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Aneurysms pose life-threatening risks due to the dilatation of the arteries and carry a high risk of rupture. Despite continuous research efforts, there are still no satisfactory or clinically effective pharmaceutical treatments for this condition. Accelerated inflammatory processes during aneurysm development lead to increased levels of matrix metalloproteinases (MMPs) and destabilization of the vessel wall through the degradation of the structural components of the extracellular matrix (ECM), mainly collagen and elastin. Tissue inhibitors of metalloproteinases (TIMPs) directly regulate MMP activity and consequently inhibit ECM proteolysis. In this work, the synthesis of TIMP-1 protein was increased by the exogenous delivery of synthetic TIMP-1 encoding mRNA into aortic vessel tissue in an attempt to inhibit MMP-9. In vitro, TIMP-1 mRNA transfection resulted in significantly increased TIMP-1 protein expression in various cells. The functionality of the expressed protein was evaluated in an appropriate ex vivo aortic vessel model. Decreased MMP-9 activity was detected using in situ zymography 24 h and 48 h post microinjection of 5 µg TIMP-1 mRNA into the aortic vessel wall. These results suggest that TIMP-1 mRNA administration is a promising approach for the treatment of aneurysms. [ABSTRACT FROM AUTHOR]

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.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo. Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications.

Owing to messenger RNA's unique biological advantages, it has received increasing attention to be used as a therapeutic, known as mRNA-based gene therapy. It is critical to have an ideal strategy of mRNA gene therapy for glioma, which grows in a special environment. In the present study, we screened out a safe and efficient transfection reagent for intracranial delivery of synthetic mRNA in mouse brain. First, in order to analyze the effect of different transfection reagents on the intracranial delivery of mRNA, the synthetic luciferase mRNA was wrapped with two different transfection reagents and microinjected into the brain at the fixed point. The expression status of delivered mRNA was monitored by a small animal imaging system. The possible reagent-induced biological toxicity was evaluated by behavioral and blood biochemical measurements. Then, to test the therapeutic effect of our intracranial delivery mRNA model on glioma, synthetic modified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA was used as an example of therapeutic application. This model demonstrated that synthetic mRNA could be successfully delivered into the brain using commercially available transfection reagents, and TransIT-mRNA showed better results than in vivo-jetPEI kit. This model can be applied in precise targeting and personalized gene therapy of glioma.

Glioblastoma (GBM) is characterized as having high molecular heterogeneity and complexity, which can be well revealed by genomic study. A truly effective treatment for GBM should flexibly address its heterogeneities, complexity, and strong drug resistance. This study was performed to explore the effectiveness of an mRNA-based therapeutic strategy using in vitro synthesized PTEN-mRNA and TRAIL-mRNA in tumor cells derived from PTEN-deletion patients. The PTEN gene alterations were revealed by whole-exome sequencing of three paired clinical GBMs and selected as the therapy target. Patient-derived primary glioblastoma stem cells (GBM2) and a DBTRG-cell-derived xenograft were used to detect mRNA's cytotoxicity in vitro and tumor suppression in vivo. Following the successful in vitro synthesis of PTEN-mRNA and TRAIL-mRNA, the combinational treatment of PTEN-mRNA and TRAIL-mRNA significantly suppressed tumor growth compared with treatment with PBS (96.4%), PTEN-mRNA (89.7%), and TRAIL-mRNA (84.5%). The combinational application of PTEN-mRNA and TRAIL-mRNA showed synergistic inhibition of tumor growth, and the JNK pathway might be the major mechanism involved. This study provided a basis for an mRNA-based therapeutic strategy to be developed into an effective patient-tailored treatment for GBM.

Abstract Background Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. Methods We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. Results We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. Conclusions This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases. Graphical abstract

Endothelial progenitor cells (EPCs) are one of the most important stem cells for the neovascularization of tissues damaged by ischemic diseases such as myocardial infarction, ischemic stroke, or critical limb ischemia. However, their low homing efficiency in the treatment of ischemic tissues limits their potential clinical applications. The use of synthetic messenger RNA (mRNA) for cell engineering represents a novel and promising technology for the modulation of cell behavior and tissue regeneration. To improve the therapeutic potential of EPCs, in this study, murine EPCs were engineered with synthetic mRNAs encoding C-X-C chemokine receptor 4 (CXCR4) and P-selectin glycoprotein ligand 1 (PSGL-1) to increase the homing and migration efficiency of EPCs to inflamed endothelium. Flow cytometric measurements revealed that the transfection of EPCs with CXCR4 and PSGL-1 mRNA resulted in increased expressions of CXCR4 and PSGL-1 on the cell surface compared with the unmodified EPCs. The transfection of EPCs with mRNAs did not affect cell viability. CXCR4-mRNA-modified EPCs showed significantly higher migration potential than unmodified cells in a chemotactic migration assay. The binding strength of the EPCs to inflamed endothelium was determined with single-cell atomic force microscopy (AFM). This showed that the mRNA-modified EPCs required a three-fold higher detachment force to be released from the TNF-α-activated endothelium than unmodified EPCs. Furthermore, in a dynamic flow model, significantly increased binding of the mRNA-modified EPCs to inflamed endothelium was detected. This study showed that the engineering of EPCs with homing factors encoding synthetic mRNAs increases the homing and migration potentials of these stem cells to inflamed endothelium. Thus, this strategy represents a promising strategy to increase the therapeutic potential of EPCs for the treatment of ischemic tissues. [ABSTRACT FROM AUTHOR]

Background: Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy. Methods: We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy. Results: We generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment. Conclusions: This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Endothelial progenitor cells (EPCs) are one of the most important stem cells for the neovascularization of tissues damaged by ischemic diseases such as myocardial infarction, ischemic stroke, or critical limb ischemia. However, their low homing efficiency in the treatment of ischemic tissues limits their potential clinical applications. The use of synthetic messenger RNA (mRNA) for cell engineering represents a novel and promising technology for the modulation of cell behavior and tissue regeneration. To improve the therapeutic potential of EPCs, in this study, murine EPCs were engineered with synthetic mRNAs encoding C-X-C chemokine receptor 4 (CXCR4) and P-selectin glycoprotein ligand 1 (PSGL-1) to increase the homing and migration efficiency of EPCs to inflamed endothelium. Flow cytometric measurements revealed that the transfection of EPCs with CXCR4 and PSGL-1 mRNA resulted in increased expressions of CXCR4 and PSGL-1 on the cell surface compared with the unmodified EPCs. The transfection of EPCs with mRNAs did not affect cell viability. CXCR4-mRNA-modified EPCs showed significantly higher migration potential than unmodified cells in a chemotactic migration assay. The binding strength of the EPCs to inflamed endothelium was determined with single-cell atomic force microscopy (AFM). This showed that the mRNA-modified EPCs required a three-fold higher detachment force to be released from the TNF-α-activated endothelium than unmodified EPCs. Furthermore, in a dynamic flow model, significantly increased binding of the mRNA-modified EPCs to inflamed endothelium was detected. This study showed that the engineering of EPCs with homing factors encoding synthetic mRNAs increases the homing and migration potentials of these stem cells to inflamed endothelium. Thus, this strategy represents a promising strategy to increase the therapeutic potential of EPCs for the treatment of ischemic tissues.

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.

In recent years, synthetic mRNA-based applications to produce desired exogenous proteins in cells have been gaining importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs and, thereby, fail to target desired cells. Thus, local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables patient-friendly, painless, and efficient delivery of synthetic mRNAs into the dermis; thus, this method could be applied for local treatment of different skin diseases as well as for vaccination and immunotherapy.

The structure of synthetic mRNAs as used in vaccination against cancer and infectious diseases contain specifically designed caps followed by sequences of the 5′ untranslated repeats of β-globin gene. The strategy for successful design of synthetic mRNAs by chemically modifying their caps aims to increase resistance to the enzymatic deccapping complex, offer a higher affinity for binding to the eukaryotic translation initiation factor 4E (elF4E) protein and enforce increased translation of their encoded proteins. However, the cellular homeostasis is finely balanced and obeys to specific laws of thermodynamics conferring balance between complexity and growth rate in evolution. An overwhelming and forced translation even under alarming conditions of the cell during a concurrent viral infection, or when molecular pathways are trying to circumvent precursor events that lead to autoimmunity and cancer, may cause the recipient cells to ignore their differential sensitivities which are essential for keeping normal conditions. The elF4E which is a powerful RNA regulon and a potent oncogene governing cell cycle progression and proliferation at a post-transcriptional level, may then be a great contributor to disease development. The mechanistic target of rapamycin (mTOR) axis manly inhibits the elF4E to proceed with mRNA translation but disturbance in fine balances between mTOR and elF4E action may provide a premature step towards oncogenesis, ignite pre-causal mechanisms of immune deregulation and cause maturation (aging) defects.

The field of gene therapy has evolved over the past two decades after the first introduction of nucleic acid drugs, such as plasmid DNA (pDNA). With the development of in vitro transcription (IVT) methods, synthetic mRNA has become an emerging class of gene therapy. IVT mRNA has several advantages over conventional pDNA for the expression of target proteins. mRNA does not require nuclear localization to mediate protein translation. The intracellular process for protein expression is much simpler and there is no potential risk of insertion mutagenesis. Having these advantages, the level of protein expression is far enhanced as comparable to that of viral expression systems. This makes IVT mRNA a powerful alternative gene expression system for various applications in regenerative medicine. In this review, we highlight the synthesis and preparation of IVT mRNA and its therapeutic applications. The article includes the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in cellular reprogramming, stem cell engineering, and protein replacement therapy. Finally, future perspectives and challenges of IVT mRNA are discussed. [ABSTRACT FROM AUTHOR]

Direct reprogramming of pancreatic beta cells is of great interest due to its possible use in diabetes treatment. Various studies have demonstrated direct reprogramming of insulin expressing beta cells from other somatic cells by overexpressing important pancreatic transcription factors through viral vectors. However, concerns about using viral vectors in the clinic have prompted scientists to find new approaches for gene delivery and expression. In vitro synthesis and transfection of mRNAs is a safer strategy for ectopic gene expression. In this study we aimed to design and in vitro synthesize human pancreatic transcription factors' mRNAs: Pdx1, Ngn3, and MafA. We also aimed to assess their expression efficiency in cultured cells. First we ligated the open reading frames of these genes to modified 5′ and 3′ UTRs. Ligation products were then cloned into a plasmid and subjected to in vitro transcription to get their corresponding mRNAs. Modified mRNAs were subsequently examined for their quality, quantity, and protein translation efficiency. In vitro-transcribed mRNAs were successfully transfected to the human fibroblast cells. Efficiently translated proteins were correctly localized into the nucleus. It is concluded that pancreatic mRNAs designed and in vitro-transcribed in this study can be used in further direct beta cell reprogramming studies. [ABSTRACT FROM AUTHOR]

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 me 1 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 me 1 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., Competing Interests: The authors have no competing interests to declare., (© 2023 The Authors.)

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation'Pdx1, Neurogenin3, and MafA'efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2′-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2′-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

Synthetic mRNAs are rising rapidly as alternative therapeutic agents for delivery of proteins. However, the practical use of synthetic mRNAs has been restricted by their low cellular stability as well as poor protein production efficiency. The key roles of poly(A) tail on mRNA biology inspire us to explore the optimization of tail sequence to overcome the aforementioned limitations. Here, the systematic substitution of non-A nucleotides in the tails revealed that cytidine-containing tails can substantially enhance the protein production rate and duration of synthetic mRNAs both in vitro and in vivo . Such C-containing tails shield synthetic mRNAs from deadenylase CCR4-NOT transcription complex, as the catalytic CNOT proteins, especially CNOT6L and CNOT7, have lower efficiency in trimming of cytidine. Consistently, these enhancement effects of C-containing tails were observed on all synthetic mRNAs tested and were independent of transfection reagents and cell types. As the C-containing tails can be used along with other mRNA enhancement technologies to synergically boost protein production, we believe that these tails can be broadly used on synthetic mRNAs to directly promote their clinical applications., Competing Interests: All authors hereby declare no conflict of interest relating to this manuscript., (© 2022 The Author(s).)

Advances in mRNA synthesis and lipid nanoparticles technologies have helped make mRNA therapeutics and vaccines a reality. The 5' cap structure is a crucial modification required to functionalize synthetic mRNA for efficient protein translation in vivo and evasion of cellular innate immune responses. The extent of 5' cap incorporation is one of the critical quality attributes in mRNA manufacturing. RNA cap analysis involves multiple steps: generation of predefined short fragments from the 5' end of the kilobase-long synthetic mRNA molecules using RNase H, a ribozyme or a DNAzyme, enrichment of the 5' cleavage products, and LC-MS intact mass analysis. In this paper, we describe (1) a framework to design site-specific RNA cleavage using RNase H; (2) a method to fluorescently label the RNase H cleavage fragments for more accessible readout methods such as gel electrophoresis or high-throughput capillary electrophoresis; (3) a simplified method for post-RNase H purification using desthiobiotinylated oligonucleotides and streptavidin magnetic beads followed by elution using water. By providing a design framework for RNase H-based RNA 5' cap analysis using less resource-intensive analytical methods, we hope to make RNA cap analysis more accessible to the scientific community., (© 2022 Chan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Glioblastoma (GBM) is characterized as having high molecular heterogeneity and complexity, which can be well revealed by genomic study. A truly effective treatment for GBM should flexibly address its heterogeneities, complexity, and strong drug resistance. This study was performed to explore the effectiveness of an mRNA-based therapeutic strategy using in vitro synthesized PTEN-mRNA and TRAIL-mRNA in tumor cells derived from PTEN-deletion patients. The PTEN gene alterations were revealed by whole-exome sequencing of three paired clinical GBMs and selected as the therapy target. Patient-derived primary glioblastoma stem cells (GBM2) and a DBTRG-cell-derived xenograft were used to detect mRNA's cytotoxicity in vitro and tumor suppression in vivo . Following the successful in vitro synthesis of PTEN-mRNA and TRAIL-mRNA, the combinational treatment of PTEN-mRNA and TRAIL-mRNA significantly suppressed tumor growth compared with treatment with PBS (96.4%), PTEN-mRNA (89.7%), and TRAIL-mRNA (84.5%). The combinational application of PTEN-mRNA and TRAIL-mRNA showed synergistic inhibition of tumor growth, and the JNK pathway might be the major mechanism involved. This study provided a basis for an mRNA-based therapeutic strategy to be developed into an effective patient-tailored treatment for GBM., Competing Interests: The authors have declared that no competing interest exists., (© 2022 The Authors.)

The structure of synthetic mRNAs as used in vaccination against cancer and infectious diseases contain specifically designed caps followed by sequences of the 5′ untranslated repeats of β-globin gene. The strategy for successful design of synthetic mRNAs by chemically modifying their caps aims to increase resistance to the enzymatic deccapping complex, offer a higher affinity for binding to the eukaryotic translation initiation factor 4E (elF4E) protein and enforce increased translation of their encoded proteins. However, the cellular homeostasis is finely balanced and obeys to specific laws of thermodynamics conferring balance between complexity and growth rate in evolution. An overwhelming and forced translation even under alarming conditions of the cell during a concurrent viral infection, or when molecular pathways are trying to circumvent precursor events that lead to autoimmunity and cancer, may cause the recipient cells to ignore their differential sensitivities which are essential for keeping normal conditions. The elF4E which is a powerful RNA regulon and a potent oncogene governing cell cycle progression and proliferation at a post-transcriptional level, may then be a great contributor to disease development. The mechanistic target of rapamycin (mTOR) axis manly inhibits the elF4E to proceed with mRNA translation but disturbance in fine balances between mTOR and elF4E action may provide a premature step towards oncogenesis, ignite pre-causal mechanisms of immune deregulation and cause maturation (aging) defects. [ABSTRACT FROM AUTHOR]

Owing to messenger RNA's unique biological advantages, it has received increasing attention to be used as a therapeutic, known as mRNA-based gene therapy. It is critical to have an ideal strategy of mRNA gene therapy for glioma, which grows in a special environment. In the present study, we screened out a safe and efficient transfection reagent for intracranial delivery of synthetic mRNA in mouse brain. First, in order to analyze the effect of different transfection reagents on the intracranial delivery of mRNA, the synthetic luciferase mRNA was wrapped with two different transfection reagents and microinjected into the brain at the fixed point. The expression status of delivered mRNA was monitored by a small animal imaging system. The possible reagent-induced biological toxicity was evaluated by behavioral and blood biochemical measurements. Then, to test the therapeutic effect of our intracranial delivery mRNA model on glioma, synthetic modified tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA was used as an example of therapeutic application. This model demonstrated that synthetic mRNA could be successfully delivered into the brain using commercially available transfection reagents, and TransIT-mRNA showed better results than in vivo -jetPEI kit. This model can be applied in precise targeting and personalized gene therapy of glioma., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The photo-regulation of transgene expression is one effective approach in mammalian synthetic biology due to its high spatial and temporal resolution. While DNAs are mainly used as vectors, modified RNAs (modRNAs) are also useful for medical applications of synthetic biology, because they can avoid insertional mutagenesis and immunogenicity. However, the optogenetic control of modRNA-delivered transgenes is much more difficult than that of DNA-delivered transgenes. Here, we develop two types of photo-controllable translational activation systems that are compatible with modRNAs. One is composed of a heterodimerization domain-fused split translational activator protein and a photocaged heterodimerizer. The other is composed of a destabilizing domain-fused translational activator protein and a photocaged stabilizer. The destabilized type can be used for not only translational activation but also translational repression of the modRNAs. These photo-controllable translation systems will expand the application of mammalian synthetic biology research. [Display omitted] • Two photo-controllable translational activators for synthetic mRNAs are developed • One is a split translational activator that is reconstituted by light • The other is a destabilized translational activator that is stabilized by light • The destabilized type enables both translational activation and repression Nakanishi et al. develop photo-controllable translational regulation systems for synthetic mRNAs containing modified nucleosides. These systems are composed of photocaged ligands, ligand-responsive domain-fused translational regulatory proteins, and target synthetic mRNAs containing a protein binding motif. They enable light-inducible translational activation or repression of the target mRNAs in mammalian cells. [ABSTRACT FROM AUTHOR]

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Musculoskeletal disorders, such as osteoarthritis and intervertebral disc degeneration are causes of morbidity, which concomitantly burdens the health and social care systems worldwide, with massive costs. Link N peptide has recently been described as a novel anabolic stimulator for intervertebral disc repair. In this study, we analyzed the influence on anabolic response, by delivering synthetic Link N encoding mRNA into primary human chondrocytes and mesenchymal stromal cells (SCP1 cells). Furthermore, both cell types were seeded on knitted titanium scaffolds, and the influence of Link N peptide mRNA for possible tissue engineering applications was investigated. Synthetic modified Link N mRNA was efficiently delivered into both cell types and cell transfection resulted in an enhanced expression of aggrecan, Sox 9, and type II collagen with a decreased expression of type X collagen. Interestingly, despite increased expression of BMP2 and BMP7, BMP signaling was repressed and TGFβ signaling was boosted by Link N transfection in mesenchymal stromal cells, suggesting possible regulatory mechanisms. Thus, the exogenous delivery of Link N peptide mRNA into cells augmented an anabolic response and thereby increased extracellular matrix synthesis. Considering these findings, we suppose that the cultivation of cells on knitted titanium scaffolds and the exogenous delivery of Link N peptide mRNA into cells could mechanically support the stability of tissue-engineered constructs and improve the synthesis of extracellular matrix by seeded cells. This method can provide a potent strategy for articular cartilage and intervertebral disc regeneration. [ABSTRACT FROM AUTHOR]

The discovery that ordinary skin cells can be turned into pluripotent stem cells by the forced expression of defined factors has raised hopes that personalized regenerative treatments based on immunologically compatible material derived from a patient's own cells might be realized in the not-too-distant future. A major barrier to the clinical use of induced pluripotent stem cells (iPSCs) was initially presented by the need to employ integrating viral vectors to express the factors that induce an embryonic gene expression profile, which entails potentially oncogenic alteration of the normal genome. Several "non-integrating" reprogramming systems have been developed over the last decade to address this problem. Among these techniques, mRNA reprogramming is the most unambiguously "footprint-free," most productive, and perhaps the best suited to clinical production of stem cells. Herein, we discuss the origins of the mRNA-based reprogramming system, its benefits and drawbacks, recent technical improvements that simplify its application, and the status of current efforts to industrialize this approach to mass-produce human stem cells for the clinic., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The 4-vinylpyrimidin-2-one nucleoside (T-vinyl) forms a cross-link with the RNA containing uracil at the complementary site at a high reaction rate. To obtain the stable T-vinyl derivative so that its reactivity is protected until it access to the target site, several derivatives were investigated, and the 2-thiopyridinyl- and 2-thiopyrimidinyl T-vinyl derivatives were determined to be good candidates. The 2-thiopyrimidinyl T-vinyl derivative was found to more efficiently cross-link with mRNA albeit having a better stability than the 2-thiopyridinyl T-vinyl derivative. The investigation using the luciferase (Luc) mRNA, the synthetic mRNA and non-cellular translation system revealed that the translation is terminated at the end of the cross-linked duplex between the mRNA and the oligoribonucleotide (ORN). Thus, the 2-thiopyrimidinyl T-vinyl derivative has successfully demonstrated both a good stability and high efficiency for the cross-linking reaction, and expanded its applicability in biological applications.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Neural stem cells (NSCs) are a prominent cell source for understanding neural pathogenesis and for developing therapeutic applications to treat neurodegenerative disease because of their regenerative capacity and multipotency. Recently, a variety of cellular reprogramming technologies have been developed to facilitate in vitro generation of NSCs, called induced NSCs (iNSCs). However, the genetic safety aspects of established virus-based reprogramming methods have been considered, and non-integrating reprogramming methods have been developed. Reprogramming with in vitro transcribed (IVT) mRNA is one of the genetically safe reprogramming methods because exogenous mRNA temporally exists in the cell and is not integrated into the chromosome. Here, we successfully generated expandable iNSCs from human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via transfection with IVT mRNA encoding SOX2 (SOX2 mRNA) with properly optimized conditions. We confirmed that generated human UCB-MSC-derived iNSCs (UM-iNSCs) possess characteristics of NSCs, including multipotency and self-renewal capacity. Additionally, we transfected human dermal fibroblasts (HDFs) with SOX2 mRNA. Compared with human embryonic stem cell-derived NSCs, HDFs transfected with SOX2 mRNA exhibited neural reprogramming with similar morphologies and NSC-enriched mRNA levels, but they showed limited proliferation ability. Our results demonstrated that human UCB-MSCs can be used for direct reprogramming into NSCs through transfection with IVT mRNA encoding a single factor, which provides an integration-free reprogramming tool for future therapeutic application.

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, synthetic mRNA-based applications to produce desired exogenous proteins in cells have been gaining importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs and, thereby, fail to target desired cells. Thus, local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables patient-friendly, painless, and efficient delivery of synthetic mRNAs into the dermis; thus, this method could be applied for local treatment of different skin diseases as well as for vaccination and immunotherapy., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)