflammation, immune response, and biodistribution. The proposed work is innovative because it advances a peptide based strategy to efficiently deliver RNPs. Completion of the proposed studies will provide insights into the utility and safety of this versatile peptide based RNP delivery strategy to modify airway epithelial cells in vivo.

Project Narrative The proposed research is relevant to the public health because genetic and acquired diseases affecting the airways pose major disease and economic burdens. By advancing the delivery of gene editing tools, it may be possible to therapeutically modify the cells lining the airways. This novel strategy has implications for the treatment of both monogenetic and acquired lungs disease, and may have applications for other somatic cell therapies.