Nanoparticles delivered to primary human airway epithelial cells allows for targeted systemic lung delivery

Introduction::
Current treatments for cystic Fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CFTR protein.¹ However, these modulators are ineffective for the 10% of CF individuals with variants that do not allow protein production. Gene editing of these variants would allow production of CFTR protein and restoration of chloride channel function. We have screened various poly(beta-amino ester) (PBAE) nanoparticles against primary airway epithelial cells cultured from both CF and healthy individuals to aid in a formulation that will target these cells following systemic delivery. Systemic delivery using an optimized NP formulation to the Ai9 mouse model transfects both endothelial and epithelial cell types. In vitro, electroporation of our modified CRISPR-Cas9 base editor mRNA to primary human nasal epithelial (HNE) cells carrying 3120+1G >A/G480S achieves 81% correction of the 3120+1 G >A allele. As this nonviral transfection results in CFTR function within or above the range of WT values observed in WT HNEs,² we conducted in-depth single cell RNA (scRNA) sequencing across edited and unedited samples. This gene editing approach is promising for new, durable CF treatment.




Materials and Methods::
PBAEs were synthesized via Michael addition, fabricated into nanoparticles by electrostatic self-assembly with mRNA, and evaluated for gene delivery by flow cytometry. mRNA-encoded GFP served as a reporter to evaluate transfection efficiency, cell viability and mean fluorescence intensity (MFI) across three dosages (150, 75, 32.5 ng mRNA), four polymer-to-mRNA weight to weight (w/w) ratios (60, 40, 30, 20) and four different polymers. Primary human airway epithelial cells from CF and healthy donors were seeded in 96-well plates then transfected with nanoparticles before undergoing microscopy and flow 24 hours post transfection. Nanoparticles carrying Cre mRNA were delivered via tail vein injection to Ai9 mice at three different intervals (1x, 3x, 9x). Lungs were dissociated and antibody stained before undergoing flow. All flow analysis was completed in FlowJo. Primary HNE cells (described above) were co-electroporated with ABE8e/sgRNA, then allowed to differentiate for 21 days on ALI culture before being dissociated for single cell library preparation. This was performed using 10X Genomics v3.1 3’ Library Preparation chemistry with dual indexing system. Sequencing was performed by the Johns Hopkins Genomic Resources Core Facility via paired-end sequencing 2x100 cycles using a NovaSeq 6000 Illumina sequencing machine. Analysis of scRNA-seq results was performed using 10X Genomics Cell Ranger 3.1.0 followed by the Seurat package (version 4.1) in R created by the Satija Lab.⁴ Subsetting was determined based on comparisons of the number of genes expressed, UMI count, and percentage of reads mapped to mitochondrial RNA. A log normalization with a scale factor of 10,000 was used.




Results, Conclusions, and Discussions:: Primary human bronchial epithelial cells from a CF and healthy individual achieved 40-60% GFP transfection, with primary human nasal epithelial cells from a CF individual achieving ~35% transfection and that from a healthy donor achieving ~85% transfection. Viability for the two lowest dosages was consistently between 70-85%. Systemic delivery to the Ai9 mouse model showed consistent increase in tdTomato+ cells across all lung cells as the number of injections increased, from 0.8 to 1.9 to 9% at 1x, 3x and 9x dosing respectively. Additionally, the number of epithelial cells transfected consistently increased across dosing, with the number of endothelial and epithelial tdTomato+ cells ranging from 15-40% and 15-35% respectively (Figure 1). Analysis of scRNA sequencing of 8,791 cells across two samples of edited and unedited primary HNEs compared CFTR-positive cells as well as expression levels. There was a consistent increase in CFTR+ cells between unedited and edited cells across all defined cell type clusters, most noticeably in ciliated/deuterosomal (500% increase) and cycling basal cells (175%) (Figure 2A). Cells expressing CFTR increase from 2.5% of total cells in unedited to 3.96% in edited and normalized read count per sample increased from 0.049 to 0.081. Editing increased the % of basal and cycling basal cells expressing CFTR, indicating correction was achieved in airway progenitor cells (Figure 2B).



Our optimized PBAE nanoparticle formulation achieved up to 85% transfection rate in healthy primary human nasal epithelial cells while maintaining around 75% viability. This same polymer achieving 20-40% transfection of endothelial and epithelial tissues with systemic delivery in an Ai9 model. Base editing of primary HNE cells carrying the 3120+1G >A variant show levels of CFTR function after editing equivalent to >50% WT,¹ exceeding the established clinically significant threshold of ~10%. Here we demonstrate that editing is occurring in progenitor cells after electroporation-mediated delivery of RNA-encoded base editing reagents to undifferentiated primary HNEs. In ongoing work, we are preparing more samples for scRNA analysis of edited and unedited primary cells, as well as further investigating targeted cell types during in vivo delivery to the Ai9 mouse model using FACS and scRNASeq.


Acknowledgements (Optional): : We would like to acknowledge the Cystic Fibrosis Foundation [CUTTIN22G0] and NIH grant R01CA228133 for research support.

References (Optional): : ¹Cutting GR. Cystic fibrosis genetics: from molecular understanding to clinical application. Nat Rev Genet. 2015;16(1):45-56. doi:10.1038/nrg3849

²Joynt AT, Kavanagh EW, Newby GA, Mitchell S, Eastman AC, Paul KC, Bowling AD, Osorio DL, Merlo CA, Patel SU, Raraigh KS, Liu DR, Sharma N, Cutting GR. Protospacer modification improves base editing of a canonical splice site variant and recovery of CFTR function in human airway epithelial cells. Mol. Ther. Nucleic Acids 2023 [in review]

³Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, Papalexi E, Mimitou EP, Jain J, Srivastava A, Stuart T, Fleming LM, Yeung B, Rogers AJ, McElrath JM, Blish CA, Gottardo R, Smibert P, Satija R. Integrated analysis of multimodal single-cell data. Cell 2021; Vol. 184,13: 3573-3587.e29. doi:10.1016/j.cell.2021.04.048