(T1230-05-28) Discovery of Peptides to Target Lung Epithelia from Cystic Fibrosis Patients

Purpose: Patients with Cystic Fibrosis (CF) can have one of many known mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein, which often leads to abnormal and excessive build-up of mucus in the lungs. Lung related complications are often the main cause of death for CF patients, which is why lung targeted gene therapy shows promise for being a potential curative treatment for patients with CF. However, the hostile environment found in the lungs of CF patients, which includes hyper-concentrated mucus and immune cells, such as macrophages, greatly hinders locally delivered treatments from reaching intended target sites, i.e., bronchial epithelial cells, secretory cells, basal cells.¹ To properly tackle the multiple barriers associated with gene delivery, it is critical to identify gene delivery vehicles that can cross the CF mucus barrier and achieve targeted cellular uptake. Peptides are attractive delivery systems since they can shuttle therapeutics to their intended target site, are non-inflammatory , enhance cell internalization, and be easily incorporated into non-viral delivery systems where larger payloads can be delivered.² To address the aforementioned challenges, we have leveraged phage display technology to screen and identify peptides that achieve mucus and cell penetration against primary cells from CF patients. Screening against patient cells cultured at air-liquid interface allows us to mimic the disease pathology to identify lead candidates that can be translated to the clinic.

Methods: To screen for bacteriophages (phage) with the ability to enhance mucus penetration and cell uptake, we introduced a cysteine constrained random 7-amino acid peptide T7 phage display library (CX7C) against differentiated primary human bronchial epithelial cells (pHBECs) through iterative, high-throughput screening for a total of four rounds.  The pHBECs were pooled from seven different CF patients with the prevalent deltaF508 mutation and grown at Air-Liquid-Interface (ALI) according to Pneumacult^TM Ex-Plus and Pneumacult^TM ALI media protocols (StemCell Technologies; Vancouver, BC). pHBECs were fully differentiated by 21-days post airlift, which was indicated by ciliation, and mucus production and visualized by microscopy. After each round of selection, DNA was isolated from amplified phage internalized by the cells, DNA was prepared for next generation sequencing, and peptide sequence analysis was performed as previously reported.¹ The top 30 most abundant sequences in each round of screening were identified and their physicochemical properties were analyzed. Selected sequences were identified and engineered into T7 for further validation studies. After confirming uptake in pHBECs, peptide-presenting phage were tested for uptake due to sequence specificity by redoing our cell uptake assay against controls with scrambled sequences against the pooled CF patient cell model grown at ALI. Phage were also incubated against differentiated THP-1 macrophages to confirm whether uptake is selective to patient CF cells.

Results: After four rounds of iterative screening against pHBEs, our CX7C library was enriched for both mucus penetration and cellular uptake. From the first to fourth round, we observed a 231-fold enrichment, which was statistically higher than that seen with a “wild-type” (WT) peptide-less T7 phage control, which showed no enrichment (Figure 1). After our iterative screening process, we completed next-generation sequencing to analyze thousands of phage displayed peptide sequences present after each round of screening. From this, we identified the top 30 most abundant sequences in each round. Select sequences were further validated to determine whether they possessed both cell and mucus penetration ability. When compared to our WT control and an unselected T7 library, four clones demonstrated a significant difference in cell uptake in differentiated CF pHBEs (Figure 2; p < 0.05). Next, we sought to determine if our screening process was successful in enriching for peptide-displaying phage that were selective for primary CF HBEs versus THP-1 differentiated macrophages. Of the four clones, two demonstrated significantly higher uptake in CF HBEs at ALI vs. macrophage cells (Figure 3). Lastly, we wanted to see if cellular uptake was due to sequence order specificity, and as a result, we designed peptides with the same amino acids but scrambled order for each clone and performed a cell uptake study with our differentiated pHBEC CF model. From this study, we found that two clones demonstrated improved uptake compared to their scrambled counterparts.

Conclusion: A peptide-presenting phage library was used to screen and identify novel peptides that can cross the problematic CF mucus barrier and into CF epithelia. From validation studies, clones were identified that had targeted uptake for bronchial epithelia vs macrophages and peptide sequence specificity for cellular uptake. Future work includes using these candidate peptides to decorate nanoparticle systems for use in gene delivery.

References: 1. Leal et al. J Controlled Release. 2020; 322: 457-469
2. Ghosh et al. J Pharm Investig. 2018; 48: 89-111

Acknowledgments: Funding for this research was provided by National Institutes of Health National Heart, Lung and Blood Institute (R01HL138251) and Cystic Fibrosis Foundation (GHOSH19XX0). We would also like to acknowledge the Marisco Lung Institute/Cystic Fibrosis and Pulmonary Diseases Research and Treatment Center at the University of North Carolina at Chapel Hill for providing the primary cells used in this study.

Figure 1. Enrichment of CX7C phage library for enhanced CF pHBEC uptake. Cout represents the concentration of phage collected after each round and Cin is the initial input phage concentration. Phage were added at 1000 viral genomes/cell and incubated for 16 hours at 37°C for round 1 and 1 hour at 37°C for round 4. WT = wild-type (peptide-less phage).


Figure 2. Validation of selected clones for enhanced CF pHBEC uptake. Cout represents the concentration of phage collected after each round and Cin is the initial input phage concentration. a: significant difference from all controls, b: significant difference from Naïve library (phage library without selection) only. Phage were incubated against pHBECs at ALI for 1 hour at 37°C.


Figure 3. Clones 14 and 26 demonstrate significantly higher uptake in primary CF cells than a human macrophage cell line, M0 (differentiated from THP-1 cells). % Phage uptake: [(plaque forming units (pfu)/µL collected * volume collected)/(input pfu/µL * volume input)] * 100 . * denotes p-value <0.05 and *** denotes < p-value <0.0005. Phage were incubated against either pHBECs or M0 for 1 hour at 37°C.