Improved tropoelastin synthesis in the skin by codon optimization and nucleotide modification of tropoelastin-encoding synthetic mRNA.

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.

Delivery of synthetic mRNAs for tissue regeneration.

In recent years, nucleic acid-based therapeutics have gained increasing importance as novel treatment options for disease prevention and treatment. Synthetic messenger RNAs (mRNAs) are promising nucleic acid-based drugs to transiently express desired proteins that are missing or defective. Recently, synthetic mRNA-based vaccines encoding viral proteins have been approved for emergency use against COVID-19. Various types of vehicles, such as lipid nanoparticles (LNPs) and liposomes, are being investigated to enable the efficient uptake of mRNA molecules into desired cells. In addition, the introduction of novel chemical modifications into mRNAs increased the stability, enabled the modulation of nucleic acid-based drugs, and increased the efficiency of mRNA-based therapeutic approaches. In this review, novel and innovative strategies for the delivery of synthetic mRNA-based therapeutics for tissue regeneration are discussed. Moreover, with this review, we aim to highlight the versatility of synthetic mRNA molecules for various applications in the field of regenerative medicine and also discuss translational challenges and required improvements for mRNA-based drugs.

Ubiquitin-specific protease USP36 knockdown impairs Parkin-dependent mitophagy via downregulation of Beclin-1-associated autophagy-related ATG14L.

Parkin is an ubiquitin ligase regulating mitochondrial quality control reactions, including the autophagic removal of depolarized mitochondria (mitophagy). Parkin-mediated protein ubiquitinations may be counteracted by deubiquitinating enzymes (DUBs). We conducted a high-content imaging screen of Parkin translocation to depolarized mitochondria after siRNA mediated silencing of each DUB in Parkin overexpressing HeLa cells. Knockdown of the ubiquitin-specific protease USP36 led to delayed Parkin translocation while only slightly disturbing the ubiquitination of mitochondrial proteins, but final autophagic elimination of mitochondria was severely disrupted. The localization of the nucleolar USP36 was not altered during mitophagy. However, the marker for transcriptional active chromatin, histone 2B Lys120 mono-ubiquitination was found reduced in USP36-silenced cells undergoing mitophagy. We observed a reduction of the mRNA and protein levels of Beclin-1 and its associated autophagy-related key regulator ATG14L in USP36 knockdown cells. Importantly, transfection of active ATG14L into USP36-silenced cells significantly restored Parkin-dependent mitophagy. We propose USP36 as regulator for the Parkin-dependent mitophagy at least in part via the Beclin-1-ATG14L pathway.

Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA.

The application of synthetic modified messenger RNA (mRNA) is a promising approach for the treatment of a variety of diseases and vaccination. In the past few years, different modifications of synthetic mRNA were applied to render the mRNA more stable and less immunogenic. However, the repeated application of synthetic mRNA still requires the suppression of immune activation to avoid cell death and to allow a sufficient production of exogenous proteins. Thus, the addition of type I interferon (IFN) inhibiting recombinant protein B18R is often required to avoid IFN response. In this study, the ability of B18R encoding mRNA to prevent the immune response of cells to the delivered synthetic mRNA was analyzed. The co-transfection of enhanced green fluorescent protein (eGFP) mRNA transfected fibroblasts with B18R encoding mRNA over 7-days resulted in comparable cell viability and eGFP protein expression as in the cells transfected with eGFP mRNA and incubated with B18R protein. Using qRT-PCR, significantly reduced expression of interferon-stimulated gene Mx1 was detected in the cells transfected with B18R mRNA and stimulated with IFNß compared to the cells without B18R mRNA transfection. Thereby, it was demonstrated that the co-transfection of synthetic mRNA transfected cells with B18R encoding mRNA can reduce the IFN response-related cell death and thus, improve the protein expression.

Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs-The Potential Role in Increasing Anabolic Response.

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 TGFß 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.

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.

Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility.

Hemocompatibility of blood-contacting biomaterials is one of the most important criteria for their successful in vivo applicability. Thus, extensive in vitro analyses according to ISO 10993-4 are required prior to clinical applications. In this review, we summarize essential aspects regarding the evaluation of the hemocompatibility of biomaterials and the required in vitro analyses for determining the blood compatibility. Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed. Furthermore, the surface of biomaterials are evaluated concerning attachment of blood cells, adsorption of proteins, and generation of thrombus and fibrin networks.

Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin.

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.

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.

Incorporation of Synthetic mRNA in Injectable Chitosan-Alginate Hybrid Hydrogels for Local and Sustained Expression of Exogenous Proteins in Cells.

The application of synthetic messenger RNA (mRNA) exhibits various advantages, such as expression of desired proteins in cells without genomic integration. In the field of tissue engineering, synthetic mRNAs could be also used to modulate the protein expression in implanted cells. Therefore, in this study, we incorporated synthetic humanized Gaussia luciferase (hGLuc) mRNA into alginate, chitosan, or chitosan-alginate hybrid hydrogels and analyzed the release of hGLuc mRNA from these hydrogels. After 3 weeks, 79% of the incorporated mRNA was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels. Due to the injectability, chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded hGLuc mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis. Therefore, HEK293 cells were incorporated into chitosan-alginate hydrogels containing mRNA transfection complexes and the luciferase activity in the supernatants was detected for up to 3 weeks. These results showed that the biodegradable chitosan-alginate hybrid hydrogels are promising delivery systems for sustained delivery of synthetic mRNAs into cells. Since chitosan-alginate hybrid hydrogels are injectable, the hydrogels can be simultaneously loaded with cells and the desired synthetic mRNA for exogenous protein synthesis and can be administered by minimally invasive local injection for tissue engineering applications.

The ubiquitin-conjugating enzymes UBE2N, UBE2L3 and UBE2D2/3 are essential for Parkin-dependent mitophagy.

Depolarized mitochondria are degraded by mitophagy in a process that depends on the Parkinson's disease gene products PINK1 and Parkin. This is accompanied by ubiquitylation of several mitochondrial substrates. The roles of E2 ubiquitin-conjugating enzymes (UBE2) in mitophagy are poorly understood. Here, we investigate a set of UBE2 enzymes that might regulate Parkin-mediated mitophagy. Knockdown of the E2 enzymes UBE2N, UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly reduced autophagic clearance of depolarized mitochondria. However, this did not interfere with mitochondrial PINK1 stabilization and Parkin translocation. UBE2N knockdown prevented specifically K63-linked ubiquitylation at mitochondrial sites. Nevertheless, polyubiquitin and p62 (officially known as SQSTM1) were still found on mitochondria after individual UBE2 knockdown. Knockdown of all of these UBE2s together significantly reduced mitochondrial polyubiquitylation and p62 recruitment. Moreover, reduced ubiquitylation of mitofusins, the mitochondrial import receptor subunits TOM20 and TOM70, the voltage-dependent anion channel protein 1 and Parkin was observed in cells silenced for all of these UBE2s. A version of Parkin with a mutation in the active site (C431S) failed to ubiquitylate these mitochondrial substrates even in the presence of UBE2s. We conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically contribute to Parkin-mediated mitophagy.