Exogenous mRNA Research Articles

Hematogenous Routing of Exogenous Messenger RNA Delivered Into the Amniotic Fluid

IntroductionTherapies based on exogenous messenger RNA (mRNA) administration have emerged as a powerful novel strategy for the actual or potential treatment of an assortment of diseases, including congenital surgical pathologies. We sought to determine whether the minimally invasive transamniotic route could be an alternative for prenatal mRNA delivery. MethodsPregnant Sprague–Dawley dams underwent laparotomy followed by volume-matched intra-amniotic injections in all their fetuses (n = 120) of either a suspension of a custom firefly luciferase mRNA encapsulated by a lipid- and synthetic cationic polymer-based composite, or of a suspension of the same encapsulation components without mRNA, on gestational day 17 (E17; term = E21-22). On E18, E19, E20, and E21, samples from 14 fetal anatomical sites and maternal serum were procured for the screening of mRNA incorporation by host cells by measurement of luciferase activity via microplate luminometry. Statistical analysis was by Mann–Whitney U-test, including Bonferroni-adjustment. ResultsOverall survival was 87.5% (105/120). Controlled by the encapsulating composite without mRNA, luciferase activity was detected in the animals that received encapsulated mRNA in the following fetal annexes: amniotic fluid, amnion, chorion, umbilical cord, and placenta (P = 0.033 to <0.001), as well as in the following fetal sites: liver, stomach, intestines, and lungs (P = 0.043-0.002). ConclusionsPackaged exogenous mRNA can be incorporated by the fetus at least at select anatomical sites after simple intra-amniotic administration in a rodent model. The pattern and chronology of mRNA incorporation are compatible with transplacental hematogenous routing, as well as with fetal swallowing/aspiration. Further study of transamniotic mRNA administration is warranted.

Canine adipose tissue-derived MSCs engineered with mRNA to overexpress TSG-6 and enhance the anti-inflammatory effects in canine macrophages.

Mesenchymal stem cells (MSCs) are useful agents in the treatment of various inflammatory diseases. The immunomodulatory effects of MSCs are largely related to their secretory properties. mRNA engineering emerged as a safe alternative to enhance the secretory function of MSCs. Optimization of the untranslated region (UTR) sequence is important for enhancing the translational efficiency of exogenous mRNAs. However, research on the optimization of UTR in canine MSCs has not yet been conducted. We aimed to identify the UTR sequence related to the expression efficiency of in vitro transcription (IVT) mRNA in canine MSCs and investigate whether mRNA-engineered MSCs that overexpress TSG-6 exhibit enhanced anti-inflammatory effects. Canine adipose tissue-derived (cAT)-MSCs were transfected with green fluorescence protein (GFP) mRNA with three different UTRs: canine hemoglobin subunit alpha-like 1 (HBA1), HBA2, and hemoglobin subunit beta-like (HBB). The translation efficacy of each mRNA was evaluated using relative fluorescence. TSG-6 mRNA was produced with the UTR optimized according to relative fluorescence results. cAT-MSCs were transfected with TSG-6 mRNA (MSCTSG-6), and TSG-6 expression was analyzed using real-time quantitative PCR, ELISA, and western blotting. To evaluate the anti-inflammatory effects of MSCsTSG-6, DH82 cells were co-cultured with MSCsTSG-6 or treated with dexamethasone, and changes in the expression of inflammatory cytokines were analyzed using qPCR. The highest fluorescence level was observed in the HBA1 UTR at 24 h post-transfection. TSG-6 mRNA transfection yielded high levels of TSG-6 in the cAT-MSCs. In DH82 cells co-cultured with MSCsTSG-6, the expression of inflammatory cytokines decreased compared to that in co-culturing with naïve MSCs and dexamethasone treatment. Optimization of the HBA1 UTR improved the translation efficiency of IVT mRNA in canine MSCs. cAT-MSCs engineered with TSG-6 mRNA effectively enhanced the anti-inflammatory effects of the MSCs when co-cultured with LPS-activated DH82 cells.

Recent Advances in the Lipid Nanoparticle-Mediated Delivery of mRNA Vaccines.

Lipid nanoparticles (LNPs) have recently emerged as one of the most advanced technologies for the highly efficient in vivo delivery of exogenous mRNA, particularly for COVID-19 vaccine delivery. LNPs comprise four different lipids: ionizable lipids, helper or neutral lipids, cholesterol, and lipids attached to polyethylene glycol (PEG). In this review, we present recent the advances and insights for the design of LNPs, as well as their composition and properties, with a subsequent discussion on the development of COVID-19 vaccines. In particular, as ionizable lipids are the most critical drivers for complexing the mRNA and in vivo delivery, the role of ionizable lipids in mRNA vaccines is discussed in detail. Furthermore, the use of LNPs as effective delivery vehicles for vaccination, genome editing, and protein replacement therapy is explained. Finally, expert opinion on LNPs for mRNA vaccines is discussed, which may address future challenges in developing mRNA vaccines using highly efficient LNPs based on a novel set of ionizable lipids. Developing highly efficient mRNA delivery systems for vaccines with improved safety against some severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains difficult.

Terminal hairpins improve protein expression in IRES-initiated mRNA in the absence of a cap and polyadenylated tail

Synthesizing mRNA in vitro is a standard and simple procedure. Adding the 5′ cap and 3′ polyadenylated (poly(A)) tail to make this mRNA functional for use as a vaccine or therapy increases the time and cost of production and usually decreases the yield, however. We designed mRNA that lacked a cap and poly(A) tail but included an internal ribosomal entry site (IRES) to initiate protein translation. To protect the 5′ and 3′ ends of mRNA from exonucleases, we added stable terminal hairpins. When compared against typical mRNA (i.e., mRNA that contained a cap and poly(A) tail but lacked hairpins), expression of the delivered reporter protein in HEK293 cells was similar. Using a triple instead of a single hairpin at each end increased protein expression even more. This method has the potential to simplify the production and reduce the cost of synthesizing exogenous mRNA for use as biologics or vaccines.

Pluripotency retention and exogenous mRNA introduction in planarian stem cells in culture

Planarians possess naturally occurring pluripotent adult somatic stem cells (neoblasts) required for homeostasis and whole-body regeneration. However, no reliable neoblast culture methods are currently available, hindering mechanistic studies of pluripotency and the development of transgenic tools. We report robust methods for neoblast culture and delivery of exogenous mRNAs. We identify optimal culture media for the short-term maintenance of neoblasts invitro and show via transplantation that cultured stem cells retain pluripotency for two days. We developed a procedure that significantly improves neoblast yield and purity by modifying standard flow cytometry methods. These methods enable the introduction and expression of exogenous mRNAs in neoblasts, overcoming a key hurdle impeding the application of transgenics in planarians. The advances in cell culture reported here create new opportunities for mechanistic studies of planarian adult stem cell pluripotency, and provide a systematic framework to develop cell culture techniques in other emerging research organisms.

Branching Ionizable Lipids Can Enhance the Stability, Fusogenicity, and Functional Delivery of mRNA

mRNA Delivery In article number 2200071, Kazuki Hashiba, Yusuke Sato, and co-workers investigate the impact of branched ionizable lipids on lipid-nanoparticle-based mRNA delivery. Systematic examination of the chemical structures of branched ionizable lipids reveals that branched structures play important roles in endosomal escape via membrane fusion and in delivery of exogenous mRNAs to the cytoplasm to be translated.

Ftr82 is necessary for hair cell morphogenesis and auditory function during zebrafish development

Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, while the pathological mechanism remains not fully understood due to the many potential deafness genes unidentified. ftr82, a member of the largely TRIMs family in fish, has been found specifically expressed in the otic vesicle while its function is still unclear. Here, we investigate the roles of ftr82 in HC development and hearing function utilizing the zebrafish model. The results of in situ hybridization illustrate that ftr82 is always restricted to localize in otic vesicles at different stages. The defects of HCs are observed both in ftr82 morphants and mutants, including significantly decreased crista HCs, shortened cilia as well as remarkably reduced functional HCs in neuromasts, which could be successfully rescued by co-injection of exogenous ftr82 mRNA. The behavior assay of startle response indicates that larvae lacking of ftr82 exhibits lower sensitivity to external sound stimuli. Further research reveals that the loss of HCs is mainly caused by cell apoptosis mediated by caspase-3 activation. Our study demonstrates that ftr82 is a crucial hearing-related gene that regulates the HC morphogenesis and auditory function performing, which provides new insight into the rapid identification of the deafness gene.

Lipid Nanoparticle Delivery of Chemically Modified NGFR100W mRNA Alleviates Peripheral Neuropathy.

Messenger RNA (mRNA) carries genetic instructions to the cell machinery for the transient production of antigens or therapeutic proteins and shows enormous potential in vaccine development, cancer immunotherapy, protein replacement therapy, and genome engineering. Here, the synthesis of chemically modified nerve growth factor mutant (NGFR100W ) mRNA through in vitro transcription is described. After the replacement of the original signal peptide sequence with the Ig Kappa leader sequence, codon-optimized NGFR100W mRNA yielded high secretion of mature NGFR100W , which promotes axon growth in PC12 cells. Using lipid nanoparticle (LNP)-delivery of N1-methylpseudouridine-modified mRNA in mice, NGFR100W -mRNA-LNPs result in the successful expression of NGFR100W protein, which significantly reduces nociceptive activity compared to that of NGFWT . This indicates that NGFR100W derived from exogenous mRNA elicited "painless" neuroprotective activity. Additionally, the therapeutic value of NGFR100W mRNA is established in a paclitaxel-induced peripheral neuropathy model by demonstrating the rapid recovery of intraepidermal nerve fibers. The results show that in vitro-transcribed mRNA has significant flexibility in sequence design and fast in vivo functional validation of target proteins. Furthermore, the results highlight the therapeutic potential of mRNA as a supplement to beneficial proteins for preventing or reversing some chronic medical conditions, such as peripheral neuropathy.

Immediate protein expression from exogenous mRNAs in embryonic brain

mRNA vaccines for SARS-CoV-2 have been widely used and saving millions of people in the world. How efficiently proteins are produced from exogenous mRNAs in the embryonic brain, however, is less known. Here we show that protein expression occurs highly efficiently in neural stem cells, in a very narrow time window after mRNA electroporation in the embryonic mouse brain, where plasmids have been successfully transfected. Protein expression is detected 1 h and 12 h after the electroporation of mRNAs and plasmids, respectively. The delivery of exogenous mRNAs may be useful for not only vaccines but also functional analysis in the brain.

Intracellular delivery of messenger RNA to macrophages with surfactant-derived lipid nanoparticles

The broad biomedical applications of messenger RNA (mRNA)-based therapeutics rely heavily on the rapid development of mRNA delivery systems. In this study, dual-component lipid nanoparticles (LNPs) were developed for intracellular delivery of mRNA to macrophages. By screening a panel of quaternary ammonium compounds with varying alkyl or aryl side chains, a cationic surfactant containing two hexadecyl tails was found to be effective for mRNA delivery with the assistance of fusogenic lipids. Further investigating structure alterations and optimization of formulation parameters, we found that a surfactant-derived ionizable lipid mixed with a fusogenic lipid was capable of condensing mRNA and self-assembling into LNPs. Without using the PEGylated lipid, the resulting LNPs were able to render the exogenous mRNA resistant to hydrolysis by nucleases and displayed excellent biocompatibility, along with the capacity to deliver mRNA to hard-to-transfect. Overall, the dual-component nanocarrier provides a novel mRNA delivery platform for genetic engineering of macrophages.

Sema4C Is Required for Vascular and Primary Motor Neuronal Patterning in Zebrafish.

Endothelial cells (ECs) and neurons share a number of common signaling pathways and molecular mediators to orchestrate directional migration and guide the pattern of the vascular network and nervous system. So far, research concerning the functional coupling between vascular and neuronal pathfinding remains insufficient. Semaphorin4C (sema4C), a member of class 4 semaphorins, is initially described in the nervous system, whose role has been demonstrated in diverse biological developments. The present study focused on the role of sema4C in the vascular and neural development process in zebrafish embryos. It confirmed that sema4C is expressed in both the nervous system and intersegmental vessels (ISVs) in zebrafish embryos by diverse expression analysis. It also showed that the knockdown of sema4C caused a serious pathfinding anomaly both in the ISVs and primary motor neurons (PMNs) of zebrafish embryos. In addition, overexpressing exogenous sema4C mRNA in sema4C morphants remarkably neutralized the defective pattern of the vascular and neural system. Collectively, this report suggests that sema4C acts as a dual guiding factor regulating vascular and neuronal development. These findings elucidate a new molecular mechanism underlying blood vessel and nerve development and might serve as groundwork for future research on functional coupling between both systems.

Chemically modified in-vitro-transcribed mRNA encoding thrombopoietin stimulates thrombopoiesis in mice

The use of messenger RNA (mRNA) enables the transient production of therapeutic proteins with stable and predictable translational kinetics and without the risk of insertional mutagenesis. Recent findings highlight the enormous potential of mRNA-based therapeutics. Here, we describe the synthesis of chemically modified thrombopoietin (TPO) mRNA through in vitro transcription and in vivo delivery via lipid nanoparticles (LNPs). After delivery of TPO mRNA in mice, compared with normal physiological values, plasma TPO protein levels increased over 1000-fold in a dose-dependent manner. Moreover, through a single intravenous dose of TPO mRNA-loaded LNPs, both reticulated and total platelet count increased significantly in mice, demonstrating that TPO protein derived from the exogenous mRNA was able to maintain normal activity. Submicrogram quantity of N1-methylpseudouridine-modified TPO mRNA showed a similar effect in promoting thrombopoiesis as that by the TPO receptor agonist romiplostim. In addition, a therapeutic value was established in anti-GPIbα (CD42b) antibody-induced thrombocytopenia mouse models that showed a fast recovery of platelet count. Our study demonstrated chemically modified in-vitro-transcribed TPO mRNA as a potentially safe therapeutic intervention to stimulate thrombopoiesis.

Fluorescent Platforms for RNA Chemical Biology Research.

Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating.

The Future of Tissue-Targeted Lipid Nanoparticle-Mediated Nucleic Acid Delivery.

The earliest example of in vivo expression of exogenous mRNA is by direct intramuscular injection in mice without the aid of a delivery vehicle. The current state of the art for therapeutic nucleic acid delivery is lipid nanoparticles (LNP), which are composed of cholesterol, a helper lipid, a PEGylated lipid and an ionizable amine-containing lipid. The liver is the primary organ of LNP accumulation following intravenous administration and is also observed to varying degrees following intramuscular and subcutaneous routes. Delivery of nucleic acid to hepatocytes by LNP has therapeutic potential, but there are many disease indications that would benefit from non-hepatic LNP tissue and cell population targeting, such as cancer, and neurological, cardiovascular and infectious diseases. This review will concentrate on the current efforts to develop the next generation of tissue-targeted LNP constructs for therapeutic nucleic acids.

Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency.

Diabetes mellitus is prevalent among women of reproductive age, and many women are left undiagnosed or untreated1. Gestational diabetes has profound and enduring effects on the long-term health of the offspring2,3. However, the link between pregestational diabetes and disease risk into adulthood in the next generation has not been sufficiently investigated. Here we show that pregestational hyperglycaemia renders the offspring more vulnerable to glucose intolerance. The expression of TET3 dioxygenase, responsible for 5-methylcytosine oxidation and DNA demethylation in the zygote4, is reduced in oocytes from a mouse model of hyperglycaemia (HG mice) and humanswith diabetes. Insufficient demethylation by oocyte TET3 contributes to hypermethylation at the paternal alleles of several insulin secretion genes, including the glucokinase gene (Gck), that persists from zygote to adult, promoting impaired glucose homeostasis largely owing to the defect in glucose-stimulated insulin secretion. Consistent with these findings, mouse progenies derived from the oocytes of maternal heterozygous and homozygous Tet3 deletion display glucose intolerance and epigenetic abnormalities similar to those from the oocytes of HG mice. Moreover, the expression of exogenous Tet3 mRNA in oocytes from HG mice ameliorates the maternal effect in offspring. Thus, our observations suggest an environment-sensitive window in oocyte development that confers predisposition to glucose intolerance in the next generation through TET3 insufficiency rather than through a direct perturbation of the oocyte epigenome. This finding suggests a potential benefit of pre-conception interventions in mothers to protect the health of offspring.

Exploiting exercise and mTOR enhanced protein synthesis to augment mRNA vaccine translation

Exercise has been previously proposed as an adjuvant for traditional vaccines through mechanisms of lymph movement and skeletal muscle damage. However, the novel mRNA technology being used in COVID‐19 and other novel vaccines fundamentally changes how we should consider muscle’s active role in protein production and subsequent protective immunity. Following exercise, mammalian target of rapamycin (mTOR) activation drives total protein translation increases in skeletal muscle. We hypothesize that this 4‐24 hour window of opportunity can be exploited to augment exogenous mRNA translation. To evaluate the feasibility of this approach, we first interrogated mTOR using fully differentiated C2C12 mouse skeletal muscle myotubes that were transfected with mRNA encoding for enhanced green fluorescent protein (eGFP) or FLAG‐tagged Comirnaty SARS‐CoV‐2 Spike sequence in mTOR modulating conditions. Myotube transfection in the presence of rapamycin (Figure 1A) and resveratrol (Figure 1B) showed significant decreases in protein translation as measured by eGFP fluorescence, indicating that exogenous mRNA translation can be decreased pharmacologically by inhibiting the mTOR pathway. To simulate exercise enhanced protein synthesis, we treated myotubes with insulin‐like growth‐factor 1 (IGF‐1), as this protein is not only upregulated following exercise but also signals through an overlapping pathway with mechanoreceptors. Transfections with mRNA encoding for either eGFP or Comirnaty‐FLAG during IGF‐1 treatment showed a dose dependent increase in eGFP fluorescence (Figure 1C) and a significant increase in Comirnaty‐FLAG translation as measured by ELISA (Figure 1D). Current studies are investigating the use of electrical stimulation induced contraction on C2C12 myotubes prior to transfection as well as in vivo models of exercise and intramuscular mRNA injections. The implications of these results may impact future mRNA therapies to utilize exercise, or modulation of intracellular signaling pathways affected by exercise, as a method to improve vaccine‐induced exogenous protein production.

Kinesin motor KIFC1 is required for tubulin acetylation and actin-dependent spindle migration in mouse oocyte meiosis.

Mammalian oocyte maturation is a unique asymmetric division, which is mainly because of actin-based spindle migration to the cortex. In the present study, we report that a kinesin motor KIFC1, which is associated with microtubules for the maintenance of spindle poles in mitosis, is also involved in actin dynamics in murine oocyte meiosis, co-localizing with microtubules during mouse oocyte maturation. Depletion of KIFC1 caused the failure of polar body extrusion, and we found that meiotic spindle formation and chromosome alignment were disrupted. This might be because of the effects of KIFC1 on HDAC6 and NAT10-based tubulin acetylation, which further affected microtubule stability. Mass spectroscopy analysis revealed that KIFC1 also associated with several actin nucleation factors and we found that KIFC1 was essential for the distribution of actin filaments, which further affected spindle migration. Depletion of KIFC1 leaded to aberrant expression of formin 2 and the ARP2/3 complex, and endoplasmic reticulum distribution was also disturbed. Exogenous KIFC1 mRNA supplement could rescue these defects. Taken together, as well as its roles in tubulin acetylation, our study reported a previously undescribed role of kinesin KIFC1 on the regulation of actin dynamics for spindle migration in mouse oocytes.

Delivery Strategies for mRNA Vaccines.

The therapeutic potential for messenger RNA (mRNA) in infectious diseases and cancer was first realized almost three decades ago, but only in 2018 did the first lipid nanoparticle-based small interfering RNA (siRNA) therapy reach the market with the United States Food and Drug Administration (FDA) approval of patisiran (Onpattro™) for hereditary ATTR amyloidosis. This was largely made possible by major advances in the formulation technology for stabilized lipid-based nanoparticles (LNPs). Design of the cationic ionizable lipids, which are a key component of the LNP formulations, with an acid dissociation constant (pKa) close to the early endosomal pH, would not only ensure effective encapsulation of mRNA into the stabilized lipoplexes within the LNPs, but also its subsequent endosomal release into the cytoplasm after endocytosis. Unlike other gene therapy modalities, which require nuclear delivery, the site of action for exogenous mRNA vaccines is the cytosol where they get translated into antigenic proteins and thereby elicit an immune response. LNPs also protect the mRNA against enzymatic degradation by the omnipresent ribonucleases (RNases). Cationic nano emulsion (CNE) is also explored as an alternative and relatively thermostable mRNA vaccine delivery vehicle. In this review, we have summarized the various delivery strategies explored for mRNA vaccines, including naked mRNA injection; ex vivo loading of dendritic cells; CNE; cationic peptides; cationic polymers and finally the clinically successful COVID-19 LNP vaccines (Pfizer/BioNTech and Moderna vaccines)—their components, design principles, formulation parameter optimization and stabilization challenges. Despite the clinical success of LNP-mRNA vaccine formulations, there is a specific need to enhance their storage stability above 0 °C for these lifesaving vaccines to reach the developing world.

Maternal EHMT2 is essential for homologous chromosome segregation by regulating Cyclin B3 transcription in oocyte meiosis.

During oocyte growth, various epigenetic modifications are gradually established, accompanied by accumulation of large amounts of mRNAs and proteins. However, little is known about the relationship between epigenetic modifications and meiotic progression. Here, by using Gdf9-Cre to achieve oocyte-specific ablation of Ehmt2 (Euchromatic-Histone-Lysine-Methyltransferase 2) from the primordial follicle stage, we found that female mutant mice were infertile. Oocyte-specific knockout of Ehmt2 caused failure of homologous chromosome separation independent of persistently activated SAC during the first meiosis. Further studies revealed that lacking maternal Ehmt2 affected the transcriptional level of Ccnb3, while microinjection of exogenous Ccnb3 mRNA could partly rescue the failure of homologous chromosome segregation. Of particular importance was that EHMT2 regulated ccnb3 transcriptions by regulating CTCF binding near ccnb3 gene body in genome in oocytes. In addition, the mRNA level of Ccnb3 significantly decreased in the follicles microinjected with Ctcf siRNA. Therefore, our findings highlight the novel function of maternal EHMT2 on the metaphase I-to-anaphase I transition in mouse oocytes: regulating the transcription of Ccnb3.

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale.

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.