The clinical management of acute lung injury (ALI) remains constrained by pulmonary mucus barriers that impede therapeutic penetration and complex pathophysiology involving oxidative stress amplification and dysregulated inflammation. We report an engineered nanotherapeutic platform featuring poloxamer 124-functionalized cerium-tannic acid coordination metal-organic frameworks-based nanozymes (Ce-TA@124) that surmount the aforementioned biological barriers through a multi-tiered therapeutic strategy. Demonstrating superior efficacy to dexamethasone benchmarks, this system achieves: (1) synergistic reactive oxygen species scavenging enabled by Ce3+/Ce4+ interconversion and excellent reducing capacity from polyphenolic TA; (2) macrophage repolarization toward M2 pro-healing phenotypes via cytokine storm mitigation (IL-1β, IL-6, TNF-α); and (3) dual cell death pathway inhibition through apoptosis suppression and GPX4-mediated ferroptosis blockade. The mucus-penetrating poloxamer 124 corona enables rapid diffusion (4-fold higher) and alveolar deposition (2.67-fold enhancement) while preserving biocompatibility. Mechanistic profiling reveals TLR4-MAPK-NF-κB signaling axis modulation as the cornerstone of anti-inflammatory action, coupled with Nrf2 pathway activation for redox homeostasis restoration. Through comprehensive in vivo validation demonstrating a 65% reduction in lung injury scores and complete survival protection in ALI-acute respiratory distress syndrome models, this work establishes Ce-TA@124 as a paradigm-shifting nanoplatform that addresses both pathological drivers and delivery challenges in pulmonary inflammation management, with significant clinical translation potential.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the atypical ATP-binding cassette (ABC) family that functions as a phosphorylation-regulated epithelial anion channel. Cystic fibrosis (CF) is characterised by variants in the CFTR gene that lead to impaired epithelial chloride-ion transport and increased mucus viscosity. Although CFTR modulators such as Trikafta® have transformed the care of many CF patients, individuals harbouring rare CFTR variants still have no effective treatment options. In this study, we used primary air-liquid interface (ALI) airway cultures obtained from 21 CF patients (pwCF) and 21 healthy controls (HC) to evaluate the therapeutic efficacy of CFTR restoration based on chitosan-mediated CFTR mRNA and modulators. While modulators restored CFTR channel function in most cultures derived from CF patients, those with class I or other rare variants showed no improvement. Chitosan-mediated CFTR mRNA delivery successfully restored CFTR function in ALI cultures of patients carrying rare CFTR variants with limited or no observed clinical response to modulator therapy, assessed by electrophysiology using our newly developed Multi Transepithelial Current Clamp (MTECC) Ussing chamber. This was then confirmed by morphological visualisation of CFTR protein expression in modulator-responsive patient samples using immunofluorescence (IF) staining. IF revealed an increase in CFTR signal and the restoration of epithelial barrier integrity following chitosan-mRNA and modulator treatment as a secondary outcome alongside CFTR functional measurements. Notably, MUC5AC expression, a major gel-forming mucin expressed by airway goblet cells and mucus viscosity were elevated in CF cultures, but were markedly reduced following successful intervention, approaching the levels seen in HCs. These findings establish the potential of chitosan-mRNA delivery as a therapeutic approach for CF patients, particularly those who do not respond to modulators. They also provide a practical, comparative evaluation of advanced mRNA-based treatments in patient-derived airway models.

The ongoing emergence of novel SARS-CoV-2 variants due to viral mutations poses a persistent challenge to the efficacy of existing vaccines. To address this challenge, we engineered and comprehensively tested three optimized mRNA vaccine candidates, evaluating the kinetics, quality, and magnitude of antibody responses as well as antigen-specific T cell immunity during a prime-boost vaccination regimen in mice. Among the tested candidates, TP2A encoding secreted receptor-binding domains (RBDs) derived from SARS-CoV-2 wild type (WT), Delta and Omicron variants demonstrated superior immunogenicity, inducing an early IgG2a-dominated antibody response against distinct SARS-CoV-2 spike (S) glycoprotein variants. In addition, TP2A elicited IFN-γ-producing T cells in both spleen and draining lymph nodes and antigen-specific cytotoxic T lymphocytes. Notably, beyond broad immunity induced by the vaccine, TP2A functions as a modular platform, thus enabling flexible antigen assembly and rapid vaccine adaptation to newly emerging variants or even other viral pathogens. These findings position TP2A as a promising next-generation mRNA vaccine candidate.

The development of mucosal mRNA vaccines is promising but extremely challenging. Major efforts have been focused on optimizing delivery systems, but it is still unknown whether the intrinsic quality of mRNA components significantly impacts the potency of airway-inoculated mRNA vaccines. Here, we systematically demonstrate that mucosal mRNA vaccine requires higher standards of purification and a tailor-designed sequence to fulfill its potency compared to its parenteral-route-inoculated counterpart. Double-stranded RNA (dsRNA) contaminants are prone to trigger the innate immune response in the airway that activates the mRNA degradation mechanism, thereby diminishing mRNA expression and subsequent antigen-specific immune responses. To address these challenges, we developed a strategy that combines optimized untranslated regions (UTRs) screened from endogenous genes of pulmonary cells with affinity chromatography-based purification, which effectively removed dsRNA contaminants. The optimized mRNA administered via the airway route not only demonstrated superior protein expression (30-fold increase) and reduced inflammation in the lung but also promoted robust adaptive immunity comprising significantly elevated systemic, cellular, and mucosal immune responses. This was in stark contrast to the intramuscular-injected counterpart that displayed less-pronounced benefits. Our findings offer new insights into the development of mucosal mRNA therapeutics from an overlooked but crucial perspective of optimizing mRNA components.

The COVID-19 pandemic has spotlighted the potential of in vitro transcribed (IVT) mRNA vaccines with their demonstrated efficacy, safety, cost-effectiveness, and rapid manufacturing. Numerous IVT mRNA vaccines are now under clinical trials for a range of targets, including infectious diseases, cancers, and genetic disorders. Despite their promise, IVT mRNA vaccines face hurdles such as limited expression levels, nonspecific targeting beyond the liver, rapid degradation, and unintended immune activation. Overcoming these challenges is crucial to harnessing the full therapeutic potential of IVT mRNA vaccines for global health advancement. This review provides a comprehensive overview of the latest research progress and optimization strategies for IVT mRNA molecules and delivery systems, including the application of artificial intelligence (AI) models and deep learning techniques for IVT mRNA structure optimization and mRNA delivery formulation design. We also discuss recent development of the delivery platforms, such as lipid nanoparticles (LNPs), polymers, and exosomes, which aim to address challenges related to IVT mRNA protection, cellular uptake, and targeted delivery. Lastly, we offer insights into future directions for improving IVT mRNA vaccines, with the hope to spur further progress in IVT mRNA vaccine research and development.

There continues to be a dearth of competent inhalable mRNA delivery although it holds great potential for addressing a wide variety of refractory diseases. The huge advances seen with parenteral-administered lipid nanoparticle (LNP) have not been translated into nebulized mRNA delivery due to the aggressive nebulization process and insurmountable barriers inherent to respiratory mucosa. Here, we show amphiphilic block copolymers revealed by machine learning (ML) can spontaneously form stabilized nanoparticles (PoLixNano) with the lipids components of LNP and simultaneously impart the PoLixNano with "shield" (shear force-resistant) and "spear" (pulmonary barriers-penetrating abilities) capabilities. We present a ML approach that leverages physicochemical properties and inhaled mRNA transfection profiles of a chemically diverse library of polymeric components to validate the integration of "shield" and "spear" properties as highly predictive indicators of transfection efficiency. This quantitative structure-mRNA transfection prediction (QSMTP) model identifies top-performing amphiphilic-copolymers from more than 10000 candidates and suggests their mucus-penetrating ability outweights the shear force-resistant property in contributing to efficient mRNA transfection. The optimized PoLixNano substantially outperforms the LNP counterpart and mediates up to 1114-times higher levels of mRNA transfection in animal models with negligible toxicities. The PoLixNano promotes overwhelming SARS-CoV-2 antigen-specific sIgA antibody secretion and expansion of TRM cells which collectively confers 100% protection in mice against lethal SARS-CoV-2 challenges and blocks the transmission of Omicron variant between hamsters. PoLixNano also displays versatile therapeutic potential in lung carcinoma and cystic fibrosis models. Our study provides new insights for designing delivery platforms of aerosol-inhaled mRNA therapeutics with clinical translation potential. ### Competing Interest Statement The authors have declared no competing interest.

The coronavirus disease 2019 (COVID-19) pandemic poses a disruptive impact on public health and the global economy. Fortunately, the development of COVID-19 vaccines based on in vitro-transcribed messenger RNA (IVT mRNA) has been a breakthrough in medical history, benefiting billions of people with its high effectiveness, safety profile, and ease of large-scale production. This success is the result of decades of continuous RNA research, which has led to significant improvements in the stability and expression level of IVT mRNA through various approaches such as sequence optimization and improved preparation processes. IVT mRNA sequence optimization has been shown to have a positive effect on enhancing the mRNA expression level. The innovation of IVT mRNA purification technology is also indispensable, as the purity of IVT mRNA directly affects the success of downstream vaccine preparation processes and the potential for inducing unwanted side effects in therapeutic applications. Despite the progress made, challenges related to IVT mRNA sequence design and purification still require further attention to enhance the quality of IVT mRNA in the future. In this review, we discuss the latest innovative progress in IVT mRNA design and purification to further improve its clinical efficacy.

The ongoing SARS-CoV-2 pandemic represents a brutal reminder of the continual threat of mucosal infectious diseases. Mucosal immunity may provide robust protection at the predominant sites of SARS-CoV-2 infection. However, it remains unclear whether respiratory mucosal administration of DNA vaccines could confer protective immune responses against SARS-CoV-2 challenge due to insurmountable barriers posed by the airway. Here, we applied self-assembled peptide-poloxamine nanoparticles with mucus-penetrating properties for pulmonary inoculation of a COVID-19 DNA vaccine (pSpike/PP-sNp). The pSpike/PP-sNp not only displays superior gene transfection and favorable biocompatibility in the mouse airway, but also promotes a tripartite immunity consisting of systemic, cellular, and mucosal immune responses that are characterized by mucosal IgA secretion, high levels of neutralizing antibodies, and resident memory phenotype T-cell responses in the lungs of mice. Most importantly, immunization with pSpike/PP-sNp completely eliminates SARS-CoV-2 infection in both upper and lower respiratory tracts and enables 100% survival rate of mice following lethal SARS-CoV-2 challenge. Our findings indicate PP-sNp is a promising platform in mediating DNA vaccines to elicit all-around mucosal immunity against SARS-CoV-2.

Recent advancements in the field of in vitro transcribed mRNA (IVT-mRNA) vaccination have attracted considerable attention to such vaccination as a cutting-edge technique against infectious diseases including COVID-19 caused by SARS-CoV-2. While numerous pathogens infect the host through the respiratory mucosa, conventional parenterally administered vaccines are unable to induce protective immunity at mucosal surfaces. Mucosal immunization enables the induction of both mucosal and systemic immunity, efficiently removing pathogens from the mucosa before an infection occurs. Although respiratory mucosal vaccination is highly appealing, successful nasal or pulmonary delivery of nucleic acid-based vaccines is challenging because of several physical and biological barriers at the airway mucosal site, such as a variety of protective enzymes and mucociliary clearance, which remove exogenously inhaled substances. Hence, advanced nanotechnologies enabling delivery of DNA and IVT-mRNA to the nasal and pulmonary mucosa are urgently needed. Ideal nanocarriers for nucleic acid vaccines should be able to efficiently load and protect genetic payloads, overcome physical and biological barriers at the airway mucosal site, facilitate transfection in targeted epithelial or antigen-presenting cells, and incorporate adjuvants. In this review, we discuss recent developments in nucleic acid delivery systems that target airway mucosa for vaccination purposes.

Hintergrund Seit Beginn des Ukrainekrieges mussten über sechs Millionen Ukrainer:innen ihr Land verlassen. Die Tuberkulose-Inzidenz ist dort mit 73/100.000 Einwohnern eine der höchsten innerhalb Europas. Nicht selten handelt es sich hierbei um Patienten mit einer MDR (multidrug resistant)- oder XDR (extensively drug-resistant)-Tuberkulose.

In vitro transcribed messenger RNA (mRNA) vaccines have displayed enormous potential in fighting against the coronavirus disease 2019 (COVID-19) pandemic. Efficient and safe delivery systems must be included in the mRNA vaccines due to the fragile properties of mRNA. A self-assembled peptide-poloxamine nanoparticle (PP-sNp) gene delivery system is specifically designed for the pulmonary delivery of nucleic acids and displays promising capabilities in mediating successful mRNA transfection. Here, an improved method for preparing PP-sNp is described to elaborate on how the PP-sNp encapsulates Metridia luciferase (MetLuc) mRNA and successfully transfects cultured cells. MetLuc-mRNA is obtained by an in vitro transcription process from a linear DNA template. A PP-sNp is produced by mixing synthetic peptide/poloxamine with mRNA solution using a microfluidic mixer, allowing for the self-assembly of PP-sNp. The charge of PP-sNp is subsequently evaluated by measuring the zeta potential. Meanwhile, the polydispersity and hydrodynamic size of PP-sNp nanoparticles are measured using dynamic light scattering. The mRNA/PP-sNp nanoparticles are transfected into cultured cells, and supernatants from the cell culture are assayed for luciferase activity. The representative results demonstrate their capacity for in vitro transfection. This protocol may shed light on developing next-generation mRNA vaccine delivery systems.

In vitro-transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The nebulized formulations of IVT-mRNA-encoding alpha-1-antitrypsin (A1AT-mRNA) would be a highly acceptable and tolerable remedy for the protein replacement therapy for alpha-1-antitrypsin deficiency in the future. Here we show that lipoplexes containing A1AT-mRNA prepared in optimum conditions could successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial number of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and could successfully inhibit the enzyme activity of trypsin as well as elastase. Our data indicate that aerosolization of A1AT-mRNA therapy constitutes a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT, while bringing along the unique advantages of IVT-mRNA.

Pediatric and adolescent rehabilitation plays a numerically subordinate role in the medical care of children and adolescents; however, in addition to inpatient and outpatient acute care it is a very important pillar of the treatment for chronically ill children and adolescents and their families. This is particularly true for complex allergic diseases, such as severe bronchial asthma, atopic dermatitis or food allergies. The aim of this article is to show the framework conditions and possibilities of rehabilitation for children and adolescents based on the valid regulations and to illustrate the treatment of patients with allergic diseases based on case examples. In addition to the time factor and the search for trigger substances, intensive education measures by an interdisciplinary team and nonpharmaceutical aspects of the treatment play a decisive role in coping with these diseases.

Background: In vitro transcribed (IVT) mRNA has come into focus in recent years as a potential therapeutic approach for the treatment of genetic diseases. The pulmonary delivery of IVT-mRNA encoding alpha-1-antitrypsin (A1AT) is a promising strategy for protein replacement therapy of alpha-1-antitrypsin deficiency (AATD). The nebulized A1AT-mRNA formulations would be a highly acceptable and tolerable remedy for the AATD patients in the future. Method: we first optimized parameters that were influencing the transfection efficiency of formulations containing IVT-mRNA and Lipofectamine2000 based on human bronchial epithelial cells transfection. Cell viability was evaluated by performing MTT assay after transfection with different IVT-mRNA lipoplexes. Functional analysis was employed to assess the biological function of A1AT proteins produced from optimized formulations using anti-trypsin assay and anti-elastase assay. Results: Lipoplexes prepared by IVT-mRNA encoding A1AT (A1AT-mRNA) in optimum conditions can successfully transfect human bronchial epithelial cells without significant toxicity. A reduction in transfection efficiency was observed for aerosolized lipoplexes that can be partially overcome by increasing the initial amount of components. A1AT produced from cells transfected by nebulized A1AT-mRNA lipoplexes is functional and can successfully inhibit the enzyme activity of trypsin as well as elastase. Conclusion: Aerosolization of A1AT-mRNA therapeutic constitute a potentially powerful means to transfect airway epithelial cells with the purpose of producing functional A1AT while bringing along the unique advantages of IVT-mRNA.

In Deutschland werden etwa 43.000 Kinder und Jugendliche/Jahr mit chronischen Krankheiten in rund 50 Rehabilitationskliniken stationär betreut (durchschnittliche Behandlungsdauer etwa 30 Tage). Die therapeutischen Maßnahmen richten sich neben der Optimierung der medikamentösen Therapie auf den verbesserten Umgang mit der chronischen Erkrankung, um die Teilhabe an Schule und späterer Erwerbstätigkeit zu ermöglichen bzw. zu sichern. Inhalte sind aktive und passive Behandlungselemente, Schulungen, ergänzende und Verlaufsdiagnostik und ein in den Hauptfächern begleitender Schulunterricht. Mitaufgenommene Familienangehörige/Begleitpersonen sind in den therapeutischen Prozess einzubeziehen. Studien zeigen, dass stationäre Rehabilitationsmaßnahmen die klinische Symptomatik chronisch kranker Kinder und Jugendlicher deutlich verbessern. Diese stellen neben ambulanter und akutmedizinischer Versorgung eine wichtige Säule in der Versorgung chronisch kranker Kinder und Jugendlicher in Deutschland dar.

Die Gentherapie als Behandlungskonzept bei menschlichen Erkrankungen hat in den letzten Jahren Einzug in die klinische Medizin gehalten. Bislang sind in Europa bereits 6 Gentherapeutika zugelassen worden. Weitere Präparate, die in den USA bereits zugelassen sind, stehen auch in Europa kurz davor. Durch die Gentherapie konnten Verfahren der onkologischen Immuntherapie verbessert oder erst möglich gemacht werden. Kausale Therapien für eine große Zahl angeborener Erkrankungen werden entwickelt. Gentherapien für die Adenosindesaminasedefizienz und die β‑Thalassämie sind bereits verfügbar. Bei der großen Zahl derzeit in der Durchführung befindlicher klinischer Gentherapiestudien, die bereits in der Phase II/III sind, ist in naher Zukunft mit der Zulassung weiterer Gentherapeutika zu rechnen. Die schweren unerwarteten Nebenwirkungen, die im Rahmen der ersten Gentherapiestudien auftraten, konnten durch verbesserte Genvektorsysteme vermieden werden. Trotzdem stehen Patienten, die mit Gentherapeutika behandelt wurden, unter engmaschiger Kontrolle, da bestimmte virale Genvektoren zu einer insertionellen Mutagenese führen können. Neben der Genersatztherapie werden durch die Entwicklung sog. Genscheren neue Therapiekonzepte möglich, die zukünftig eine Genomeditierung erlauben, bis hin zur Korrektur einzelner Mutationen.

Every year approximately 43,000 children and adolescents with chronic diseases are treated as inpatients in around 50 rehabilitation clinics in Germany (average duration of treatment ca. 30 days). The therapeutic measures are based on the optimization of drug treatment and improvement of coping with the chronic disease in order to enable or ensure participation in school and later in gainful employment. The contents are active and passive treatment elements, patient education, supplementary and follow-up diagnostics and school lessons in the main subjects. Accompanying persons or relatives should be included in the therapeutic process. Studies have shown that inpatient rehabilitation measures can significantly improve the clinical symptoms in chronically ill children and adolescents. In addition to outpatient and acute medical care these represent an important pillar in the treatment of chronically ill children and adolescents in Germany.