Dimiou, Savvas; (2024) ‘‘Inhaling Genes’’: Development and Characterization of Polyplexes Based on Glycol Chitosan Derivatives for Pulmonary Gene Delivery via Inhalation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Pulmonary gene delivery is desirable due to its non-invasive nature and the possibility of achieving an increased local drug concentration. While formulating genes as inhalable dry powders could provide additional benefits such as longer shelf-life, it is challenging as genes are prone to degradation due to thermal and shear stress. This work focuses on developing a biocompatible and transfection-competent polymeric gene dry powder system for pulmonary delivery. Ten polymers were synthesised based on the glycol chitosan (GC) backbone to systematically investigate the effect of polymer chain length (molecular weight) and chemical modification (hydrophobicity, hydrophilicity) on gene transfer and thus identify the optimal polymer/polyplex properties for efficient gene transfer. Following physicochemical characterisation and in vitro screening, a GC polymer of 60 kDa (GC60) was selected for its superior transfection efficiency (comparable to the positive control, Lipofectamine) and low cytotoxicity (GC60 IC50 = 2,420 ± 210 μg/mL, Lipofectamine IC50 = 1.4 ± 0.2 μg/mL). GC60 gene nanoparticles were then converted into microparticles via spray-drying generating a dry powder with low moisture content, a microparticle size appropriate for deep lung targeting, and similar physicochemical properties to the fresh liquid formulation after powder reconstitution. Most importantly, the nucleic acid integrity was preserved post-spray-drying with the gene dry powder exhibiting similar transfection efficiency to the liquid formulation and remaining room-temperature-stable up to 3 months and refrigeration-temperature-stable up to 5 months. Preliminary in vivo studies showed high variability in pulmonary gene expression and off-target expression effects in brain regions likely due to the administration route chosen (intranasal) and the formulation viscosity. On repeated (4 doses; 0.8 mg/kg dose) nose-to-lung administration with a modified intranasal technique, GC60 yielded a 3-fold higher pulmonary gene expression compared to naked DNA with minimal off-target expression. Taken together, these data showcase the potential of GC60 as a non-viral vector for inhalable pulmonary gene transfer for either therapeutic or prophylactic applications.

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