Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA

Sonia Golombek; Thomas Hoffmann; Ludmilla Hann; Markus Mandler; Sabine Schmidhuber; Josefin Weber; Young-Tae Chang; Roman Mehling; Andrea Ladinig; Christian Knecht; Johanna Leyens; Christian Schlensak; Hans Peter Wendel; Achim Schneeberger; Meltem Avci-Adali

doi:10.1016/j.omtn.2023.07.035

Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA

Mol Ther Nucleic Acids. 2023 Aug 2:33:642-654. doi: 10.1016/j.omtn.2023.07.035. eCollection 2023 Sep 12.

Authors

Affiliations

¹ Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Calwerstraße 7/1, 72076 Tübingen, Germany.
² Accanis Biotech, Karl-Farkas-Gasse 22, Vienna 1030, Austria.
³ Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea.
⁴ Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, Röntgenweg 13, 72076 Tübingen, Germany.
⁵ University Clinic for Swine, Department of Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Veterinärplatz 1, Vienna 1210, Austria.

Abstract

Loss of elastin due to aging, disease, or injury can lead to impaired tissue function. In this study, de novo tropoelastin (TE) synthesis is investigated in vitro and in vivo using different TE-encoding synthetic mRNA variants after codon optimization and nucleotide modification. Codon optimization shows a strong effect on protein synthesis without affecting cell viability in vitro, whereas nucleotide modifications strongly modulate translation and reduce cell toxicity. Selected TE mRNA variants (3, 10, and 30 μg) are then analyzed in vivo in porcine skin after intradermal application. Administration of 30 μg of native TE mRNA with a me¹ Ψ modification or 10 and 30 μg of unmodified codon-optimized TE mRNA is required to increase TE protein expression in vivo. In contrast, just 3 μg of a codon-optimized TE mRNA variant with the me¹ Ψ modification is able to increase protein expression. Furthermore, skin toxicity is investigated in vitro by injecting 30 μg of mRNA of selected TE mRNA variants into a human full-thickness skin model, and no toxic effects are observed. Thereby, for the first time, an increased dermal TE synthesis by exogenous administration of synthetic mRNA is demonstrated in vivo. Codon optimization of a synthetic mRNA can significantly increase protein expression and therapeutic outcome.

Keywords: MT: Oligonucleotides: Therapies and Applications; coding sequence; de novo synthesis; mRNA optimization; nucleotide modification; protein replacement therapy; synthetic mRNA; tissue regeneration; tropoelastin.