Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Transient delivery of modified mRNA hTERT reactivates telomerase activity and is a promising therapeutic candidate
J. L. Ye1, M. Juchem1, S. Chatterjee1, T. Thum1, C. Bär1, für die Studiengruppe: IMTTS
1Institut für Molekulare und Translationale Therapiestrategien, OE-8886, Medizinische Hochschule Hannover, Hannover;
Introduction: 
Telomere attrition is an important hallmark of aging and is increased in various age-related diseases such as cardiovascular diseases (CVDs). Moreover, patients with telomerase haplo-insufficiency suffer a broad spectrum of fatal diseases summarized as the telomere syndromes, which are caused as a result of accelerated telomere attrition. The telomerase enzyme complex contains the telomerase reverse transcriptase (TERT) that is responsible for maintaining the telomere lengths. However, TERT is silenced after birth in somatic cells. In mouse models of myocardial infarction and aplastic anaemia, pharmacological activation or gene delivery of telomerase was reported to be beneficial. Currently, viral-based TERT gene therapy is most widely used for extending telomeres, providing constitutive expression after transduction. In contrast, the application of modified mRNA (modRNA) represents a faster way to transiently reactivate TERT, which can further replenish telomeres and hold off aging-associated deteriorations in somatic cells devoid of telomerase expression. ModRNA avoids the risk of insertional mutagenesis and one dose of modRNA TERT enables potential long-term therapeutic effects overcoming continuous treatment.

Methods and Results:
Before investigating the potential exogenous TERT therapy we produced optimized modRNA by in vitro transcription (IVT). The modRNA was subsequently optimized for TERT activation through precise modifications of multiple parameters such as the IVT template, 3’ UTR, 5’ UTR, polyA tail length, and nucleotide modification. We confirmed the successful production of high quality modRNA, which was then ultimately tested via transfection of the modRNA-TERT into various TERT-negative cell types (HUVEC, MRC-5).  The efficiency of modRNA-TERT was validated by performing qPCR to observe TERT expression and Telomeric Repeat Amplification Protocol (TRAP) to detect enhanced telomerase activity in modRNA-TERT treated cells. HUVEC and MRC-5 treated with modRNA-TERT revealed a dose-dependent increase of TERT expression (qPCR) and telomerase activity (TRAP). Moreover, this dose-dependent increase of TERT expression was proven in a translational model of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs).    

Conclusion:
Taken everything together, we have successfully proven that modRNA-TERT can be applied to different cell types, making it attractive as an efficient therapeutic strategy for various diseases like myocardial infarction, aplastic anaemia or idiopathic pulmonary fibrosis. To further test the safety of modRNA-TERT as a therapeutic strategy, pre-clinical cytotoxicity assessments will be performed. Subsequently, the modRNA-TERT will be tested in 3D ex-vivo model of living human precision tissue slices from the heart and lungs, as well as in 3D organoids.
 
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