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.

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).

Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening.

Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3' UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3' UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) the delivery of vaccine antigens in order to mount T cell immune responses and (2) the introduction of reprogramming factors into differentiated cells in order to induce pluripotency, that mRNAs tagged with the 3' UTR elements discovered in this study outperform those with commonly used 3' UTRs. This approach further leverages the utility of mRNA as a gene therapy drug format.

Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference.

RNA interference defends against RNA viruses and retro-elements within an organism's genome. It is triggered by duplex siRNAs, of which one strand is selected to confer sequence-specificity to the RNA induced silencing complex (RISC). In Drosophila, Dicer-2 (Dcr-2) and the double-stranded RNA binding domain (dsRBD) protein R2D2 form the RISC loading complex (RLC) and select one strand of exogenous siRNAs according to the relative thermodynamic stability of base-pairing at either end. Through genome editing we demonstrate that Loqs-PD, the Drosophila homolog of human TAR RNA binding protein (TRBP) and a paralog of R2D2, forms an alternative RLC with Dcr-2 that is required for strand choice of endogenous siRNAs in S2 cells. Two canonical dsRBDs in Loqs-PD bind to siRNAs with enhanced affinity compared to miRNA/miRNA* duplexes. Structural analysis, NMR and biophysical experiments indicate that the Loqs-PD dsRBDs can slide along the RNA duplex to the ends of the siRNA. A moderate but notable binding preference for the thermodynamically more stable siRNA end by Loqs-PD alone is greatly amplified in complex with Dcr-2 to initiate strand discrimination by asymmetry sensing in the RLC.

Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones.

We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.

Transcription-dependent spatial arrangements of CFTR and conserved adjacent loci are not conserved in human and murine nuclei.

The human genes CFTR, ASZ1/GASZ, and CTTNBP2/CORTBP2 map to adjacent loci on chromosome 7q31 and display characteristic patterns of nuclear positioning, which strictly correlate with the state of activity. To address the evolutionary conservation of gene positioning, we investigated transcriptional activity and nuclear positioning of the highly conserved murine orthologs and of additional murine genes mapping to the region of conserved synteny on mouse chromosome 6. The results showed that all murine loci investigated constitutively localized in the nuclear interior irrespective of their functional state. Silenced loci did not display preferential association with the nuclear periphery or with chromocenters, respectively, and no differential positioning with respect to the chromosome 6 territory could be observed. This positional behavior of the murine loci was in striking contrast to the positioning of the human orthologs, and the results show that the transcription-dependent positioning of CFTR and adjacent loci has not been conserved. The findings reveal that the nuclear organization of conserved chromosomal regions can change rapidly during evolution and is not always as highly conserved as other features of chromosome organization. Furthermore, the results suggest that the way how nuclear positioning contributes to the regulation of conserved loci can be different in different vertebrate species.