Defining Optimized Properties of Modified mRNA to Enhance Virus- and DNA- Independent Protein Expression in Adult Stem Cells and Fibroblasts

Abstract

Background: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. Methods: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins. Results: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. Conclusion: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

In VitroEvaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency

“…Similar results were shown in previous studies, where protein expression levels and mRNA stability could be increased and immune activation was reduced through the introduction of modifications [26,33,34].…”

“…Until now, various studies exist, where the advantages and disadvantages of differently modified mRNAs were studied [16,26,27,34]. Hence, we produced AAT mRNA with different modifications and compared the efficiency in AAT protein expression in A549 cells.…”

“…Furthermore, the mRNA can be modified through the incorporation of modified nucleotides, like pseudouridine and 5-methylcytidine, which significantly increases protein expression level [23,25,26], biological stability [23] as well as suppresses the potential to activate the immune system [23,27,28]. Furthermore, the modified cap structure [antireverse cap analog (ARCA)] prevents the change in orientation during the in vitro transcription (IVT) [29], results in significantly higher translational efficiency [30], and increases mRNA stability against hydrolases [31].…”

Individual Variation in Conditional β Cell Ablation Mice Contributes Significant Biases in Evaluating β Cell Functional Recovery

“…Therefore, in this study, we attempted to utilize the transient nature of chemically modRNA and examine the beneficial effect of VEGF A modRNA in β cell regeneration after injury. Previous reports have demonstrated that gene expression is transient yet highly efficient following modRNA transfection ( 15 , 25 , 26 ), so it has been used as a therapeutic delivery agent in many studies ( 27 – 29 ). We have also successfully applied hVEGF A modRNA in promoting murine cardiac regeneration after myocardial infarction ( 14 , 16 ).…”

Alterations in Cardiomyocyte Differentiation-Related Proteins in Rat Mesenchymal Stem Cells Exposed to Hypoxia

“…In addition, they have been found in almost all tissues and are mostly located in perivascular niches [3]. Therefore, they are more readily availability, pose fewer ethical problems, and have lower immunogenicity and immunomodulatory effects than do embryonic stem cells; thus, MSCS play an important role as a candidate for clinical applications in regenerative medicine [4,5]. However, a problem with MSC therapy is that a large majority of transplanted cells undergo apoptosis following transplantation [6], so overcoming this limitation is urgent.…”