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Argininosuccinic aciduria (ASA) is a metabolic disorder caused by a deficiency in argininosuccinate lyase (ASL), which cleaves argininosuccinic acid to arginine and fumarate in the urea cycle. ASL deficiency (ASLD) leads to hepatocyte dysfunction, hyperammonemia, encephalopathy, and respiratory alkalosis. Here we describe a novel therapeutic approach for treating ASA, based on nucleoside-modified messenger RNA (modRNA) formulated in lipid nanoparticles (LNP). To optimize ASL-encoding mRNA, we modified its cap, 5′ and 3′ untranslated regions, coding sequence, and the poly(A) tail. We tested multiple optimizations of the formulated mRNA in human cells and wild-type C57BL/6 mice. The ASL protein showed robust expression in vitro and in vivo and a favorable safety profile, with low cytokine and chemokine secretion even upon administration of increasing doses of ASL mRNA-LNP. In the ASLNeo/Neo mouse model of ASLD, intravenous administration of the lead therapeutic candidate LNP-ASL CDS2 drastically improved the survival of the mice. When administered twice a week lower doses partially protected and 3 mg/kg LNP-ASL CDS2 fully protected the mice. These results demonstrate the considerable potential of LNP-formulated, modified ASL-encoding mRNA as an effective alternative to AAV-based approaches for the treatment of ASA.

As mRNA vaccines have proved to be very successful in battling the coronavirus disease 2019 (COVID-19) pandemic, this new modality has attracted widespread interest for the development of potent vaccines against other infectious diseases and cancer. Cervical cancer caused by persistent human papillomavirus (HPV) infection is a major cause of cancer-related deaths in women, and the development of safe and effective therapeutic strategies is urgently needed. In the present study, we compared the performance of three different mRNA vaccine modalities to target tumors associated with HPV-16 infection in mice. We generated lipid nanoparticle (LNP)–encapsulated self-amplifying mRNA as well as unmodified and nucleoside-modified non-replicating mRNA vaccines encoding a chimeric protein derived from the fusion of the HPV-16 E7 oncoprotein and the herpes simplex virus type 1 glycoprotein D (gDE7). We demonstrated that single low-dose immunizations with any of the three gDE7 mRNA vaccines induced activation of E7-specific CD8 + T cells, generated memory T cell responses capable of preventing tumor relapses, and eradicated subcutaneous tumors at different growth stages. In addition, the gDE7 mRNA-LNP vaccines induced potent tumor protection in two different orthotopic mouse tumor models after administration of a single vaccine dose. Last, comparative studies demonstrated that all three gDE7 mRNA-LNP vaccines proved to be superior to gDE7 DNA and gDE7 recombinant protein vaccines. Collectively, we demonstrated the immunogenicity and therapeutic efficacy of three different mRNA vaccines in extensive comparative experiments. Our data support further evaluation of these mRNA vaccines in clinical trials.

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target.MethodsWe investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing.ResultsWe identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2–4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed.ConclusionTaken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) that encodes the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy in preventing coronavirus disease-19 (COVID-19)1. Here we extend our previous phase 1/2 trial report2 and present BNT162b2 prime/boost induced immune response data from a second phase 1/2 trial in healthy adults (18-55 years of age). BNT162b2 elicited strong antibody responses, with SARS-CoV-2 serum 50% neutralizing geometric mean titers up to 3.3-fold above those observed in COVID-19 human convalescent samples (HCS) one week post-boost. BNT162b2-elicited sera neutralized 22 pseudoviruses bearing SARS-CoV-2 S variants. Most participants had a strong IFNγ- or IL-2-positive CD8+ and CD4+ T helper type 1 (TH1) T cell response, detectable throughout the full observation period of nine weeks following the boost. pMHC multimer technology identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week post-boost, epitope-specific CD8+ T cells of the early differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes conserved in a broad range of variants at well tolerated doses.

RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. It remains unclear whether pseudouridylation of mRNA can be snoRNA-guided, bearing important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at relatively low efficiencies, and is constrained by mRNA localization, snoRNA expression levels and the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously shown to suppress translational termination. However, in this and follow-up experiments in human cells we observe no evidence for significant levels of readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, ‘design rules’, constraints and consequences of snoRNA-mediated pseudouridylation.

An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein1. Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18–55 years of age. Two doses of 1–50 μg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-μg dose) to 3.5-fold (50-μg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (TH1)-skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms. In a phase I/II dose-escalation clinical trial, the mRNA COVID-19 vaccine BNT162b1 elicits specific T cell and antibody responses that suggest it has protective potential.

Messenger RNA was discovered in 1961 and it took 60 years until the first mRNA became FDA-approved product in the form of COVID-19 mRNA vaccine. During those years a lot of progress has been made by hundreds of scientists. It was 1978 when the first-time isolated mRNA delivered into mammalian cells produced the encoded protein. In vitro transcription introduced in 1984 made it possible to generate any desired mRNA from the encoding plasmid using phage RNA polymerases. In the early 90s mRNA was used for therapy as well as vaccine against infectious diseases and cancer. Inflammatory nature of the mRNAs limited their in vivo use. Replacing uridine with pseudouridine made the mRNA non-immunogenic, more stable and highly translatable. Delivery of the lipid nanoparticle-formulated nucleoside-modified mRNA encoding viral antigens became a platform for effective vaccine. Labile nature of the mRNA is ideal for transient production of the viral antigen, to generate effective antibody and cellular immune response. The mRNA platform is revolutionizing the delivery of effective and safe vaccines, therapeutics and gene therapies.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B

Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the cell surface oncoprotein glypican 2 (GPC2) is highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as definable subsets of highly malignant gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment. First, we validated and prioritized these CARs using in vitro cytotoxicity and T cell degranulation assays with GPC2-expressing neuroblastoma cells. Next, we expanded the testing of the two most potent GPC2-directed CAR constructs prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, locoregional delivery bi-weekly of two to four million GPC2-directed mRNA CAR T cells induced significant and sustained tumor regression in two orthotopic medulloblastoma models, and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells.One Sentence SummaryGlypican 2 is expressed on the surface of multiple pediatric brain tumors and can be successfully targeted with mRNA chimeric antigen receptor T cells.

Adoptive T cell therapy is a form of cellular therapy that utilizes human immune cells, often empowered by the expression of recombinant proteins, to attack selected targets present on tumor or infected cells. T cell-based immunotherapy has been progressing over the past several decades, and reached a milestone with the recent US Food and Drug Administration (FDA) approval of chimeric antigen receptor T cell therapy for relapsed and refractory leukemia and lymphoma. Although most studies have used viral vectors, a growing number of researchers have come to appreciate in vitro-transcribed (IVT) mRNA for the development, testing, and application of T cell-based immunotherapeutics. IVT mRNA offers inherent safety features, highly efficient recombinant protein translation, and the ability to control pharmacokinetic properties of the therapy. In this review, we discuss the history of IVT mRNA in adoptive T cell therapy, from tumor-infiltrating lymphocytes and T cell receptor-based therapies to chimeric antigen receptor therapy and gene-editing techniques, as well as prior and ongoing clinical trials.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against SARS-CoV-2, but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes the induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP outperformed a widely used MF59-like adjuvant, AddaVax™. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction, but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of both traditional and next-generation vaccine platforms., Graphical Abstract, The mechanism of action of nucleoside-modified mRNA-LNP vaccines is unknown. Alameh, Tombácz, Bettini et al. demonstrate that LNPs can possess adjuvant activity and promote a robust induction of Tfh cell, B cell and humoral responses when utilized in mRNA and protein subunit vaccines in mice. IL-6 induction and the ionizable lipid component are critical for the adjuvant activity of LNPs.

Summary SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19., Highlights • mRNA vaccines induce robust type 1 CD4+ and CD8+ T cells in the spleen and lung • Vaccine-induced T cells readily exit the vasculature and enter the lung parenchyma • mRNA vaccines elicit strong long-lived plasma cell and memory B cell responses • mRNA vaccines induce antibodies with potent anti-SARS-CoV-2 neutralization activity, SARS-CoV-2 mRNA-based vaccines are among the most promising vaccine candidates against COVID-19. Laczkó et al. evaluate two nucleoside-modified mRNA vaccine candidates in mice and demonstrate that they induce potent T and B cell immune responses and high levels of neutralizing antibodies after administration of a single vaccine dose.

An effective vaccine is needed to halt the spread of the SARS-CoV-2 pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 COVID-19 vaccine trial with BNT162b1, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Here we present antibody and T cell responses after BNT162b1 vaccination from a second, non-randomized open-label phase 1/2 trial in healthy adults, 18-55 years of age. Two doses of 1 to 50 µg of BNT162b1 elicited robust CD4+ and CD8+ T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those in a COVID-19 convalescent human serum panel (HCS). Day 43 SARS-CoV-2 serum neutralising geometric mean titers were 0.7-fold (1 µg) to 3.5-fold (50 µg) those of HCS. Immune sera broadly neutralised pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had TH1 skewed T cell immune responses with RBD-specific CD8+ and CD4+ T cell expansion. Interferon (IFN)γ was produced by a high fraction of RBD-specific CD8+ and CD4+ T cells. The robust RBD-specific antibody, T-cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest multiple beneficial mechanisms with potential to protect against COVID-19.

Currently available influenza virus vaccines have inadequate effectiveness and are reformulated annually due to viral antigenic drift. Thus, development of a vaccine that confers long-term protective immunity against antigenically distant influenza virus strains is urgently needed. The highly conserved influenza virus hemagglutinin (HA) stalk represents one of the potential targets of broadly protective/universal influenza virus vaccines. Here, we evaluate a potent broadly protective influenza virus vaccine candidate that uses nucleoside-modified and purified mRNA encoding full-length influenza virus HA formulated in lipid nanoparticles (LNPs). We demonstrate that immunization with HA mRNA-LNPs induces antibody responses against the HA stalk domain of influenza virus in mice, rabbits, and ferrets. The HA stalk-specific antibody response is associated with protection from homologous, heterologous, and heterosubtypic influenza virus infection in mice., The highly conserved influenza virus hemagglutinin (HA) stalk represents a potential target for a broadly protective vaccine. Here, the authors show that immunization with nucleoside-modified mRNA encoding full-length HA formulated in lipid nanoparticles elicits HA stalk-specific antibodies and protects from heterosubtypic virus infection.

Pardi and colleagues report on a vaccine platform in which purified, antigen-encoding, nucleoside-modified mRNA is encapsulated in lipid nanoparticles. Immunization with this vaccine elicits potent T follicular helper cell, germinal center B cell, and protective, neutralizing antibody responses., T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Katalin Kariko describes the discovery that replacing uridine with pseudouridine renders RNA non-immunogenic. This paved the way for developing mRNA for protein replacement therapy and, surprisingly, also for mRNA-based vaccine development.

Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases., Monoclonal antibodies are highly effective therapeutics that can be delivered as proteins or encoded DNA or mRNA. Here the authors develop lipid nanoparticle-formulated nucleoside-modified mRNA encoding an HIV-1 neutralizing antibody and see sustained and protective antibody levels in treated mice.