Abstract
The ongoing SARS-CoV-2 pandemic necessitates the fast development of vaccines. Recently, viral mutants termed variants of concern (VOC) which may escape host immunity have emerged. The ...efficacy of spike encoding mRNA vaccines (CVnCoV and CV2CoV) against the ancestral strain and the VOC B.1.351 was tested in a K18-hACE2 transgenic mouse model. Naive mice and mice immunized with a formalin-inactivated SARS-CoV-2 preparation were used as controls. mRNA-immunized mice develop elevated SARS-CoV-2 RBD-specific antibody and neutralization titers which are readily detectable, but significantly reduced against VOC B.1.351. The mRNA vaccines fully protect from disease and mortality caused by either viral strain. SARS-CoV-2 remains undetected in swabs, lung, or brain in these groups. Despite lower neutralizing antibody titers compared to the ancestral strain BavPat1, CVnCoV and CV2CoV show complete disease protection against the novel VOC B.1.351 in our studies.
Post‐transcriptional gene silencing often limits the over‐expression of transgenes in transgenic plants. It involves RNA‐DEPENDENT RNA POLYMERASE 6 (RDR6), which recognizes aberrant transcripts, such ...as inaccurately processed or uncapped mRNA, and triggers silencing of target transcripts. Here, we describe the isolation and characterization of an Arabidopsis mutant displaying increased transgene silencing (its1). Reduced accumulation of transgene mRNA in the its1 mutant background was accompanied by accumulation of transgene‐specific siRNAs and was overcome by potyvirus infection. We therefore speculated that ITS1 is a suppressor of post‐transcriptional gene silencing. Map‐based cloning and subsequent complementation revealed that ITS1 encodes DECAPPING 2 (DCP2), which is crucial for decapping, a prerequisite for mRNA degradation. In agreement with the proposed function of DCP2, we found a reduced accumulation of uncapped mRNA in the its1 mutant. Furthermore, silencing in the its1 mutant was dependent on RDR6 function, suggesting that reduced decapping leads to accumulation of aberrant capped mRNA. Hence, we provide evidence for a class of aberrant mRNA that accumulates upon impaired mRNA decapping and triggers post‐transcriptional gene silencing in Arabidopsis. As DCP2 knockouts cause post‐embryo lethality, we isolated a hypomorphic dcp2 allele, providing insights into mRNA degradation and its interplay with post‐transcriptional gene silencing.
The incidence of Clostridioides difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. Here ...we report on the development of mRNA-LNPs encoding camelid-derived VHH-based neutralizing agents (VNAs) targeting toxins A and/or B of C. difficile. In preclinical models, intravenous administration of the mRNA-LNPs provided serum VNA levels sufficient to confer protection of mice against severe disease progression following toxin challenge. Furthermore, we employed an mRNA-LNP encoded effector antibody, a molecular tool designed to specifically bind an epitopic tag linked to the VNAs, to prolong VNA serum half-life. Co-administration of VNA-encoding mRNA-LNPs and an effector antibody, either provided as recombinant protein or encoded by mRNA-LNP, increased serum VNA half-life in mice and in gnotobiotic piglets. Prolonged serum half-life was associated with higher concentrations of serum VNA and enhanced prophylactic protection of mice in challenge models.
More than two years after the emergence of SARS-CoV-2, 33 COVID-19 vaccines, based on different platforms, have been approved in 197 countries. Novel variants that are less efficiently neutralised by ...antibodies raised against ancestral SARS-CoV-2 are circulating, highlighting the need to adapt vaccination strategies. Here, we compare the immunogenicity of a first-generation mRNA vaccine candidate, CVnCoV, with a second-generation mRNA vaccine candidate, CV2CoV, in rats. Higher levels of spike (S) protein expression were observed in cell culture with the CV2CoV mRNA than with the CVnCoV mRNA. Vaccination with CV2CoV also induced higher titres of virus neutralising antibodies with accelerated kinetics in rats compared with CVnCoV. Significant cross-neutralisation of the SARS-CoV-2 variants, Alpha (B.1.1.7), Beta (B.1.351), and the ‘mink’ variant (B1.1.298) that were circulating at the time in early 2021 were also demonstrated. In addition, CV2CoV induced higher levels of antibodies at lower doses than CVnCoV, suggesting that dose-sparing could be possible with the next-generation SARS-CoV-2 vaccine, which could improve worldwide vaccine supply.
While active immunization elicits a lasting immune response by the body, passive immunotherapy transiently equips the body with exogenously generated immunological effectors in the form of either ...target-specific antibodies or lymphocytes functionalized with target-specific receptors. In either case, administration or expression of recombinant proteins plays a fundamental role. mRNA prepared by in vitro transcription (IVT) is increasingly appreciated as a drug substance for delivery of recombinant proteins. With its biological role as transient carrier of genetic information translated into protein in the cytoplasm, therapeutic application of mRNA combines several advantages. For example, compared to transfected DNA, mRNA harbors inherent safety features. It is not associated with the risk of inducing genomic changes and potential adverse effects are only temporary due to its transient nature. Compared to the administration of recombinant proteins produced in bioreactors, mRNA allows supplying proteins that are difficult to manufacture and offers extended pharmacokinetics for short-lived proteins. Based on great progress in understanding and manipulating mRNA properties, efficacy data in various models have now demonstrated that IVT mRNA constitutes a potent and flexible platform technology. Starting with an introduction into passive immunotherapy, this review summarizes the current status of IVT mRNA technology and its application to such immunological interventions.
First attempts to use exogenous mRNA for protein expression in vivo were made more than 25 years ago. However, widespread appreciation of in vitro transcribed mRNA as a powerful technology for ...supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such in vivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient in vivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.
The delivery of genetic information has emerged as a valid therapeutic approach. Various reports have demonstrated that mRNA, besides its remarkable potential as vaccine, can also promote expression ...without inducing an adverse immune response against the encoded protein. In the current study, we set out to explore whether our technology based on chemically unmodified mRNA is suitable for passive immunization. To this end, various antibodies using different designs were expressed and characterized in vitro and in vivo in the fields of viral infections, toxin exposure, and cancer immunotherapies. Single injections of mRNA–lipid nanoparticle (LNP) were sufficient to establish rapid, strong, and long‐lasting serum antibody titers in vivo, thereby enabling both prophylactic and therapeutic protection against lethal rabies infection or botulinum intoxication. Moreover, therapeutic mRNA‐mediated antibody expression allowed mice to survive an otherwise lethal tumor challenge. In conclusion, the present study demonstrates the utility of formulated mRNA as a potent novel technology for passive immunization.
Synopsis
As a transient carrier of information, exogenous mRNA is considered a possibly powerful tool to instruct a body to produce its own therapeutics. Antibody‐encoding, sequence‐optimized but otherwise unmodified mRNA in LNPs empowered mice to successfully fight various biological threats.
Sequence‐optimization enables efficient antibody expression from in vitro transcribed mRNA.
Exogenous mRNA can instruct cells to produce various types of antibodies.
Optimized mRNA in lipid nanoparticles provides efficient antibody expression in hepatocytes upon intravenous administration.
mRNA‐mediated antibody expression can protect mice against toxins, viruses, and tumors.
As a transient carrier of information, exogenous mRNA is considered a possibly powerful tool to instruct a body to produce its own therapeutics. Antibody‐encoding, sequence‐optimized but otherwise unmodified mRNA in LNPs empowered mice to successfully fight various biological threats.
Post-transkriptionelles gene silencing (PTGS) kann ein limitierender Faktor für die Expression von Transgenen sein. Transgene mRNAs können von der RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) erkannt ...werden, was die Synthese doppelsträngiger RNA (dsRNA) initiiert und die Akkumulation von small interfering RNAs (siRNAs) zur Folge hat. Diese vermitteln die sequenzspezifische endonukleolytische Spaltung einer Ziel-mRNA und verhindern somit deren Akkumulation. Für die Initiierung des Transgen Silencing spielen quantitative aber auch vor allem qualitative Faktoren eine Rolle. Mutationen im mRNA Metabolismus können die Balance zwischen Synthese und Degradation von mRNAs stören und folglich das Transgen Silencing verursachen. Aufgrund eines gestörten mRNA Metabolismus können sogenannte aberrante mRNAs akkumulieren, die durch RDR6 erkannt werden und die Silencing Kaskade in Gang bringen. Im Zuge dieser Doktorarbeit sollten neue Erkenntnisse zur Initiierung des Transgen Silencing gewonnen werden. Hierbei stand die Identifizierung und Charakterisierung von Genen, deren Mutation zu verstärktem Transgen Silencing führt, im Vordergrund. Im Vorfeld wurde eine transgene Arabidopsis-Linie EMSmutagenisiert und nach Mutanten mit verstärkten Transgen Silencing durchmustert. Geeignete Mutanten zeichneten sich durch einen Verlust der MP17:GFP mRNA Akkumulation aus. Es konnte die MP17:GFP Suppressormutante tss4e (transgene silencing suppressor) mit einer eindeutig genetisch nachvollziehbaren Vererbung isoliert werden. In dieser akkumulieren Transgen-spezifische siRNAs, was durch Infektion mit dem Potyvirus TuMV überkommen werden konnte. Daher wurde angenommen, dass es in der tss4e Mutante zum PTGS der Transgene gekommen war. Durch die Marker-gestützte Kartierung konnte eine Mutation im AtDCP2 Gen identifiziert werden. Die Genidentität wurde durch Komplementation des Mutanten Phänotyps und der Analyse eines weiteren dcp2 Allels, das nicht in der Lage war die Mutante zu komplementieren, bestätigt. Anhand einer Protein-Modellierung wurde postuliert, dass die tss4e spezifische R138C Mutation vermutlich die katalytische Funktion des DCP2 Proteins in den Mutanten beeinträchtigt, was zu einer Veränderung des mRNA Metabolismus führt. Durch in vivo Analysen konnte bestätigt werden, dass die Menge ungecappter mRNA in den Mutanten reduziert war, was diese Vermutung bekräftigte. Durch Kreuzung in einen RDR6-defizienten Hintergrund konnte bestätigt werden, dass das Transgen Silencing in den tss4e Mutanten RDR6-abhängig initiiert wird und vermutlich auf die Akkumulation aberranter mRNA zurückzuführen ist. Daher kommt es in der tss4e Mutante vermutlich zur Akkumulation gecappter mRNA ohne Poly-(A) Schwanz. Ein weiteres charakteristisches Merkmal der tss4e Mutante ist die Akkumulation von Anthocyanen in Source-Blättern. Durch Transkriptom-Analysen konnte beobachtet werden, dass dies möglicherweise auf eine Veränderung der TAS4 ta-siRNA Biogenese zurückzuführen ist. Diese bedingt eine Induktion der PAP1/2 Transkriptionsfaktoren und Anthocyan-Syntheseassoziierter Genen. Somit konnte durch die Analyse der tss4e Mutante ein experimenteller Hinweis auf die postulierte Assoziation von TAS4 und seiner Regulation der AnthocyanBiosynthese erbracht werden. Der äußerliche Phänotyp der tss4e Mutante ließ sich verstärkt in der dcp2-1/tss4e Mutante beobachten, was demonstriert, dass mit tss4e erstmals ein hypomorphes dcp2 Allel isoliert werden konnte. Im Gegensatz zu post-embryoletalen dcp Nullmutanten kann mit tss4e im Folgenden die Relevanz der DCP2 Funktion für die Entwicklung von Arabidopsis untersucht werden.
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