Optimizing mRNA vaccines and virusmediated CAR T-cell therapy for immuno-oncology: Synthetic tools for gene modification

With the success of mRNA vaccines during the COVID-19 pandemic and CAR T-cell therapies in clinical trials, there is growing opportunity for immunotherapies in the treatment of many types of cancers. Lentiviral vectors have proven effective at delivery of genetic material or gene editing technology for ex vivo processing, but the benefits and promise of Adeno-associated virus (AAV) and mRNA tools for in vivo immunotherapy have garnered recent interest. Here we describe complete synthetic solutions for immuno-oncology research programs using either mRNA-vaccines or virus-mediated cell and gene engineering. These solutions optimize workflows to minimize screening time while maximizing successful research results through: (1) Efficiency in lentiviral packaging with versatility in titer options for high-quality particles. (2) A highthroughput viral packaging process to enable rapid downstream screening. (3) Proprietary plasmid synthesis and preparation techniques to maintain ITR integrity through AAV packaging and improve gene delivery. (4) Rapid synthesis, in vitro transcription, and novel sequencing of mRNA constructs for complete characterization of critical components such as the polyA tail. The reported research demonstrates a streamlined approach that improves data quality through innovative synthesis and sequencing methodologies as compared to current standard practices.

Evolving ribonucleocapsid assembly/packaging signals in the genomes of the human and animal coronaviruses: targeting, transmission and evolution.

A world-wide COVID-19 pandemic intensified strongly the studies of molecular mechanisms related to the coronaviruses. The origin of coronaviruses and the risks of human-to-human, animal-to-human and human-to-animal transmission of coronaviral infections can be understood only on a broader evolutionary level by detailed comparative studies. In this paper, we studied ribonucleocapsid assembly-packaging signals (RNAPS) in the genomes of all seven known pathogenic human coronaviruses, SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63 and compared them with RNAPS in the genomes of the related animal coronaviruses including SARS-Bat-CoV, MERS-Camel-CoV, MHV, Bat-CoV MOP1, TGEV and one of camel alphacoronaviruses. RNAPS in the genomes of coronaviruses were evolved due to weakly specific interactions between genomic RNA and N proteins in helical nucleocapsids. Combining transitional genome mapping and Jaccard correlation coefficients allows us to perform the analysis directly in terms of underlying motifs distributed over the genome. In all coronaviruses, RNAPS were distributed quasi-periodically over the genome with the period about 54 nt biased to 57 nt and to 51 nt for the genomes longer and shorter than that of SARS-CoV, respectively. The comparison with the experimentally verified packaging signals for MERS-CoV, MHV and TGEV proved that the distribution of particular motifs is strongly correlated with the packaging signals. We also found that many motifs were highly conserved in both characters and positioning on the genomes throughout the lineages that make them promising therapeutic targets. The mechanisms of encapsidation can affect the recombination and co-infection as well.Communicated by Ramaswamy H. Sarma.

CryoEM of Viral Ribonucleoproteins and Nucleocapsids of Single-Stranded RNA Viruses.

Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication. Structural determination of RNPs can provide crucial information on the molecular mechanisms of these processes, paving the way for the development of new, more effective strategies to control and prevent the spread of ssRNAv diseases. In this scenario, cryogenic electron microscopy (cryoEM), relying on the technical and methodological revolution it has undergone in recent years, can provide invaluable help in elucidating how these macromolecular complexes are organized, packaged within the virion, or the functional implications of these structures. In this review, we summarize some of the most prominent achievements by cryoEM in the study of RNP and nucleocapsid structures in lipid-enveloped ssRNAv.

Ribonucleocapsid assembly/packaging signals in the genomes of the coronaviruses SARS-CoV and SARS-CoV-2: detection, comparison and implications for therapeutic targeting.

The genomic ssRNA of coronaviruses is packaged within a helical nucleocapsid. Due to transitional symmetry of a helix, weakly specific cooperative interaction between ssRNA and nucleocapsid proteins leads to the natural selection of specific quasi-periodic assembly/packaging signals in the related genomic sequence. Such signals coordinated with the nucleocapsid helical structure were detected and reconstructed in the genomes of the coronaviruses SARS-CoV and SARS-CoV-2. The main period of the signals for both viruses was about 54 nt, that implies 6.75 nt per N protein. The complete coverage of the ssRNA genome of length about 30,000 nt by the nucleocapsid would need 4.4 × 103 N proteins, that makes them the most abundant among the structural proteins. The repertoires of motifs for SARS-CoV and SARS-CoV-2 were divergent but nearly coincided for different isolates of SARS-CoV-2. We obtained the distributions of assembly/packaging signals over the genomes with nonoverlapping windows of width 432 nt. Finally, using the spectral entropy, we compared the load from point mutations and indels during virus age for SARS-CoV and SARS-CoV-2. We found the higher mutational load on SARS-CoV. In this sense, SARS-CoV-2 can be treated as a 'newborn' virus. These observations may be helpful in practical medical applications and are of basic interest. Communicated by Ramaswamy H. Sarma.