Search

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.)

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).)

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.)

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.)

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 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.)

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.)