Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges.

Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79.

Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance in the fields of regenerative medicine, tissue engineering, and disease modeling. Patient-specific iPSCs have as an unlimited cell source a tremendous potential for generating various types of autologous cells. For the future clinical applicability of these iPSC-derived cells, the generation of iPSCs via nongenome integrating methods and the efficient reprogramming of patients' somatic cells are required. In this study, 2 different RNA-based footprint-free methods for the generation of iPSCs were compared: the use of synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors and GFP. Using both RNA-based methods, integration-free iPSCs without genomic alterations were obtained. The pluripotency characteristics identified by specific marker detection and the in vitro and in vivo trilineage differentiation capacity were comparable. Moreover, the incorporation of a GFP encoding sequence into the srRNA enabled a direct and convenient monitoring of the reprogramming procedure and the successful detection of srRNA translation in the transfected cells. Nevertheless, the use of a single srRNA to induce pluripotency was less time consuming, faster, and more efficient than the daily transfection of cells with synthetic mRNAs. Therefore, we believe that the srRNA-based approach might be more appropriate and efficient for the reprogramming of different types of somatic cells for clinical applications.

Reprogramming of Urine-Derived Renal Epithelial Cells into iPSCs Using srRNA and Consecutive Differentiation into Beating Cardiomyocytes.

The generation of induced pluripotent stem cells (iPSCs) from patient's somatic cells and the subsequent differentiation into desired cell types opens up numerous possibilities in regenerative medicine and tissue engineering. Adult cardiomyocytes have limited self-renewal capacity; thus, the efficient, safe, and clinically applicable generation of autologous cardiomyocytes is of great interest for the treatment of damaged myocardium. In this study, footprint-free iPSCs were successfully generated from urine-derived renal epithelial cells through a single application of self-replicating RNA (srRNA). The expression of pluripotency markers and the in vitro as well as in vivo trilineage differentiation were demonstrated. Furthermore, the resulting iPSCs contained no residual srRNA, and the karyotyping analysis demonstrated no detectable anomalies. The cardiac differentiation of these iPSCs resulted in autologous contracting cardiomyocytes after 10 days. We anticipate that the use of urine as a non-invasive cell source to obtain patient cells and the use of srRNA for reprogramming into iPSCs will greatly improve the future production of clinically applicable cardiomyocytes and other cell types. This could allow the regeneration of tissues by generating sufficient quantities of autologous cells without the risk of immune rejection.

Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs.

The application of endothelial progenitor cells (EPCs) for the revascularization of ischemic tissues, such as after myocardial infarction, stroke, and acute limb ischemia, has a huge clinical potential. However, the low retention and engraftment of EPCs as well as the poor survival of migrated stem cells in ischemic tissues still hamper the successful clinical application. Thus, in this study, we engineered, for the first time, murine EPCs with synthetic mRNAs to transiently produce proangiogenic factors vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1α (SDF-1α), and angiopoietin-1 (ANG-1). After the transfection of cells with synthetic mRNAs, significantly increased VEGF-A, SDF-1α, and ANG-1 protein levels were detected compared to untreated EPCs. Thereby, mRNA-engineered EPCs showed significantly increased chemotactic activity versus untreated EPCs and resulted in significantly improved attraction of EPCs. Furthermore, ANG-1 mRNA-transfected EPCs displayed a strong wound-healing capacity. Already after 12 hr, 94% of the created wound area in the scratch assay was closed compared to approximately 45% by untreated EPCs. Moreover, the transfection of EPCs with ANG-1 or SDF-1α mRNA also significantly improved the in vitro tube formation capacity; however, the strongest effect could be detected with EPCs simultaneously transfected with VEGF-A, SDF-1α, and ANG-1 mRNA. In the in vivo chicken chorioallantoic membrane (CAM) assay, EPCs transfected with ANG-1 mRNA revealed the strongest angiogenetic potential with significantly elevated vessel density and total vessel network length. In conclusion, this study demonstrated that EPCs can be successfully engineered with synthetic mRNAs encoding proangiogenic factors to improve their therapeutic angiogenetic potential in patients experiencing chronic or acute ischemic disease.

De Novo Synthesis of Elastin by Exogenous Delivery of Synthetic Modified mRNA into Skin and Elastin-Deficient Cells.

Elastin is one of the most important and abundant extracellular matrix (ECM) proteins that provide elasticity and resilience to tissues and organs, including vascular walls, ligaments, skin, and lung. Besides hereditary diseases, such as Williams-Beuren syndrome (WBS), which results in reduced elastin synthesis, injuries, aging, or acquired diseases can lead to the degradation of existing elastin fibers. Thus, the de novo synthesis of elastin is required in several medical conditions to restore the elasticity of affected tissues. Here, we applied synthetic modified mRNA encoding tropoelastin (TE) for the de novo synthesis of elastin and determined the mRNA-mediated elastin synthesis in cells, as well as ex vivo in porcine skin. EA.hy926 cells, human fibroblasts, and mesenchymal stem cells (MSCs) isolated from a patient with WBS were transfected with 2.5 µg TE mRNA. After 24 hr, the production of elastin was analyzed by Fastin assay and dot blot analyses. Compared with untreated cells, significantly enhanced elastin amounts were detected in TE mRNA transfected cells. The delivered synthetic TE mRNA was even able to significantly increase the elastin production in elastin-deficient MSCs. In porcine skin, approximately 20% higher elastin amount was detected after the intradermal delivery of synthetic mRNA by microinjection. In this study, we demonstrated the successful applicability of synthetic TE encoding mRNA to produce elastin in elastin-deficient cells as well as in skin. Thus, this auspicious mRNA-based integration-free method has a huge potential in the field of regenerative medicine to induce de novo elastin synthesis, e.g., in skin, blood vessels, or alveoli.

RNA-Eluting Surfaces for the Modulation of Gene Expression as A Novel Stent Concept.

Presently, a new era of drug-eluting stents is continuing to improve late adverse effects such as thrombosis after coronary stent implantation in atherosclerotic vessels. The application of gene expression-modulating stents releasing specific small interfering RNAs (siRNAs) or messenger RNAs (mRNAs) to the vascular wall might have the potential to improve the regeneration of the vessel wall and to inhibit adverse effects as a new promising therapeutic strategy. Different poly (lactic-co-glycolic acid) (PLGA) resomers for their ability as an siRNA delivery carrier against intercellular adhesion molecule (ICAM)-1 with a depot effect were tested. Biodegradability, hemocompatibility, and high cell viability were found in all PLGAs. We generated PLGA coatings with incorporated siRNA that were able to transfect EA.hy926 and human vascular endothelial cells. Transfected EA.hy926 showed significant siICAM-1 knockdown. Furthermore, co-transfection of siRNA and enhanced green fluorescent protein (eGFP) mRNA led to the expression of eGFP as well as to the siRNA transfection. Using our PLGA and siRNA multilayers, we reached high transfection efficiencies in EA.hy926 cells until day six and long-lasting transfection until day 20. Our results indicate that siRNA and mRNA nanoparticles incorporated in PLGA films have the potential for the modulation of gene expression after stent implantation to achieve accelerated regeneration of endothelial cells and to reduce the risk of restenosis.

An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA.

In the last decades, many efforts have been made to counteract adverse effects after stenting atherosclerotic coronary arteries. A breakthrough in better vascular wall regeneration was noted in the new era of drug-eluting stents. A novel personalized approach is the development of gene-eluting stents promising an alteration in gene expression involved in regeneration. We investigated a coating system consisting of the polymer atelocollagen (ATCOL) and a specific small interfering RNA (siRNA) for intercellular adhesion molecule-1 (ICAM-1) found on the surface of defective endothelial cells (ECs). We demonstrated very high cell viability, in which EA.hy926 grew on 0.008% or 0.032% ATCOL layers. Additionally, hemocompatibility assays proved the biocompatibility of this coating. The highest transfection efficiency with EA.hy926 was achieved with 5 µg siRNA immobilized in ATCOL after 2 days. The release of fluorescent-labeled siRNA was about 9 days. Long-term knockdown of ICAM-1 was analyzed by flow cytometry, revealing that the coating with 0.008% ATCOL and 5 µg siICAM-1 provoked gene silencing up to 8 days. 5'-RNA ligase-mediated rapid amplification of cDNA ends PCR (RLM-RACE-PCR) demonstrated the specificity of our established ATCOL gene-silencing coating, meaning that our coating is well suited for further investigations in in vivo studies. Herein, we would like to demonstrate that our ATCOL is well-suited for better artery wall regeneration after stent implantation.

In vitro Study of a Novel Stent Coating Using Modified CD39 Messenger RNA to Potentially Reduce Stent Angioplasty-Associated Complications.

BACKGROUND: Stent angioplasty provides a minimally invasive treatment for atherosclerotic vessels. However, no treatment option for atherosclerosis-associated endothelial dysfunction, which is accompanied by a loss of CD39, is available, and hence, adverse effects like thromboembolism and restenosis may occur. Messenger RNA (mRNA)-based therapy represents a novel strategy, whereby de novo synthesis of a desired protein is achieved after delivery of a modified mRNA to the target cells. METHODS AND FINDINGS: Our study aimed to develop an innovative bioactive stent coating that induces overexpression of CD39 in the atherosclerotic vessel. Therefore, a modified CD39-encoding mRNA was produced by in vitro transcription. Different endothelial cells (ECs) were transfected with the mRNA, and CD39 expression and functionality were analyzed using various assays. Furthermore, CD39 mRNA was immobilized using poly(lactic-co-glycolic-acid) (PLGA), and the transfection efficiency in ECs was analyzed. Our data show that ECs successfully translate in vitro-generated CD39 mRNA after transfection. The overexpressed CD39 protein is highly functional in hydrolyzing ADP and in preventing platelet activation. Furthermore, PLGA-immobilized CD39 mRNA can be delivered to ECs without losing its functionality. SUMMARY: In summary, we present a novel and promising concept for a stent coating for the treatment of atherosclerotic blood vessels, whereby patients could be protected against angioplasty-associated complications.

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

In single-gene disorders, like alpha-1-antitrypsin deficiency (AATD), a gene mutation causes missing or dysfunctional protein synthesis. This, in turn, can lead to serious complications for the patient affected. Furthermore, single-gene disorders are associated with severe early-onset conditions and necessitate expensive lifelong care. Until nowadays, therapeutic treatment options are still limited, cost-intensive, or lack effectiveness. For these reasons, we aim to develop a novel mRNA-based therapeutic strategy for the treatment of single-gene disorders, such as AATD, which is based on the induction of de novo synthesis of the functional proteins. Therefore, an alpha-1-antitrypsin (AAT) encoding mRNA was generated by in vitro transcription. After in vitro delivery of the mRNA to different cells, protein expression and functionality, as well as adverse effects and mRNA serum stability, were analyzed. Our results show that the AAT mRNA-transfected cells express the AAT protein in high amounts within the first 24 h. Moreover, the expressed AAT protein is highly functional, since the activity of elastase is significantly inhibited. Our data also show that mRNA concentrations up to 1 µg per 150,000 cells have no adverse effects on cell viability and immune activation. Furthermore, the encapsulated AAT encoding mRNA is stable and functional in human serum for up to 30 min. Overall, the proposed project provides an innovative, highly promising, and safe therapeutic approach and, thus, promises a novel progress in the treatment of single-gene disorders, whereby affected patients could greatly benefit.

Optimized conditions for successful transfection of human endothelial cells with in vitro synthesized and modified mRNA for induction of protein expression.

BACKGROUND: The induction of protein synthesis by exogenous delivery of coding synthetic mRNA in desired cells is an auspicious strategy in the fields of basic cell biology, regenerative medicine, treatment of diseases, and reprogramming of cells. Here, we produced modified messenger RNA (mRNA) with reduced immune activation potential and increased stability and performed transfection experiments with different cells, HEK293 cells, BJ fibroblasts, and endothelial cells (ECs). RESULTS: The mRNA induced protein expression in cells was analyzed after transfection with different mRNA amounts and transfection reagents using flow cytometry. Different cell types showed different degrees of eGFP expression. HEK293 cells exhibited the highest eGFP expression compared to the BJ fibroblasts and ECs. However, the mRNA induced eGFP expression was detected in all cell types until 3 days after transfection. Already, the use of 0.5 µg of the synthesized mRNA led to the significant expression of eGFP in ECs. From all analyzed ECs approximately 87% were eGFP positive, which showed a high transfection efficiency. CONCLUSIONS: The synthesis of stabilized mRNA and the high transfection efficiency will enable the mRNA engineering of ECs as well as other somatic cells. The delivery of synthetic exogenous mRNA into cells allows the transient expression of desired proteins, which would be normally not expressed by the cells.

In vitro synthesis of modified mRNA for induction of protein expression in human cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.