Recombinant influenza vectors with a polII promoter and ribozymes for vaccines and gene therapy

A composition comprising a plurality of vectors, comprising

• a) vectors for vRNA production including a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein at least one vector comprises a RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, wherein any vector for vRNA production which does not have a RNA polymerase II promoter, has a RNA polymerase I promoter;
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• b) vectors for vRNA production including a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, or a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP;
• and
• wherein transcription from the RNA polymerase II promoter of the at least one vector comprising the RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, results in a transcript that is a template for influenza virus polymerase, and wherein transfection of a vertebrate cell with the plurality of vectors yields infectious influenza virus.

A method to prepare influenza virus, comprising contacting a vertebrate cell with a vector for vRNA production comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, so as to yield infectious virus, wherein the promoter in at least one vector for vRNA production comprising a viral cDNA comprises a RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to a transcription termination sequence, wherein any vector for vRNA production which does not have a RNA polymerase II promoter, has a RNA polymerase I promoter;

• and
• wherein transcription from the RNA polymerase II promoter of the at least one vector comprising the RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, results in a transcript that is a template for influenza virus polymerase.

A method to prepare influenza virus, comprising contacting a vertebrate cell with a vector for vRNA production comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus cDNA for NB and NA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, wherein at least one vector for vRNA production comprises a RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, wherein any vector for vRNA production which does not have a RNA polymerase II promoter, has a RNA polymerase I promoter;

• and
• wherein transcription from the RNA polymerase II promoter of the at least one vector comprising the RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, results in a transcript that is a template for influenza virus polymerase.

A vertebrate cell comprising a plurality of vectors, comprising

• a) vectors for vRNA production including a vector comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, wherein at least one vector comprises a RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to the transcription termination sequence, wherein any vector for vRNA production which does not have a RNA polymerase II promoter, has a RNA polymerase I promoter;
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• b) vectors for mRNA production including a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, or a vector comprising a promoter operably linked to a DNA segment encoding influenza virus NP;
• and
• wherein the vectors are in present in the cell in an amount that yields titers of infectious influenza virus of at least 5×103 pfu/mL.

A method to prepare influenza virus, comprising contacting a vertebrate cell with a vector for vRNA production comprising a promoter operably linked to an influenza virus PA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB1 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PB2 cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA cDNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M cDNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter operably linked to an influenza virus NS cDNA linked to a transcription termination sequence, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PA, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB1, a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus PB2, and a vector for mRNA production comprising a promoter operably linked to a DNA segment encoding influenza virus NP, wherein the promoter in at least one vector for vRNA production comprises a RNA polymerase II promoter 5′ to a first ribozyme sequence which is 5′ to the viral cDNA which is 5′ to a second ribozyme sequence which is 5′ to a transcription termination sequence, wherein any vector for vRNA production which does not have a RNA polymerase II promoter, has a RNA polymerase I promoter,

• so as to yield titers of infectious influenza virus that are substantially the same as those obtained from a vertebrate cell transfected with a plurality of vectors for influenza virus production comprising vectors for vRNA production for each of PA, PB1, PB2, HA, NP, NA, M and NS which comprise a PolI promoter and a PolI terminator sequence and vectors for mRNA production for at least PA, PB1, PB2 and NP.