Biodegradable polymeric nanoparticles for safe and effective delivery of mRNA to the lungs.

Introduction: Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CFTR protein. 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 function. Here, we demonstrate poly(β-amino ester) (PBAE) nanoparticle (NP) formulations allow for efficient delivery of GFP mRNA to primary human nasal epithelial (HNE) and bronchial epithelial (HBE) cells from healthy and CF individuals. Optimized PBAE endcap formulations systemically administered in vivo allow for high expression of fLuc mRNA within the lungs, with E63 demonstrating high levels of editing in an Ai9 reporter mouse model across bronchial, epithelial, and endothelial cell populations. Multiple administrations with and without targeting ligand anti-CD326 proved both efficacious and non-toxic.

Materials and

Methods: PBAEs were synthesized via Michael addition, fabricated into NPs 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 across 3 dosages (150, 75, 32.5 ng mRNA), 4 polymer-to-mRNA weight to weight ratios (60, 40, 30, 20) and 4 polymers. Primary HBE and HBE cells from CF and healthy donors were seeded in 96-well plates then transfected with NPs before undergoing microscopy and flow 24 hours post transfection. NPs encapsulating fLuc mRNA were administered systemically to mice followed by whole-body bioluminescence or lung cell dissociation. Cre mRNA was delivered intravenously to Ai9 mice at four intervals (1x, 3x, 6x, 9x). Lungs were dissociated and stained with antibodies before undergoing analysis via flow cytometry.

Results, Conclusions, and Discussions: NPs achieved 80-90% GFP transfection in primary HNEs from a CF and healthy individual, with ~70% transfection of primary HBEs from a CF individual and ~50% transfection of those from a healthy donor at 32.5 and 72ng doses. Viability at these two doses was consistently between 70-85%. Small chemical modifications to just the end-group of the PBAE structure led to significantly enhanced targeting of the lungs and successful in vivo transgene expression in BALB/c and C57BL/6 mice in a dose dependent manner (Figure 1). The Ai9 mouse model showed consistent increase in transfected cells across all lung cells as the number of injections increased, from 0.8 to 1.9 to 9.0% at 1x, 3x and 9x dosing respectively. Additionally, the number of epithelial cells transfected consistently increased across dosing, with the number of epithelial and bronchial transfected cells ranging from 15-30% and 15-40% respectively. Alveolar type I and type II transfected cells averaged anywhere between 20-40%, while CD31+ CD326+ cells averaged 50% transfection and up to 80% regardless of dosing (Figure 2).

We hereby demonstrate systemic delivery of optimized PBAE NP formulation E63 allows for over 90% transfection rate in healthy primary HNEs in vitro while maintaining at least 75% viability. This same polymer achieves 20-30% transfection of cell types relevant to CF with systemic delivery in mice. Mice injected up to 9x showed no adverse side effects or toxicity, demonstrating the ability for this platform to allow for multiple administrations of various nucleic acid products. Addition of targeting ligand CD326 did not mark significant differences in transfection. We achieved up to 50% transfection of relevant CF cell types, as well as significant transfection in alveolar and endothelial or endothelial-like cells, signaling potential therapeutic targets for other diseases outside of CF, such as pulmonary hypertension and surfactant disorders. In ongoing work, we are investigating CFTR gene correction and functional restoration in vivo delivery to a CF mouse.

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