Disclosed herein are nucleosides, nucleotides and analogs thereof, pharmaceutical compositions that include one or more of nucleosides, nucleotides and analogs thereof, and methods of synthesizing the same. Also disclosed herein are methods of ameliorating and/or treating a disease and/or a condition, including an infection from a paramyxovirus and/or an orthomyxovirus, with a nucleoside, a nucleotide and an analog thereof.

The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

Provided is a method for replicating a mirror nucleic acid, comprising: reacting a mirror nucleic acid template, a mirror nucleic acid primer and mirror dNTPs/rNTPs in the presence of a mirror nucleic acid polymerase, so as to obtain the mirror nucleic acid.

Provided is a new-type cis-replicon construct for efficiently expressing a target protein, which comprises an RNA polymerase coding unit and a target protein coding unit. The replicon construct can drive the replication of the target protein coding unit and reduce or avoid the replication and expression of the RNA polymerase, thereby effectively improving the expression of the target protein and reducing the expression of the non-target protein.

This disclosure provides, among other things, amplifiable nucleic acid constructs for expressing a gene of interest in a cell, e.g., an erythroid cell. The amplifiable nucleic acid construct may contain the gene of interest and an RNA-dependent RNA polymerase (RdRP)-responsive 5 UTR, and may optionally further contain an RdRP-responsive 3 UTR. RdRP may also be provided, e.g., on the same construct or a different construct.

Described are methods for increasing the expression of a transgene in eukaryotic cells by reducing RNA interference (RNAi), and variant cells produce by the method. The methods and variant cells are useful, for example, for the efficient production of therapeutic and industrial polypeptides in eukaryotic cells.

Provided herein are attenuated influenza viruses and methods of making attenuated influenza viruses.

The application relates to the technical field of biomedicine, in particular to a limited self-replicating mRNA molecular system, producing method and use. The limited self-replicating mRNA molecular system including: a first mRNA encoding a mutated alphavirus replicase; and at least one second mRNA encoding a target protein; by generating specific mutation adjustments in the nsP2 subunit of mutated replicase, this limited self-replicating mRNA molecular system can achieve limited self-replication and avoid cytotoxicity; by constructing different mRNA with mutated alphavirus replicase and different target proteins, the mutated alphavirus replicase encoded by the first mRNA can simultaneously replicate multiple different target proteins, achieving sustained expression of multiple target proteins.

Immunogenic compositions and methods of their use in eliciting immune responses to coronaviruses, such as SARS-CoV-2 are provided.

Described herein are cell-selective mRNA constructs that can contain a RNA of interest and one or more miRNA targets. The cell-selective mRNA constructs described herein can be used to express an RNA of interest to a cell in a cell-selective manner.