Nonreplicating synthetic mRNA vaccines: A journey through the European (Journal of Immunology) history.

The first worldwide article reporting that injections of synthetic nonreplicating mRNA could be used as a vaccine, which originated from a French team located in Paris, was published in the European Journal of Immunology (EJI) in 1993. It relied on work conducted by several research groups in a handful of countries since the 1960s, which put forward the precise description of eukaryotic mRNA and the method to reproduce this molecule in vitro as well as how to transfect it into mammalian cells. Thereafter, the first industrial development of this technology began in Germany in 2000, with the founding of CureVac, which stemmed from another description of a synthetic mRNA vaccine published in EJI in 2000. The first clinical studies investigating mRNA vaccines in humans were performed as collaboration between CureVac and the University of Tübingen in Germany as early as 2003. Finally, the first worldwide approved mRNA vaccine (an anti-COVID-19 vaccine) is based on the mRNA technologies developed by BioNTech since its 2008 foundation in Mainz, Germany, and earlier by the pioneering academic work of its founders. In addition to the past, present, and future of mRNA-based vaccines, the article aims to present the geographical distribution of the early work, how the development of the technology was implemented by several independent and internationally distributed research teams, as well as the controversies on the optimal way to design or formulate and administer mRNA vaccines.

Recruiting In Vitro Transcribed mRNA against Cancer Immunotherapy: A Contemporary Appraisal of the Current Landscape.

Over 100 innovative in vitro transcribed (IVT)-mRNAs are presently undergoing clinical trials, with a projected substantial impact on the pharmaceutical market in the near future. Τhe idea behind this is that after the successful cellular internalization of IVT-mRNAs, they are subsequently translated into proteins with therapeutic or prophylactic relevance. Simultaneously, cancer immunotherapy employs diverse strategies to mobilize the immune system in the battle against cancer. Therefore, in this review, the fundamental principles of IVT-mRNA to its recruitment in cancer immunotherapy, are discussed and analyzed. More specifically, this review paper focuses on the development of mRNA vaccines, the exploitation of neoantigens, as well as Chimeric Antigen Receptor (CAR) T-Cells, showcasing their clinical applications and the ongoing trials for the development of next-generation immunotherapeutics. Furthermore, this study investigates the synergistic potential of combining the CAR immunotherapy and the IVT-mRNAs by introducing our research group novel, patented delivery method that utilizes the Protein Transduction Domain (PTD) technology to transduce the IVT-mRNAs encoding the CAR of interest into the Natural Killer (NK)-92 cells, highlighting the potential for enhancing the CAR NK cell potency, efficiency, and bioenergetics. While IVT-mRNA technology brings exciting progress to cancer immunotherapy, several challenges and limitations must be acknowledged, such as safety, toxicity, and delivery issues. This comprehensive exploration of IVT-mRNA technology, in line with its applications in cancer therapeutics, offers valuable insights into the opportunities and challenges in the evolving landscape of cancer immunotherapy, setting the stage for future advancements in the field.

Mechanisms and research advances in mRNA antibody drug-mediated passive immunotherapy.

Antibody technology is widely used in the fields of biomedical and clinical therapies. Nonetheless, the complex in vitro expression of recombinant proteins, long production cycles, and harsh storage conditions have limited their applications in medicine, especially in clinical therapies. Recently, this dilemma has been overcome to a certain extent by the development of mRNA delivery systems, in which antibody-encoding mRNAs are enclosed in nanomaterials and delivered to the body. On entering the cytoplasm, the mRNAs immediately bind to ribosomes and undergo translation and post-translational modifications. This process produces monoclonal or bispecific antibodies that act directly on the patient. Additionally, it eliminates the cumbersome process of in vitro protein expression and extends the half-life of short-lived proteins, which significantly reduces the cost and duration of antibody production. This review focuses on the benefits and drawbacks of mRNA antibodies compared with the traditional in vitro expressed antibodies. In addition, it elucidates the progress of mRNA antibodies in the prevention of infectious diseases and oncology therapy.

Demystifying mRNA vaccines: an emerging platform at the forefront of cryptic diseases.

Messenger RNA (mRNA) vaccines have been studied for decades, but only recently, during the COVID-19 pandemic, has the technology garnered noteworthy attention. In contrast to traditional vaccines, mRNA vaccines elicit a more balanced immune response, triggering both humoral and cellular components of the adaptive immune system. However, some inherent hurdles associated with stability, immunogenicity, in vivo delivery, along with the novelty of the technology, have generated scepticism in the adoption of mRNA vaccines. Recent developments have pushed to bypass these issues and the approval of mRNA-based vaccines to combat COVID-19 has further highlighted the feasibility, safety, efficacy, and rapid development potential of this platform, thereby pushing it to the forefront of emerging therapeutics. This review aims to demystify mRNA vaccines, delineating the evolution of the technology which has emerged as a timely solution to COVID-19 and exploring the immense potential it offers as a prophylactic option for other cryptic diseases.

The infinite possibilities of RNA therapeutics.

Although the study of ribonucleic acid (RNA) therapeutics started decades ago, for many years, this field of research was overshadowed by the growing interest in DNA-based therapies. Nowadays, the role of several types of RNA in cell regulation processes and the development of various diseases have been elucidated, and research in RNA therapeutics is back with force. This short literature review aims to present general aspects of many of the molecules currently used in RNA therapeutics, including in vitro transcribed mRNA (IVT mRNA), antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), and microRNAs (miRNAs). In addition, we describe the state of the art of technologies applied for synthetic RNA manufacture and delivery. Likewise, we detail the RNA-based therapies approved by the FDA so far, as well as the ongoing clinical investigations. As a final point, we highlight the current and potential advantages of working on RNA-based therapeutics and how these could lead to a new era of accessible and personalized healthcare.

Efficient Colorectal Cancer Gene Therapy with IL-15 mRNA Nanoformulation.

Immunogene therapy is a novel method for the treatment of colorectal cancer. Cytokine IL-15 has exhibited therapeutic anticancer potential due to its immune-stimulation property. However, conventional IL-15-based cancer gene therapy studies have been performed using the plasmid DNA form, which has potential shortcomings including weak delivery efficiency and backbone effect. In this study, an IL-15 immunogene therapy study for colon cancer using in vitro transcript mRNA is described. A protamine/liposome system (CLPP) is developed to provide efficient condensation and delivery capacity for in vivo mRNA transportation. They demonstrated that the prepared CLPP system could deliver the IL-15-encoding mRNA into C26 cells with high efficacy. The secretory expressed IL-15 cytokine by the C26 cells successfully produced lymphocyte stimulation and triggered anticancer cytotoxicity upon cancer cells in vitro. Local or systemic administration of the CLPP/mIL-15 complex exhibited obvious inhibition effects on multiple C26 murine colon cancer models with inhibition rates of up to 70% in the C26 abdominal cavity metastasis tumor model, 55% in the subcutaneous model, and 69% in the pulmonary metastasis model, demonstrating high efficacy and safety. These results successfully demonstrated the high therapeutic potential of the CLPP/mIL-15 complex for colorectal cancer immunogene therapy.

IVT-mRNA reprogramming of myeloid cells for cancer immunotherapy.

In the past decade, in vitro transcribed messenger RNAs (IVT-mRNAs) have emerged as promising therapeutic molecules. The clinical success of COVID-19 mRNA vaccines developed by Pfizer-BioNTech and Moderna, have demonstrated that IVT-mRNAs can be safely and successfully used in a clinical setting, and efforts are underway to develop IVT-mRNAs for therapeutic applications. Current applications of mRNA-based therapy have been focused on (1) mRNA vaccines for infectious diseases and cancer treatment; (2) protein replacement therapy; (3) gene editing therapy; and (4) cell-reprogramming therapies. Due to the recent clinical progress of cell-based immunotherapies, the last direction-the use of IVT-mRNAs as a therapeutic approach to program immune cells for the treatment of cancer has received extensive attention from the cancer immunotherapy field. Myeloid cells are important components of our immune system, and they play critical roles in mediating disease progression and regulating immunity against diseases. In this chapter, we discussed the progress of using IVT-mRNAs as a therapeutic approach to program myeloid cells against cancer and other immune-related diseases. Towards this direction, we first reviewed the pharmacology of IVT-mRNAs and the biology of myeloid cells as well as myeloid cell-targeting therapeutics. We then presented a few cases of current IVT-mRNA-based approaches to target and reprogram myeloid cells for disease treatment and discussed the advantages and limitations of these approaches. Finally, we presented our considerations in designing mRNA-based approaches to target myeloid cells for disease treatment.

mRNA-Engineered CD5-CAR-γδTCD5- Cells for the Immunotherapy of T-Cell Acute Lymphoblastic Leukemia.

Clinical trials of Chimeric Antigen Receptor T-cell (CAR-T) therapy have demonstrated remarkable success in treating both solid tumors and hematological malignancies. Nanobodies (Nbs) have emerged as promising antigen-targeting domains for CARs, owing to their high specificity, robust stability, and strong affinity, leading to significant advancements in the field of Nb-CAR-T. In the realm of T-cell acute lymphoblastic leukemia (T-ALL) targets, CD5 stands out as a potentially excellent candidate for T-cell-based CAR therapy, due to its distinct expression on the surface of malignant T-ALL cells. To mitigate graft-versus-host disease associated with allogeneic CAR-T, γδT cells are selected and stimulated from peripheral blood mononuclear cells, and γδT cells are engineered via CRISPR/Cas9 to eliminate fratricide, enabling the creation of fratricide-resistant CAR-γδTCD5- cells. In vitro transcribed (IVT) mRNA is used to construct CAR-T, presenting a safer, faster, and cost-effective method compared to traditional viral vector approaches. In this study, a CD5-VHH library is constructed, and specific CD5-nanobodies are screened for subsequent use in CD5-CAR-γδTCD5- therapy. IVT-mRNA-CD5-CAR-γδTCD5- cells exhibited favorable functional characteristics and demonstrated antitumor efficacy against malignant T cell lines, underlining the potential for advancing mRNA-CD5-CAR-γδTCD5- therapy.

Deconvolution of synthetic mRNA expression: Nucleoside chemistry alters translatability.

Recent technological advances in the production of in vitro transcribed messenger RNA (IVT-mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT-mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT-mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT-mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell-free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification-dependent steps in the complex delivery process.

Synthetic mRNAs Containing Minimalistic Untranslated Regions Are Highly Functional In Vitro and In Vivo.

Synthetic mRNA produced by in vitro transcription (ivt mRNA) is the active pharmaceutical ingredient of approved anti-COVID-19 vaccines and of many drugs under development. Such synthetic mRNA typically contains several hundred bases of non-coding "untranslated" regions (UTRs) that are involved in the stabilization and translation of the mRNA. However, UTRs are often complex structures, which may complicate the entire production process. To eliminate this obstacle, we managed to reduce the total amount of nucleotides in the UTRs to only four bases. In this way, we generate minimal ivt mRNA ("minRNA"), which is less complex than the usual optimized ivt mRNAs that are contained, for example, in approved vaccines. We have compared the efficacy of minRNA to common augmented mRNAs (with UTRs of globin genes or those included in licensed vaccines) in vivo and in vitro and could demonstrate equivalent functionalities. Our minimal mRNA design will facilitate the further development and implementation of ivt mRNA-based vaccines and therapies.

mRNA PROTACs: engineering PROTACs for high-efficiency targeted protein degradation.

Proteolysis-targeting chimeras (PROTACs) are essential bifunctional molecules that target proteins of interest (POIs) for degradation by cellular ubiquitination machinery. Despite significant progress made in understanding PROTACs' functions, their therapeutic potential remains largely untapped. As a result of the success of highly flexible, scalable, and low-cost mRNA therapies, as well as the advantages of the first generation of peptide PROTACs (p-PROTACs), we present for the first time an engineering mRNA PROTACs (m-PROTACs) strategy. This design combines von Hippel-Lindau (VHL) recruiting peptide encoding mRNA and POI-binding peptide encoding mRNA to form m-PROTAC and promote cellular POI degradation. We then performed proof-of-concept experiments using two m-PROTACs targeting two cancer-related proteins, estrogen receptor alpha and B-cell lymphoma-extra large protein. Our results demonstrated that m-PROTACs could successfully degrade the POIs in different cell lines and more effectively inhibit cell proliferation than the traditional p-PROTACs. Moreover, the in vivo experiment demonstrated that m-PROTAC led to significant tumor regression in the 4T1 mouse xenograft model. This finding highlights the enormous potential of m-PROTAC as a promising approach for targeted protein degradation therapy.

Production and Evaluation of Nucleoside-Modified mRNA Vaccines for Infectious Diseases.

Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have demonstrated potency in multiple preclinical models against various pathogens and have recently received considerable attention due to the success of the two safe and effective COVID-19 mRNA vaccines developed by Moderna and Pfizer-BioNTech. The use of nucleoside modification in mRNA vaccines seems to be critical to achieve a sufficient level of safety and immunogenicity in humans, as illustrated by the results of clinical trials using either nucleoside-modified or unmodified mRNA-based vaccine platforms. It is well documented that the incorporation of modified nucleosides in the mRNA and stringent mRNA purification after in vitro transcription render it less inflammatory and highly translatable; these two features are likely key for mRNA vaccine safety and potency. Formulation of the mRNA into LNPs is important because LNPs protect mRNA from rapid degradation, enabling efficient delivery and high levels of protein production for extended periods of time. Additionally, recent studies have provided evidence that certain LNPs with ionizable cationic lipids (iLNPs) possess adjuvant activity that fosters the induction of strong humoral and cellular immune responses by mRNA-iLNP vaccines.In this chapter we describe the production of iLNP-encapsulated, nucleoside-modified, and purified mRNA and the evaluation of antigen-specific T cell and antibody responses elicited by this vaccine form.

Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

BACKGROUND: The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. METHODS: To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. RESULTS: Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. CONCLUSIONS: The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

Advancements of in vitro transcribed mRNA (IVT mRNA) to enable translation into the clinics.

The accelerated progress and approval of two mRNA-based vaccines to address the SARS-CoV-2 virus were unprecedented. This record-setting feat was made possible through the solid foundation of research on in vitro transcribed mRNA (IVT mRNA) which could be utilized as a therapeutic modality. Through decades of thorough research to overcome barriers to implementation, mRNA-based vaccines or therapeutics offer many advantages to rapidly address a broad range of applications including infectious diseases, cancers, and gene editing. Here, we describe the advances that have supported the adoption of IVT mRNA in the clinics, including optimization of the IVT mRNA structural components, synthesis, and lastly concluding with different classes of IVT RNA. Continuing interest in driving IVT mRNA technology will enable a safer and more efficacious therapeutic modality to address emerging and existing diseases.

Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency.

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.

Recent Advances and Innovations in the Preparation and Purification of In Vitro-Transcribed-mRNA-Based Molecules.

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.

Hybrid core-shell particles for mRNA systemic delivery.

mRNA based infectious disease vaccines have opened the venue for development of novel nucleic acids-based therapeutics. For all mRNA therapeutics dedicated delivery systems are required, where different functionalities and targeting abilities need to be optimized for the respective applications. One option for advanced formulations with tailored properties are lipid-polymer hybrid nanoparticles with complex nanostructure, which allow to combine features of several already well described nucleic acid delivery systems. Here, we explored hyaluronic acid (HA) as coating of liposome-mRNA complexes (LRCs) to investigate effects of the coating on surface charge, physicochemical characteristics and biological activity. HA was electrostatically attached to positively charged complexes, forming hybrid LRCs (HLRCs). At different N/P ratios, physico-chemical characterization of the two sets of particles showed similarity in size (around 200 nm) and mRNA binding abilities, while the presence of the HA shell conferred a negative surface charge to otherwise positive complexes. High transfection efficiency of LRCs and HLRCs in vitro has been obtained in THP-1 and human monocytes derived from PBMC, an interesting target cell population for cancer and immune related pathologies. In mice, quantitative biodistribution of radiolabeled LRC and HLRC particles, coupled with bioluminescence studies to detect the protein translation sites, hinted towards both particles' accumulation in the hepatic reticuloendothelial system (RES). mRNA translated proteins though was found mainly in the spleen, a major source for immune cells, with preference for expression in macrophages. The results showed that surface modifications of liposome-mRNA complexes can be used to fine-tune nanoparticle physico-chemical characteristics. This provides a tool for assembly of stable and optimized nanoparticles, which are prerequisite for future therapeutic interventions using mRNA-based nanomedicines.

Capillary electrophoresis methods for determining the IVT mRNA critical quality attributes of size and purity.

One result of the Covid-19 pandemic has been an increased awareness of IVT mRNA vaccines and the speed at which they can be produced for disease outbreaks. Currently the only approved IVT mRNA therapeutics are the Covid-19 vaccines, however IVT mRNA is being investigated for other non-Covid prophylactic vaccines, therapeutics, and therapeutic vaccines. IVT mRNAs can range from less than 100 nt in length to longer than 9,000 nt. When producing any IVT mRNA, quality control of the IVT mRNA is essential to ensure that the product is the correct length and does not contain truncated or degraded mRNA. Capillary gel electrophoresis provides high resolution separations of the IVT mRNA of interest from the degraded or truncated impurities allowing for the accurate purity assessment of IVT mRNA. Specialized capillary electrophoresis gels can also be used to provide analysis of purified poly(A) tails enabling characterization of multiple Critical Quality Attributes on a single platform. Here we describe methods for the purity assessment of IVT mRNAs through either 6,000 or 9,000 nt and determination of poly(A) tail length using different capillary gel electrophoresis methods.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines.

The 3' poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.

DOCK2 regulates antifungal immunity by regulating RAC GTPase activity.

Fungal infections cause ~1.5 million deaths each year worldwide, and the mortality rate of disseminated candidiasis currently exceeds that of breast cancer and malaria. The major reasons for the high mortality of candidiasis are the limited number of antifungal drugs and the emergence of drug-resistant species. Therefore, a better understanding of antifungal host defense mechanisms is crucial for the development of effective preventive and therapeutic strategies. Here, we report that DOCK2 (dedicator of cytokinesis 2) promotes indispensable antifungal innate immune signaling and proinflammatory gene expression in macrophages. DOCK2-deficient macrophages exhibit decreased RAC GTPase (Rac family small GTPase) activation and ROS (reactive oxygen species) production, which in turn attenuates the killing of intracellular fungi and the activation of downstream signaling pathways. Mechanistically, after fungal stimulation, activated SYK (spleen-associated tyrosine kinase) phosphorylates DOCK2 at tyrosine 985 and 1405, which promotes the recruitment and activation of RAC GTPases and then increases ROS production and downstream signaling activation. Importantly, nanoparticle-mediated delivery of in vitro transcribed (IVT) Rac1 mRNA promotes the activity of Rac1 and helps to eliminate fungal infection in vivo. Taken together, this study not only identifies a critical role of DOCK2 in antifungal immunity via regulation of RAC GTPase activity but also provides proof of concept for the treatment of invasive fungal infections by using IVT mRNA.