Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

The T-cell-directed vaccine BNT162b4 encoding conserved non-spike antigens protects animals from severe SARS-CoV-2 infection.

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Exposure to BA.4/5 S protein drives neutralization of Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5 in vaccine-experienced humans and mice.

The SARS-CoV-2 Omicron variant and its sublineages show pronounced viral escape from neutralizing antibodies elicited by vaccination or prior SARS-CoV-2 variant infection owing to over 30-amino acid alterations within the spike (S) glycoprotein. Breakthrough infection of vaccinated individuals with Omicron sublineages BA.1 and BA.2 is associated with distinct patterns of cross-neutralizing activity against SARS-CoV-2 variants of concern (VOCs). In continuation of our previous work, we characterized the effect of Omicron BA.4/BA.5 S glycoprotein exposure on the neutralizing antibody response upon breakthrough infection in vaccinated individuals and upon variant-adapted booster vaccination in mice. We found that immune sera from triple mRNA-vaccinated individuals with subsequent breakthrough infection during the Omicron BA.4/BA.5 wave showed cross-neutralizing activity against previous Omicron variants BA.1, BA.2, BA.2.12.1, and BA.4/BA.5 itself. Administration of a prototypic BA.4/BA.5-adapted mRNA booster vaccine to mice after SARS-CoV-2 wild-type strain-based primary immunization is associated with broader cross-neutralizing activity than a BA.1-adapted booster. Whereas the Omicron BA.1-adapted mRNA vaccine in a bivalent format (wild-type + BA.1) broadens cross-neutralizing activity relative to the BA.1 monovalent booster, cross-neutralization of BA.2 and descendants is more effective in mice boosted with a bivalent wild-type + BA.4/BA.5 vaccine. In naïve mice, primary immunization with the bivalent wild-type + Omicron BA.4/BA.5 vaccine induces strong cross-neutralizing activity against Omicron VOCs and previous variants. These findings suggest that, when administered as boosters, mono- and bivalent Omicron BA.4/BA.5-adapted vaccines enhance neutralization breadth and that the bivalent version also has the potential to confer protection to individuals with no preexisting immunity against SARS-CoV-2.

The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST.

Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.

BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization.

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln-Sm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.

Long-range Ca2+ waves transmit brain-damage signals to microglia.

Microglia are the resident phagocytes of the brain that are responsible for the clearance of injured neurons, an essential step in subsequent tissue regeneration. How death signals are controlled both in space and time to attract these cells toward the site of injury is a topic of great interest. To this aim, we have used the optically transparent zebrafish larval brain and identified rapidly propagating Ca2+ waves that determine the range of microglial responses to neuronal cell death. We show that while Ca2+-mediated microglial responses require ATP, the spreading of intercellular Ca2+ waves is ATP independent. Finally, we identify glutamate as a potent inducer of Ca2+-transmitted microglial attraction. Thus, this real-time analysis reveals the existence of a mechanism controlling microglial targeted migration to neuronal injuries that is initiated by glutamate and proceeds across the brain in the form of a Ca2+ wave.