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The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications.

A single, low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA encoding the pre-membrane and envelope glycoproteins of Zika virus protects both mice and rhesus macaques against infection and elicits rapid and long-lasting neutralizing antibody responses. mRNA vaccine beats Zika virus Public health efforts to combat Zika virus disease are hampered by lack of a safe and efficient vaccine. Drew Weissman and colleagues report the development of a candidate vaccine that is based on chemically stabilized messenger RNA (mRNA) that encodes the premembrane and envelope glycoproteins of the Zika virus. This mRNA is packaged into lipid nanoparticles that can be delivered intradermally. A single dose of the vaccine gave mice and rhesus macaques long-term immunity to the Zika virus. These findings pave the way for the development of candidate vaccines that could protect humans against Zika virus disease. Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults 1 . There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins 2 , 3 . Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30  μ g of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50  μ g was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA–LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)—a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses 1 – 5 . OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2 6 , 7 . Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear 5 , 8 . Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such ‘primary addiction’ decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens. Serum antibody responses to sequential homologous booster vaccines derive overwhelmingly from primary cohort B cells at the expense of de novo responses; this ‘primary addiction’ can be overcome by boosting with variant antigens.

Messenger RNA (mRNA) vaccines represent a new, effective vaccine platform with high capacity for rapid development. Generation of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is a necessity for reducing influenza-associated morbidity and mortality. Here we focus on the development of a universal influenza B virus vaccine based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform. We evaluate vaccine candidates based on different target antigens that afford protection against challenge with ancestral and recent influenza B viruses from both antigenic lineages. A pentavalent vaccine combining all tested antigens protects mice from morbidity at a very low dose of 50 ng per antigen after a single vaccination. These findings support the further advancement of nucleoside-modified mRNA-LNPs expressing multiple conserved antigens as universal influenza virus vaccine candidates. The public health concern caused by influenza B virus is often overlooked, yet represents a significant global burden. Here, the authors evaluate the cellular and humoral immune responses of multivalent vaccine candidates, based on the lipid nanoparticle-encapsulated nucleoside-modified mRNA platform, and demonstrate protection of mice from challenge with a broad panel of influenza B viruses.

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.

Primary human hepatocyte (PHH) transplantation is a promising alternative to liver transplantation, whereby liver function could be restored by partial repopulation of the diseased organ with healthy cells. However, currently PHH engraftment efficiency is low and benefits are not maintained long-term. Here we refine two male mouse models of human chronic and acute liver diseases to recapitulate compromised hepatocyte proliferation observed in nearly all human liver diseases by overexpression of p21 in hepatocytes. In these clinically relevant contexts, we demonstrate that transient, yet robust expression of human hepatocyte growth factor and epidermal growth factor in the liver via nucleoside-modified mRNA in lipid nanoparticles, whose safety was validated with mRNA-based COVID-19 vaccines, drastically improves PHH engraftment, reduces disease burden, and improves overall liver function. This strategy may overcome the critical barriers to clinical translation of cell therapies with primary or stem cell-derived hepatocytes for the treatment of liver diseases. Primary human hepatocyte (PHH) transplantation could be an alternative to liver transplantation. Here, the authors use the mRNA-LNP platform to express growth factors in the liver in a controlled manner to drastically improve PHH engraftment, thus, reducing disease burden and enhancing overall liver function.

Large critical-size bone defects in the oral and craniofacial region are difficult to regenerate. We evaluated the effectiveness of mRNA encoding bone morphogenic protein-2 (BMP-2) in enhancing bone regeneration using a rat calvarial defect model. Two delivery approaches were investigated: (1) in vivo application of BMP-2 mRNA encapsulated in lipid nanoparticles incorporated in a scaffold, and (2) application of ex vivo BMP-2 mRNA-transfected rat bone marrow mesenchymal stem cells (rBMSCs), loaded on a scaffold and implanted into calvarial defects. The direct application of BMP-2 mRNA encapsulated in lipid nanoparticles improved bone regeneration as indicated by micro-computed tomography analysis. The enhancement was even more pronounced with ex vivo transfected rBMSCs. rBMSCs transfected with FGF-2 mRNA did not improve bone regeneration, either alone or combined with BMP-2 mRNA-transfected rBMSCs. Similarly, PDGF-BB mRNA-transfected rBMSCs failed to enhance bone regeneration alone and notably suppressed BMP-2 mRNA-transfected rBMSCs’ effects. Interestingly, BMP-2 mRNA-transfected rat fibroblasts showed comparable bone regeneration to transfected rBMSCs. Osteogenic differentiation was absent in BMP-2 mRNA-transfected rBMSCs, implying that they may primarily serve as a source of translated BMP-2 for bone regeneration rather than undergoing osteogenic differentiation. These findings highlight the translational potential of BMP-2 mRNA for bone regeneration, particularly in oral and craniofacial applications.

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations – the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT – results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells. Compared to traditional Cas9 nucleases prime editors (PEs) are less active. Here the authors use OrthoRep, a yeast-based platform for directed protein evolution to enhance the editing efficiency of PEs: they identify mutations that have a positive effect on kinetics and use this knowledge to generate an efficient in vivo PE.

Background Adenine base editors (ABEs) enable the conversion of A•T to G•C base pairs. Since the sequence of the target locus influences base editing efficiency, efforts have been made to develop computational models that can predict base editing outcomes based on the targeted sequence. However, these models were trained on base editing datasets generated in cell lines and their predictive power for base editing in primary cells in vivo remains uncertain. Results In this study, we conduct base editing screens using SpRY-ABEmax and SpRY-ABE8e to target 2,195 pathogenic mutations with a total of 12,000 guide RNAs in cell lines and in the murine liver. We observe strong correlations between in vitro datasets generated by ABE-mRNA electroporation into HEK293T cells and in vivo datasets generated by adeno-associated virus (AAV)- or lipid nanoparticle (LNP)-mediated nucleoside-modified mRNA delivery (Spearman R  = 0.83–0.92). We subsequently develop BEDICT2.0, a deep learning model that predicts adenine base editing efficiencies with high accuracy in cell lines ( R  = 0.60–0.94) and in the liver ( R  = 0.62–0.81). Conclusions In conclusion, our work confirms that adenine base editing holds considerable potential for correcting a large fraction of pathogenic mutations. We also provide BEDICT2.0 – a robust computational model that helps identify sgRNA-ABE combinations capable of achieving high on-target editing with minimal bystander effects in both in vitro and in vivo settings.

19ISP is a nucleoside-modified mRNA-lipid nanoparticle vaccine that targets 19 Ixodes scapularis proteins. We demonstrate that adult I . scapularis have impaired fecundity when allowed to engorge on 19ISP-immunized rabbits. 19ISP, therefore, has the potential to interrupt the tick reproductive cycle, without triggering some of the other effects associated with acquired tick resistance. This may lead to the development of new strategies to reduce I. scapularis populations in endemic areas.