The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.

The present invention is concerned with linear double stranded DNA, which is coupled to a single support or a tag at the 3′ end of its non-coding strand and methods for producing said linear double stranded DNA. The present invention further relates to the use of said linear double stranded DNA in an RNA in vitro transcription reaction and also to a method for producing RNA in vitro. The present invention also relates to a bioreactor for RNA in vitro transcription.

The present invention relates to a method of obtaining an enriched population of a target polynucleotide using a synthetic single guide RNA (sgRNA) for an sgRNA-guided nucleic acid-binding protein, as well as to a method of obtaining a pool of target-irrelevant synthetic single guide RNAs (sgRNAs) for a sgRNA-guided nucleic acid-binding protein. Also provided is a target polynucleotide and sgRNAs obtainable by the methods of the invention. Further envisaged is a kit comprising a pool of sgRNAs obtainable by the method of the invention, and the use of a pool of sgRNAs obtainable by the methods of the invention.

The present invention relates to a method of obtaining an enriched population of a target polynucleotide using a synthetic single guide RNA (sgRNA) for an sgRNA-guided nucleic acid-binding protein, as well as to a method of obtaining a pool of target-irrelevant synthetic single guide RNAs (sgRNAs) for a sgRNA-guided nucleic acid-binding protein. Also provided is a target polynucleotide and sgRNAs obtainable by the methods of the invention. Further envisaged is a kit comprising a pool of sgRNAs obtainable by the method of the invention, and the use of a pool of sgRNAs obtainable by the methods of the invention.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

This invention relates to a novel composition and method for RNA/mRNA production as well as amplification using viral RNA replicase and/or RNA-dependent RNA polymerase (RdRp) enzymes and the use of associated RNA/mRNA products thereof. The present invention can be used for manufacturing and amplifying all varieties of RNA/mRNA sequences carrying at least a replicase/RdRp-binding site in the 5′- or 3′-end, or both. The RNA/mRNA so obtained is useful for not only producing mRNA vaccines and/or RNA-based medicines but for generating the mRNA-associated proteins, peptides, and/or antibodies under an in-vitro as well as in-cell translation condition. Principally, the present invention is a novel RNA replicase/RdRp-mediated RNA/mRNA amplification method, namely Replicase Cycling Reaction (RCR). The RNA replicases involved in RCR include but not limited to viral and/or bacteriophage RNA-dependent RNA polymerases (RdRp) in either modified or non-modified mRNA and/or protein compositions, particularly coronaviral (e.g. COVID-19) and hepatitis C viral (HCV) RdRp enzymes.

The present invention relates to the field of recombining production of biological molecules in host cells. The invention provides nucleic acid constructs that allow to modify expression of a desired gene using both in vitro and in vivo gene expression systems. The constructs can advantageously be used to produce a variety of biological molecules recombinantly in industrial scales, e.g. human milk oligosaccharides (HMO).

Provided herein is a nucleotide cassette comprising an inducible promoter, a nucleotide sequence that corresponds to at least one single stranded RNA diagnostic target, a nucleotide sequence that encodes artemin, a molecular switch and a nucleotide sequence that encodes a DNAse enzyme and is under control of the molecular switch, wherein the single stranded RNA diagnostic target is a sequence detected by a molecular diagnostic assay. In some embodiments the nucleotide cassette can be used to obtain an RNA expression product. Also provided are vectors and cells comprising the nucleotide cassette or the RNA expression product thereof. The nucleotide cassette can further be used to obtain a diagnostic control composition comprising a non-pathogenic recombinant bacterium having a modified genetic content comprising the nucleotide cassette and to methods of producing such recombinant bacteria.

Provided herein is a nucleotide cassette comprising an inducible promoter, a nucleotide sequence that corresponds to at least one single stranded RNA diagnostic target, a nucleotide sequence that encodes artemin, a molecular switch and a nucleotide sequence that encodes a DNAse enzyme and is under control of the molecular switch, wherein the single stranded RNA diagnostic target is a sequence detected by a molecular diagnostic assay. In some embodiments the nucleotide cassette can be used to obtain an RNA expression product. Also provided are vectors and cells comprising the nucleotide cassette or the RNA expression product thereof. The nucleotide cassette can further be used to obtain a diagnostic control composition comprising a non-pathogenic recombinant bacterium having a modified genetic content comprising the nucleotide cassette and to methods of producing such recombinant bacteria.

A human chimeric protein(1) is described, expressed by a viral vector (2) designed for treating patients affected by genetic disorders, composed of a first cDNA sequence [SEQ. 2] of a N-terminal extracellular portion of a human receptor (4) of low-density lipoproteins (5) (hLDLR), fused with a second cDNA sequence [SEQ. 3] of the human transferrin (7) (hTf).