Recent advancements in the field of in vitro transcribed mRNA (IVT-mRNA) vaccination have attracted considerable attention to such vaccination as a cutting-edge technique against infectious diseases including COVID-19 caused by SARS-CoV-2. While numerous pathogens infect the host through the respiratory mucosa, conventional parenterally administered vaccines are unable to induce protective immunity at mucosal surfaces. Mucosal immunization enables the induction of both mucosal and systemic immunity, efficiently removing pathogens from the mucosa before an infection occurs. Although respiratory mucosal vaccination is highly appealing, successful nasal or pulmonary delivery of nucleic acid-based vaccines is challenging because of several physical and biological barriers at the airway mucosal site, such as a variety of protective enzymes and mucociliary clearance, which remove exogenously inhaled substances. Hence, advanced nanotechnologies enabling delivery of DNA and IVT-mRNA to the nasal and pulmonary mucosa are urgently needed. Ideal nanocarriers for nucleic acid vaccines should be able to efficiently load and protect genetic payloads, overcome physical and biological barriers at the airway mucosal site, facilitate transfection in targeted epithelial or antigen-presenting cells, and incorporate adjuvants. In this review, we discuss recent developments in nucleic acid delivery systems that target airway mucosa for vaccination purposes.

During the last years the potential role of in vitro transcribed (IVT) mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE). A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000) in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages of IVT mRNA as compared to pDNA as transfection agent.

The coronavirus disease 2019 (COVID-19) pandemic poses a disruptive impact on public health and the global economy. Fortunately, the development of COVID-19 vaccines based on in vitro-transcribed messenger RNA (IVT mRNA) has been a breakthrough in medical history, benefiting billions of people with its high effectiveness, safety profile, and ease of large-scale production. This success is the result of decades of continuous RNA research, which has led to significant improvements in the stability and expression level of IVT mRNA through various approaches such as sequence optimization and improved preparation processes. IVT mRNA sequence optimization has been shown to have a positive effect on enhancing the mRNA expression level. The innovation of IVT mRNA purification technology is also indispensable, as the purity of IVT mRNA directly affects the success of downstream vaccine preparation processes and the potential for inducing unwanted side effects in therapeutic applications. Despite the progress made, challenges related to IVT mRNA sequence design and purification still require further attention to enhance the quality of IVT mRNA in the future. In this review, we discuss the latest innovative progress in IVT mRNA design and purification to further improve its clinical efficacy.

In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial number of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT, while bringing along the unique advantages of IVT-mRNA.