Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs

Abstract

Gene therapy is a breakthrough treatment strategy against several intractable and lethal diseases that previously lacked established treatments. Viral and non-viral vectors have been studied to realize higher gene transfection efficiencies and to suppress the degradation of gene by nucleolytic enzymes in vivo. However, it is often the case that the addition of a vector results in adverse effects. In this study, we identified formulations of dry naked plasmid DNA (pDNA) powders with no vector showing significantly higher gene expression than pDNA solutions including vectors such as polyethylenimine (PEI) in the lungs of mice. We prepared the naked pDNA powders by spray-freeze-drying with various excipients. The gene expression of naked pDNA powders exceeded those of pDNA solutions containing PEI, naked pDNA solution, and reconstituted pDNA powder. Gene expression of each naked pDNA powder was dependent on the composition of excipients. Among them, the mice administered to the pDNA powder composed of low-molecular-weight hyaluronic acid (LHA) as an excipient showed the highest gene expression. The lactate dehydrogenase activity and concentration of inflammatory cytokines in bronchoalveolar lavage fluid were comparable to those caused by ultra-pure water. The results suggest that useful dry naked nucleic acid powders for inhalation could be created by optimizing the excipients, offering new insights into the development of pulmonary gene therapy.

Development of a DNA Vaccine for Melanoma Metastasis by Inhalation Based on an Analysis of Transgene Expression Characteristics of Naked pDNA and a Ternary Complex in Mouse Lung Tissues

“…This result is in contrast to the generally accepted theory that naked pDNA achieves weak gene expression because of poor intracellular uptake and degradation by endonucleases. However, Ito et al previously reported that the inhalation of naked pDNA powder composed of hyaluronic acid achieved strong gene expression in the lungs [32]. Furthermore, an RNA interference (RNAi) effect was demonstrated following the inhalation of naked siRNA solution [33].…”

Microencapsulated Chitosan-Based Nanocapsules: A New Platform for Pulmonary Gene Delivery

“…It has been reported the administration of naked pDNA inhaled in form of dry powders prepared with different excipients, resulting in a better transfection than their counterparts in suspension. In that study, the level of transfection was highly dependent on the excipient used for the preparation of the powders, being the MS prepared with HA the ones that gave the best results [ 80 ]—which could be expected taking into account the HA transfection enhancer effect. CS NCs have already been proposed for lung delivery of genes [ 37 ], but they had not been assessed in vivo.…”

Hyaluronan as a Prominent Biomolecule with Numerous Applications in Medicine

“…The highest gene expression in mice, the lactate dehydrogenase activity and concentration of inflammatory cytokines in bronchoalveolar lavage fluid comparable to those caused by ultrapure water [172] Transferrin/HA-pDNA/nanostructured lipid carriers in lung cancer gene therapy Low cytotoxicity, enhanced gene transfer ability in vitro and in vivo [169] HA in cochlear gene therapy Facilitated expression in cells lining the scala media, atraumatic and clinically feasible method to introduce transgenes into cochlear cells [173] pDNA/HA/chitosan complexes Augmented stability and cellular transfection ability of the complexes after lyophilization-rehydration [174] Nanoparticles formed by HA, polyethyleneglycol (HA, PEG) and polyethylenimine (HA, PEI) and pDNA…”