Bioinspired cellular membrane-derived vesicles for mRNA delivery.

The rapid advancement of mRNA as vaccines and therapeutic agents in the biomedical field has sparked hope in the fight against untreatable diseases. Successful clinical application of mRNA therapeutics largely depends on the carriers. Recently, a new and exciting focus has emerged on natural cell-derived vesicles. These nanovesicles offer many functions, including enhanced drug delivery capabilities and immune evasion, thereby presenting a unique and promising platform for the effective and safe delivery of mRNA therapeutics. In this study, we summarize the characteristics and properties of biomimetic delivery systems for mRNA therapeutics. In particular, we discuss the unique features of cellular membrane-derived vesicles (CDVs) and the combination of synthetic nanovesicles with CDVs.

Dual-mRNA Delivery Using Tumor Cell Lysate-Based Multifunctional Nanoparticles as an Efficient Colon Cancer Immunogene Therapy.

Background: Messenger RNA (mRNA)-based immunogene therapy holds significant promise as an emerging tumor therapy approach. However, the delivery efficiency of existing mRNA methods and their effectiveness in stimulating anti-tumor immune responses require further enhancement. Tumor cell lysates containing tumor-specific antigens and biomarkers can trigger a stronger immune response to tumors. In addition, strategies involving multiple gene therapies offer potential optimization paths for tumor gene treatments. Methods: Based on the previously developed ideal mRNA delivery system called DOTAP-mPEG-PCL (DMP), which was formed through the self-assembly of 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and methoxypoly (ethylene glycol)-b-poly (ε-caprolactone) (mPEG-PCL), we introduced a fused cell-penetrating peptide (fCPP) into the framework and encapsulated tumor cell lysates to form a novel nanovector, termed CLSV system (CLS: CT26 tumor cell lysate, V: nanovector). This system served a dual purpose of facilitating the delivery of two mRNAs and enhancing tumor immunogene therapy through tumor cell lysates. Results: The synthesized CLSV system had an average size of 241.17 nm and a potential of 39.53 mV. The CLSV system could not only encapsulate tumor cell lysates, but also deliver two mRNAs to tumor cells simultaneously, with a transfection efficiency of up to 60%. The CLSV system effectively activated the immune system such as dendritic cells to mature and activate, leading to an anti-tumor immune response. By loading Bim-encoded mRNA and IL-23A-encoded mRNA, CLSV/Bim and CLSV/IL-23A complexes were formed, respectively, to further induce apoptosis and anti-tumor immunity. The prepared CLSV/dual-mRNA complex showed significant anti-cancer effects in multiple CT26 mouse models. Conclusion: Our results suggest that the prepared CLSV system is an ideal delivery system for dual-mRNA immunogene therapy.

Alpha-galactosylceramide improves the potency of mRNA LNP vaccines against cancer and intracellular bacteria.

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8+ T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8+ T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections.

The impact of, and expectations for, lipid nanoparticle technology: From cellular targeting to organelle targeting.

The success of mRNA vaccines against COVID-19 has enhanced the potential of lipid nanoparticles (LNPs) as a system for the delivery of mRNA. In this review, we describe our progress using a lipid library to engineer ionizable lipids and promote LNP technology from the viewpoints of safety, controlled biodistribution, and mRNA vaccines. These advancements in LNP technology are applied to cancer immunology, and a potential nano-DDS is constructed to evaluate immune status that is associated with a cancer-immunity cycle that includes the sub-cycles in tumor microenvironments. We also discuss the importance of the delivery of antigens and adjuvants in enhancing the cancer-immunity cycle. Recent progress in NK cell targeting in cancer immunotherapy is also introduced. Finally, the impact of next-generation DDS technology is explained using the MITO-Porter membrane fusion-based delivery system for the organelle targeting of the mitochondria. We introduce a successful example of the MITO-Porter used in a cell therapeutic strategy to treat cardiomyopathy.

Advances in non-viral mRNA delivery to the spleen.

Developing safe and effective delivery strategies for localizing messenger RNA (mRNA) payloads to the spleen is an important goal in the field of genetic medicine. Accomplishing this goal is challenging due to the instability, size, and charge of mRNA payloads. Here, we provide an analysis of non-viral delivery technologies that have been developed to deliver mRNA payloads to the spleen. Specifically, our review begins by outlining the unique anatomy and potential targets for mRNA delivery within the spleen. Next, we describe approaches in mRNA sequence engineering that can be used to improve mRNA delivery to the spleen. Then, we describe advances in non-viral carrier systems that can package and deliver mRNA payloads to the spleen, highlighting key advances in the literature in lipid nanoparticle (LNP) and polymer nanoparticle (PNP) technology platforms. Finally, we provide commentary and outlook on how splenic mRNA delivery may afford next-generation treatments for autoimmune disorders and cancers. In undertaking this approach, our goal with this review is to both establish a fundamental understanding of drug delivery challenges associated with localizing mRNA payloads to the spleen, while also broadly highlighting the potential to use these genetic medicines to treat disease.

Multicomponent Synthesis of Imidazole-Based Ionizable Lipids for Highly Efficient and Spleen-Selective Messenger RNA Delivery.

The spleen emerges as a pivotal target for mRNA delivery, prompting a continual quest for specialized and efficient lipid nanoparticles (LNPs) designed to enhance spleen-selective transfection efficiency. Here we report imidazole-containing ionizable lipids (IMILs) that demonstrate a pronounced preference for mRNA delivery into the spleen with exceptional transfection efficiency. We optimized IMIL structures by constructing and screening a multidimensional IMIL library containing multiple heads, tails, and linkers to perform a structure-activity correlation analysis. Following high-throughput in vivo screening, we identified A3B7C2 as a top-performing IMIL in spleen-specific mRNA delivery via the formulated LNPs, achieving a remarkable 98% proportion of splenic transfection. Moreover, A3B7C2-based LNPs are particularly potent in splenic dendritic cell transfection. Comparative analyses revealed that A3B7C2-based LNPs achieved a notable 2.8-fold and 12.9-fold increase in splenic mRNA transfection compared to SM102 and DLin-MC3-DMA lipid formulations, respectively. Additionally, our approach yielded an 18.3-fold enhancement in splenic mRNA expression compared to the SORT method without introducing additional anionic lipids. Collectively, these IMILs highlight promising avenues for further research in spleen-selective mRNA delivery. This work offers valuable insights for the swift discovery and rational design of ionizable lipid candidates tailored for spleen-selective transfection, thereby facilitating the application of mRNA therapeutics in spleen-related interventions.

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 µM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

Emerging advances in delivery systems for mRNA cancer vaccines.

The success of lipid nanoparticles (LNPs) in treating COVID-19 promotes further research of mRNA vaccines for cancer vaccination. Aiming at overcoming the constraints of currently available mRNA carriers, various alternative nano-vectors have been developed for delivering tumor antigen encoding mRNA and showed versatility to induce potent anti-tumor immunity. The rationally designed nano-vaccines increase the immune activation capacity of the mRNA vaccines by promoting crucial aspects including mRNA stability, cellular uptake, endosomal escape and targeting of immune cells or organs. Herein, we summarized the research progress of various mRNA based nano-vaccines that have been reported for cancer vaccination, including LNPs, lipid enveloped hybrid nanoparticles, polymeric nanoparticles etc. Several strategies that have been reported for further enhancing the immune stimulation efficacy of mRNA nano-vaccines, including developing nano-vaccines for co-delivering adjuvants, combination of immune checkpoint inhibitors, and optimizing the injection routes for boosting immune responses, have been reviewed. The progress of mRNA nano-vaccines in clinical trials and the prospect of the mRNA vaccines for cancer vaccination are also discussed.

Interim analyses of a first-in-human phase 1/2 mRNA trial for propionic acidaemia.

Propionic acidaemia is a rare disorder caused by defects in the propionyl-coenzyme A carboxylase α or ß (PCCA or PCCB) subunits that leads to an accumulation of toxic metabolites and to recurrent, life-threatening metabolic decompensation events. Here we report interim analyses of a first-in-human, phase 1/2, open-label, dose-optimization study and an extension study evaluating the safety and efficacy of mRNA-3927, a dual mRNA therapy encoding PCCA and PCCB. As of 31 May 2023, 16 participants were enrolled across 5 dose cohorts. Twelve of the 16 participants completed the dose-optimization study and enrolled in the extension study. A total of 346 intravenous doses of mRNA-3927 were administered over a total of 15.69 person-years of treatment. No dose-limiting toxicities occurred. Treatment-emergent adverse events were reported in 15 out of the 16 (93.8%) participants. Preliminary analysis suggests an increase in the exposure to mRNA-3927 with dose escalation, and a 70% reduction in the risk of metabolic decompensation events among 8 participants who reported them in the 12-month pretreatment period.

mRNA-responsive two-in-one nanodrug for enhanced anti-tumor chemo-gene therapy.

The combination of chemotherapy and gene therapy holds great promise for the treatment and eradication of tumors. However, due to significant differences in physicochemical properties between chemotherapeutic agents and functional nucleic acid drugs, direct integration into a single nano-agent is hindered, impeding the design and construction of an effective co-delivery nano-platform for synergistic anti-tumor treatments. In this study, we have developed an mRNA-responsive two-in-one nano-drug for effective anti-tumor therapy by the direct self-assembly of 2'-fluoro-substituted antisense DNA against P-glycoprotein (2'F-DNA) and chemo drug paclitaxel (PTX). The 2'-fluoro modification of DNA could significantly increase the interaction between the therapeutic nucleic acid and the chemotherapeutic drug, promoting the successful formation of 2'F-DNA/PTX nanospheres (2'F-DNA/PTX NSs). Due to the one-step self-assembly process without additional carrier materials, the prepared 2'F-DNA/PTX NSs exhibited considerable loading efficiency and bioavailability of PTX. In the presence of endogenous P-glycoprotein mRNA, the 2'F-DNA/PTX NSs were disassembled. The released 2'F-DNA could down-regulate the expression of P-glycoprotein, which decreased the multidrug resistance of tumor cells and enhanced the chemotherapy effect caused by PTX. In this way, the 2'F-DNA/PTX NSs could synergistically induce the apoptosis of tumor cells and realize the combined anti-tumor therapy. This strategy might provide a new tool to explore functional intracellular co-delivery nano-systems with high bioavailability and exhibit potential promising in the applications of accurate diagnosis and treatment of tumors.

Cardiac delivery of modified mRNA using lipid nanoparticles: Cellular targets and biodistribution after intramyocardial administration.

Despite research efforts being made towards preserving (or even regenerating) heart tissue after an ischemic event, there is a lack of resources in current clinical treatment modalities for patients with acute myocardial infarction that specifically address cardiac tissue impairment. Modified messenger RNA (modRNA) presents compelling properties that could allow new therapeutic strategies to tackle the underlying molecular pathways that ultimately lead to development of chronic heart failure. However, clinical application of modRNA for the heart is challenged by the lack of effective and safe delivery systems. Lipid nanoparticles (LNPs) represent a well characterized class of RNA delivery systems, which were recently approved for clinical usage in mRNA-based COVID-19 vaccines. In this study, we evaluated the potential of LNPs for cardiac delivery of modRNA. We tested how variations in C12-200 modRNA-LNP composition affect transfection levels and biodistribution after intramyocardial administration in both healthy and myocardial-infarcted mice, and determined the targeted cardiac cell types. Our data revealed that LNP-mediated modRNA delivery outperforms the current state of the art (modRNA in citrate buffer) upon intramyocardial administration in mice, with only minor differences among the formulations tested. Furthermore, we determined both in vitro and in vivo that the cardiac cells targeted by modRNA-LNPs include fibroblasts, endothelial cells and epicardial cells, suggesting that these cell types could represent targets for therapeutic interference with these LNP formulations. These outcomes may serve as a starting point for LNP development specifically for therapeutic mRNA cardiac delivery applications.

One-Component Cationic Lipids for Systemic mRNA Delivery to Splenic T Cells.

Unlocking the full potential of mRNA immunotherapy necessitates targeted delivery to specific cell subsets in the spleen. Four-component lipid nanoparticles (LNPs) utilized in numerous clinical trials are primarily limited in hepatocyte and muscular targeting, highlighting the imperative demand for targeted and simplified non-liver mRNA delivery systems. Herein, we report the rational design of one-component ionizable cationic lipids to selectively deliver mRNA to the spleen and T cells with high efficacy. Unlike the tertiary amine-based ionizable lipids involved in LNPs, the proposed cationic lipids rich in secondary amines can efficiently deliver mRNA both in vitro and in vivo as the standalone carriers. Furthermore, these vectors facilitate efficacious mRNA delivery to the T cell subsets following intravenous administration, demonstrating substantial potential for advancing immunotherapy applications. This straightforward strategy extends the utility of lipid family for extrahepatic mRNA delivery, offering new insights into vector development beyond LNPs to further the field of precise mRNA therapy.

mRNA therapies: Pioneering a new era in rare genetic disease treatment.

Rare genetic diseases, often referred to as orphan diseases due to their low prevalence and limited treatment options, have long posed significant challenges to our medical system. In recent years, Messenger RNA (mRNA) therapy has emerged as a highly promising treatment approach for various diseases caused by genetic mutations. Chemically modified mRNA is introduced into cells using carriers like lipid-based nanoparticles (LNPs), producing functional proteins that compensate for genetic deficiencies. Given the advantages of precise dosing, biocompatibility, transient expression, and minimal risk of genomic integration, mRNA therapies can safely and effectively correct genetic defects in rare diseases and improve symptoms. Currently, dozens of mRNA drugs targeting rare diseases are undergoing clinical trials. This comprehensive review summarizes the progress of mRNA therapy in treating rare genetic diseases. It introduces the development, molecular design, and delivery systems of mRNA therapy, highlighting their research progress in rare genetic diseases based on protein replacement and gene editing. The review also summarizes research progress in various rare disease models and clinical trials. Additionally, it discusses the challenges and future prospects of mRNA therapy. Researchers are encouraged to join this field and collaborate to advance the clinical translation of mRNA therapy, bringing hope to patients with rare genetic diseases.

Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.

Leveraging the extensive surface area of the lungs for gene therapy, the inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Our results demonstrated the superiority of the MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of the nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful lung-specific mRNA transfection without observable signs of toxicity. This MAP may represent an advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.

A polyamino acid-based phosphatidyl polymer library for in vivo mRNA delivery with spleen targeting ability.

A qualified delivery system is crucial for the successful application of messenger RNA (mRNA) technology. While lipid nanoparticles (LNPs) are currently the predominant platform for mRNA delivery, they encounter challenges such as high inflammation and difficulties in targeting non-liver tissues. Polymers offer a promising delivery solution, albeit with limitations including low transfection efficiency and potential high toxicity. Herein, we present a poly(L-glutamic acid)-based phosphatidyl polymeric carrier (PLG-PPs) for mRNA delivery that combines the dual advantages of phospholipids and polymers. The PLGs grafted with epoxy groups were firstly modified with different amines and then with alkylated dioxaphospholane oxides, which provided a library of PLG polymers grafted with various phosphatidyl groups. In vitro studies proved that PLG-PPs/mRNA polyplexes exhibited a significant increase in mRNA expression, peaking 14 716 times compared to their non-phosphatidyl parent polymer. Impressively, the subset PA8-PL3 not only facilitated efficient mRNA transfection but also selectively delivered mRNA to the spleen instead of the liver (resulting in 69.73% protein expression in the spleen) once intravenously administered. This type of phosphatidyl PLG polymer library provides a novel approach to the construction of mRNA delivery systems especially for spleen-targeted mRNA therapeutic delivery.

Effect of Cholesterol Content of Lipid Composition in mRNA-LNPs on the Protein Expression in the Injected Site and Liver After Local Administration in Mice.

Delivery of messenger RNA (mRNA) using lipid nanoparticles (LNPs) is expected to be applied to various diseases following the successful clinical use of the mRNA COVID-19 vaccines. This study aimed to evaluate the effect of the cholesterol molar percentage of mRNA-LNPs on protein expression in hepatocellular carcinoma-derived cells and in the liver after intramuscular or subcutaneous administration of mRNA-LNPs in mice. For mRNA-LNPs with cholesterol molar percentages reduced to 10 mol% and 20 mol%, we formulated neutral charge particles with a diameter of approximately 100 nm and polydispersity index (PDI) <0.25. After the intramuscular or subcutaneous administration of mRNA-LNPs with different cholesterol molar percentages in mice, protein expression in the liver decreased as the cholesterol molar percentage in mRNA-LNPs decreased from 40 mol% to 20 mol% and 10 mol%, suggesting that reducing the cholesterol molar percentage in mRNA-LNPs decreases protein expression in the liver. Furthermore, in HepG2 cells, protein expression decreased as cholesterol in mRNA-LNPs was reduced by 40 mol%, 20 mol%, and 10 mol%. These results suggest that the downregulated expression of mRNA-LNPs with low cholesterol content in the liver involves degradation in systemic circulating blood and decreased protein expression after hepatocyte distribution.

Rapid Generation of Circulating and Mucosal Decoy Human ACE2 using mRNA Nanotherapeutics for the Potential Treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause lethal pulmonary damage in humans. It contains spike proteins on its envelope that bind to human angiotensin-converting enzyme 2 (hACE2) expressed on airway cells, enabling entry of the virus, and causing infection. The soluble form of hACE2 binds SARS-CoV-2 spike protein, prevents viral entry into target cells, and ameliorates lung injury; however, its short half-life limits therapeutic utilities. Here, synthetic mRNA is engineered to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. A novel lipid nanoparticle (LNP) is used for packaging and delivering mRNA to cells to produce hsACE2 proteins. Intravenously administered LNP delivers mRNA to hepatocytes, leading to the production of circulatory hsACE2 initiated within 2 h and sustained over several days. Inhaled LNP results in lung transfection and secretion of mucosal hsACE2 to lung epithelia, the primary site of entry and pathogenesis for SARS-CoV-2. Furthermore, mRNA-generated hsACE2 binds to the receptor-binding domain of the viral spike protein. Finally, hsACE2 effectively inhibits SARS-CoV-2 and its pseudoviruses from infecting host cells. The proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed to neutralize SARS-CoV-2 and open new treatment opportunities for coronavirus disease 2019 (COVID-19).

A single-administration therapeutic interfering particle reduces SARS-CoV-2 viral shedding and pathogenesis in hamsters.

The high transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a primary driver of the COVID-19 pandemic. While existing interventions prevent severe disease, they exhibit mixed efficacy in preventing transmission, presumably due to their limited antiviral effects in the respiratory mucosa, whereas interventions targeting the sites of viral replication might more effectively limit respiratory virus transmission. Recently, intranasally administered RNA-based therapeutic interfering particles (TIPs) were reported to suppress SARS-CoV-2 replication, exhibit a high barrier to resistance, and prevent serious disease in hamsters. Since TIPs intrinsically target the tissues with the highest viral replication burden (i.e., respiratory tissues for SARS-CoV-2), we tested the potential of TIP intervention to reduce SARS-CoV-2 shedding. Here, we report that a single, postexposure TIP dose lowers SARS-CoV-2 nasal shedding, and at 5 days postinfection, infectious virus shed is below detection limits in 4 out of 5 infected animals. Furthermore, TIPs reduce shedding of Delta variant or WA-1 from infected to uninfected hamsters. Cohoused "contact" animals exposed to infected, TIP-treated animals exhibited significantly lower viral loads, reduced inflammatory cytokines, no severe lung pathology, and shortened shedding duration compared to animals cohoused with untreated infected animals. TIPs may represent an effective countermeasure to limit SARS-CoV-2 transmission.

Early human B cell signatures of the primary antibody response to mRNA vaccination.

Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2-naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

Intravesical delivery of KDM6A-mRNA via mucoadhesive nanoparticles inhibits the metastasis of bladder cancer.

Lysine-specific demethylase 6A (KDM6A), also named UTX, is frequently mutated in bladder cancer (BCa). Although known as a tumor suppressor, KDM6A's therapeutic potential in the metastasis of BCa remains elusive. It also remains difficult to fulfill the effective up-regulation of KDM6A levels in bladder tumor tissues in situ to verify its potential in treating BCa metastasis. Here, we report a mucoadhesive messenger RNA (mRNA) nanoparticle (NP) strategy for the intravesical delivery of KDM6A-mRNA in mice bearing orthotopic Kdm6a-null BCa and show evidence of KDM6A's therapeutic potential in inhibiting the metastasis of BCa. Through this mucoadhesive mRNA NP strategy, the exposure of KDM6A-mRNA to the in situ BCa tumors can be greatly prolonged for effective expression, and the penetration can be also enhanced by adhering to the bladder for sustained delivery. This mRNA NP strategy is also demonstrated to be effective for combination cancer therapy with other clinically approved drugs (e.g., elemene), which could further enhance therapeutic outcomes. Our findings not only report intravesical delivery of mRNA via a mucoadhesive mRNA NP strategy but also provide the proof-of-concept for the usefulness of these mRNA NPs as tools in both mechanistic understanding and translational study of bladder-related diseases.