A new study published in Pharmaceutics investigated the use of nebulized in vitro-transcribed mRNA (IVT-mRNA) in human bronchial epithelial cells as a potential treatment for alpha-1-antitrypsin deficiency (AATD). The results of the trial showed that nebulized IVT-mRNA complexes could still be used to transfect cells although at a lower efficiency than non-nebulized complexes.

The study also showed that the transfected cells not only produced alpha-1-antitrypsin (A1AT) protein but also secreted it. Based on the results, the authors stated, “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.”

IVT-mRNA-based therapies are currently being investigated in clinical trials for a number of diseases and IVT-mRNA has recently been shown effective in producing vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

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“IVT-mRNA offers a huge advantage in terms of safety [compared to DNA-based gene therapy]; it has no risk of insertional mutagenesis,” the authors wrote. “IVT-mRNA is able to rapidly express the desired protein in the cytoplasm and automatically degrade afterwards, so the protein expression could be easily controlled.” By nebulizing the IVT-mRNA, the treatment could be delivered directly to the area most affected by AATD: the lungs.

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During the trial, a number of different concentrations of A1AT-mRNA and the transfection reagent Lipofectamine 2000 were tested in cells to establish an optimal transfection process. Researchers found that the highest levels of protein production occurred 24 hours after adding the complexes, with elevated levels being detected for up to 4 days.

Cells were then tested with IVT-mRNA formulations that had been aerosolized to determine the impact of shear forces on the viability of the mRNA molecules. The nebulization process was shown to decrease the transfection potency of the IVT-mRNA lipoplexes but the authors stated that this could be partially solved by increasing the concentrations of IVT-mRNA.

While the study shows the potential of nebulized IVT-mRNA as a possible new treatment, the authors point out that since Lipofectamine 2000 is cytotoxic and would have limited in vivo use, additional delivery systems need to be developed before the technique can be translated to the clinic.


Guan S, Darmstädter M, Xu C, Rosenecker J. In vitro investigations on optimizing and nebulization of IVT-mRNA formulations for potential pulmonary-based alpha-1-antitrypsin deficiency treatment. Pharmaceutics. 2021;13(8):1281. doi:10.3390/pharmaceutics13081281