In Vitro Investigations on Optimizing and Nebulization of IVT-mRNA Formulations for Potential Pulmonary-Based Alpha-1-Antitrypsin Deficiency Treatment.

Shan Guan,Chuanfei Xu,Max Darmstädter,Joseph Rosenecker

doi:10.3390/pharmaceutics13081281

Shan Guan, Chuanfei Xu + Show 2 more

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https://doi.org/10.3390/pharmaceutics13081281

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Abstract

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.

Highlights

Nucleic acid-based therapeutics encoding specific proteins of interest have shown great potential in the treatment of devastating diseases such as genetic disorders, infectious diseases, cancer and cardiovascular diseases [1,2]
The In vitro-transcribed (IVT)-mRNA/Lipofectamine2000 complexes were prepared in OptiMEM, a medium specially designed for transfection
Dynamic light scattering measurements revealed that the size of IVT-mRNA/Lipofectamine2000 complexes prepared under different conditions and settings were typically a few hundred nanometers, as depicted in Table 1

Summary

Introduction

Nucleic acid-based therapeutics encoding specific proteins of interest have shown great potential in the treatment of devastating diseases such as genetic disorders, infectious diseases, cancer and cardiovascular diseases [1,2]. In vitro-transcribed messenger RNA (IVT-mRNA) has emerged as an alternative to the conventional DNA-based therapeutic and provides many unique features to be a promising drug candidate, for example, high efficiency in transfecting non-dividing cells and ease of production [3]. IVT-mRNA offers a huge advantage in terms of safety; it has no risk of insertional mutagenesis. IVT-mRNA is able to rapidly express the desired protein in the cytoplasm and automatically degrade afterwards, so the protein expression could be controlled [4,5]. Pioneering works initiated by Katalin Karikó et al have enabled an in-depth understanding of the relationship between IVT-mRNA structure and its immunogenicity profile [6,7]. With the utilization of chemically modified nucleotides and advanced purification methods, the stability, immunogenicity and expression efficiency of IVT-mRNA have been greatly improved [8]. A large number of IVT-mRNA-based therapeutics is tested in clinical trials [9,10,11,12,13,14], and recently IVT-mRNA has displayed great potency in developing efficacious vaccine approaches to eliminate the spread of severe acute respiratory

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