DNA expression systems based on alphaviruses
AssigneeBioption AB
InventorsHenrik Garoff, Peter Liljeström
Life of this patent
Estimating the term…
Abstract
The disclosure describes recombinant alphavirus RNA molecules and expression of heterologous proteins therefrom in animal cells. Recombinant alphaviruses of the present invention, when made to express an antigenic protein, can be administered as vaccines.
Claims60
A recombinant RNA molecule which can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said alphavirus RNA genome contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made upon introduction of said recombinant RNA into said host cell, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA is introduced into a host cell and further such that the exogenous RNA expresses its biological function in said host cell.
A DNA vector comprising a cDNA having one strand complementary to a recombinant RNA molecule that can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said alphavirus RNA genome contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made when the recombinant RNA is contained in said host cell, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA is contained in said host cell and further such that the exogenous RNA expresses its biological function in said host cell.
A method for producing recombinant alphavirus particles containing a recombinant alphavirus genome, comprising:
- (a) producing a first and a second RNA transcript by in vitro transcription;
- wherein
- (i) said first RNA transcript is made from a first vector comprising a promoter operatively linked to a cDNA encoding an alphavirus RNA that expresses at least one alphavirus structural protein and wherein said alphavirus RNA lacks sequences encoding RNA signals for packaging of RNA into alphavirus nucleocapsid particles, but contains the 5′ and 3′ nucleotides needed for replication of the alphavirus RNA in a host cell and also contains nucleotides encoding a promoter for expression of said RNA encoding said alphavirus structural protein when said first RNA transcript is contained in said host cell;
- (ii) said second RNA transcript is made from a second vector comprising a promoter operatively linked to a cDNA encoding a recombinant alphavirus RNA genome, wherein said recombinant alphavirus RNA genome contains at least one deletion or stop codon mutation in the region encoding said structural protein encoded by said first vector, such that said structural protein that is encoded by said first vector cannot be made when said second RNA transcript is contained in said host cell, and encoding all other structural proteins necessary for assembly of an alphavirus particle, so that said other structural proteins are expressed in said host cell, and further wherein an exogenous RNA sequence, encoding said exogenous protein, is operatively inserted into a region of the recombinant alphavirus RNA genome such that the exogenous RNA expresses said exogenous protein in said host cell;
- (b) transfecting a host cell with said first and second RNA transcripts produced in step (a) and allowing assembly of said recombinant alphavirus particles from structural proteins expressed from said first and second RNA transcripts;
- and
- (c) recovering said recombinant alphavirus particles from cultures of said host cell.
A recombinant alphavirus comprising an alphavirus structural protein containing an amino acid sequence which is exogenous to said alphavirus said sequence being identical to a portion of the envelope glycoprotein of HIV.
A recombinant alphavirus comprising a recombinant alphavirus genome encoding an amino acid sequence exogenous to the wild-type of said alphavirus, wherein said exogenous amino acid sequence comprises an antigenic epitope or determinant inserted into the structural protein homologous in function to the p62 spike protein of Semliki Forest Virus.
A binary vector system comprising a recombinant RNA molecule which can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said alphavirus RNA genome contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made upon introduction of said recombinant RNA into said host cell, wherein said exogenous RNA sequence is inserted at said deletion or downstream from said stop codon, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA is introduced into a host cell and further such that the exogenous RNA expresses its biological function in said host cell;
- and
- a second vector encoding a conditional-lethal mutant of said at least one structural protein of the alphavirus, wherein said second vector expresses the conditional-lethal mutant of said at least one structural protein of the alphavirus upon introduction of the second vector into said host cell.
A binary vector system comprising a first recombinant DNA vector which encodes an RNA molecule which can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said alphavirus RNA genome contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made when said recombinant RNA is contained in said host, wherein said exogenous RNA sequence is inserted at said deletion or downstream from said stop codon, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant DNA is introduced into a host cell and further such that the exogenous RNA expresses its function in said host cell;
- and
- a second recombinant DNA vector encoding a conditional-lethal mutant of said at least one structural protein of the alphavirus, wherein said second vector expresses the conditional-lethal mutant of said at least one structural protein of the alphavirus upon introduction of the second vector into said host cell.
A binary vector system comprising a recombinant RNA molecule which can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said alphavirus RNA genome contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made upon introduction of said recombinant RNA into said host cell, wherein said exogenous RNA sequence is inserted at said deletion or downstream from said stop codon, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA is introduced into a host cell and further such that the exogenous RNA expresses its biological function in said host cell, and wherein said recombinant RNA molecule further contains a conditional-lethal mutation in at least one alphavirus protein encoded by said recombinant RNA molecule;
- and
- a second vector encoding those wild-type structural proteins of the alphavirus not expressed as a result of said deletion or stop codon mutations, wherein said second vector expresses said wild-type structural proteins of the alphavirus upon introduction of the second vector into said host cell.
A recombinant RNA molecule which can be efficiently translated and replicated in an animal host cell, comprising an alphavirus RNA genome and an exogenous RNA sequence, wherein said exogenous RNA sequence is inserted into a deletion in the alphavirus RNA genome, and further wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA is introduced into a host cell and further such that the exogenous RNA expresses its biological function in said host cell.
An RNA vector comprising (i) a protein-coding ribonucleotide sequence that encodes a wild-type alphavirus structural protein or a conditional-lethal mutant thereof, (ii) 5′ and 3′ ribonucleotide sequences encoding signals for replication of an alphavirus RNA in a host cell and (iii) a ribonucleotide sequence functional as a promoter for transcription of said protein-coding ribonucleotide sequence in a host cell;
- wherein said RNA vector lacks ribonucleotide sequences encoding RNA signals for packaging of RNA into alphavirus particles.
A method for inducing an immune response in a subject comprising administering to said subject a chimeric alphavirus particle wherein said recombinant alphavirus particle comprises a chimeric alphavirus envelope protein comprising an immunogenic exogenous amino acid sequence inserted into the amino acid sequence of an envelope protein of said alphavirus.
A method for inducing an immune response in a subject comprising administering to said subject a composition comprising a recombinant RNA molecule which comprises (a) an RNA molecule genome that contains at least one deletion or stop codon mutation such that at least one structural protein of the alphavirus cannot be made upon introduction of said recombinant RNA molecule into a cell of said subject, and (b) an exogenous RNA sequence, wherein said exogenous RNA sequence is operatively inserted into a region of the alphavirus RNA genome which is non-essential to replication of the recombinant RNA molecule such that the exogenous RNA is expressed from an alphavirus transcriptional promoter when the recombinant RNA molecule is introduced into a cell of said subject and further such that the exogenous RNA expresses its biological function in said cell of said subject;
- thereby introducing said recombinant RNA molecule into a cell of said subject and eliciting an immune response in said subject.
Dependencies are inferred from the claim text.