Alphavirus replicon vector systems
AssigneeAlphaVax, Inc.
InventorsJonathan F. Smith, Kurt I. Kamrud, Jonathan O. Rayner, Sergey A. Dryga, Ian J. Caley
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Abstract
The present invention provides compositions useful in and methods for producing populations of infectious, replication-defective alphavirus replicon particles that contain no replication-competent alphavirus particles, as determined by passage on cells in culture. The compositions include helper and replicon nucleic acid molecules that can further reduce the predicted frequency for formation of replication-competent virus and can optimize manufacturing strategies and costs.
Claims65
A recombinant DNA molecule for expressing alphavirus structural proteins comprising a promoter directing the transcription of RNA from a DNA sequence comprising, in order:
- (i) a first nucleic acid sequence encoding at least one alphavirus structural protein,
- (ii) a second nucleic acid sequence encoding a ribozyme,
- (iii) a third nucleic acid sequence encoding an IRES,
- (iv) a fourth nucleic acid sequence encoding at least one alphavirus structural protein, wherein at least one alphavirus structural protein encoded by the fourth nucleic acid sequence is not encoded by the first nucleic acid sequence.
A recombinant DNA molecule for expressing alphavirus structural proteins comprising a promoter directing the transcription of RNA from a DNA sequence comprising, in order:
- (i) a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence,
- (ii) a second nucleic acid sequence encoding an RNA sequence that promotes transcription of a protein coding RNA sequence;
- (iii) a third nucleic acid sequence encoding at least one alphavirus structural protein,
- (iv) a fourth nucleic acid sequence encoding a 3′ alphavirus replication recognition sequence
- (v) a fifth nucleic acid sequence encoding a ribozyme,
- (vi) a sixth nucleic acid sequence encoding an IRES,
- (vii) a seventh nucleic acid sequence encoding at least one alphavirus structural protein,
- wherein at least one alphavirus structural protein encoded by the seventh nucleic acid sequence is not encoded by the third nucleic acid sequence.
A recombinant DNA molecule for expressing alphavirus structural proteins comprising a promoter directing the transcription of RNA from a DNA sequence comprising, in order:
- (i) a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence,
- (ii) a second nucleic acid sequence encoding an IRES,
- (iii) a third nucleic acid sequence encoding at least one alphavirus structural protein,
- (iv) a fourth nucleic acid sequence encoding a 3′ alphavirus replication recognition sequence,
- (v) a fifth nucleic acid sequence encoding a ribozyme,
- (vi) a sixth nucleic acid sequence encoding an IRES,
- (vii) a seventh nucleic acid sequence encoding at least one alphavirus structural protein,
- wherein at least one alphavirus structural protein encoded by the seventh nucleic acid sequence is not encoded by the third nucleic acid sequence.
A recombinant DNA molecule for expressing alphavirus structural proteins comprising a promoter directing the transcription of RNA from a DNA sequence comprising, in order:
- (i) a first nucleic acid sequence encoding an IRES,
- (ii) a second nucleic acid sequence encoding at least one alphavirus structural protein,
- (iii) a third nucleic acid sequence encoding a ribozyme,
- (iv) a fourth nucleic acid sequence encoding an IRES,
- (v) a fifth nucleic acid sequence encoding at least one alphavirus structural protein,
- wherein at least one alphavirus structural protein encoded by the fifth nucleic acid sequence is not encoded by the second nucleic acid sequence.
A recombinant DNA molecule for expressing alphavirus structural proteins comprising:
- (i) a DNA dependent RNA polymerase promoter,
- (ii) an IRES,
- (iii) a nucleic acid sequence encoding an alphavirus capsid protein, which is modified to remove the active site of the autoprotease,
- (iv) a non-autocatalytic protease recognition site, and
- (v) a nucleic acid sequence encoding at least one alphavirus glycoprotein.
A recombinant nucleic acid molecule comprising, in order:
- (i) a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence,
- (ii) a second nucleic acid encoding alphavirus nonstructural proteins nsp1, nsp2, and nsp3;
- (iii) a transcriptional promoter,
- (iv) a nucleic acid encoding at least one heterologous gene of interest,
- (v) an IRES,
- (vi) a third nucleic acid encoding an alphavirus nonstructural protein nsp4, and
- (vii) a fourth nucleic acid encoding a 3′ alphavirus replication recognition sequence.
A recombinant nucleic acid comprising, in order:
- (i) a first nucleic acid sequence encoding a 5′ alphavirus replication recognition sequence,
- (ii) a second nucleic acid encoding alphavirus nonstructural proteins nsp 1, nsp 2, and nsp3;
- (iii) an IRES,
- (iv) a nucleic acid encoding at least one heterologous gene of interest,
- (v) an IRES,
- (vi) a third nucleic acid encoding the alphavirus nonstructural protein nsp4, and
- (vii) a fourth nucleic acid encoding a 3′ alphavirus replication recognition sequence.
A recombinant DNA molecule for expressing alphavirus structural proteins comprising a promoter for directing the transcription of RNA from a DNA sequence operably linked to a DNA sequence encoding a complete alphavirus structural polyprotein-coding sequence, with the proviso that the DNA sequence does not encode alphaviral 5′ or 3′ replication recognition sequences or an alphavirus subgenomic promoter.
A recombinant RNA molecule comprising, in order, a 5′ alphavirus replication recognition sequence, a promoter, a nucleic acid encoding at least one alphavirus structural protein, and a 3′ alphaviral replication recognition sequence, wherein the promoter is operably linked to the nucleic acid sequence encoding at least one alphavirus structural protein, wherein the transcription-initiating sequence and the RNA polymerase recognition sequence are recognized by the nonstructural viral proteins of Venezuelan Equin Encephalitis virus, and wherein the transcription-initiating sequence and the RNA polymerase recognition sequence are derived from a virus other than the alphavirus encoding the structural protein.
An alphavirus structural protein expression system, comprising two RNA molecules, wherein
- (a) a first RNA encodes sequence for viral replicase proteins, and
- (b) a second recombinant RNA encodes sequences for (i) the 5′ replication recognition sequence for a replication complex comprising the viral replicase proteins of (a), (ii) one or more alphavirus structural proteins, and (iii) the 3′ replication recognition sequence for the replication complex comprising the viral replicase proteins of (a),
- wherein, when the first RNA and the second RNA are introduced into a helper cell, the first RNA replicates the second RNA, then the second RNA is translated to produce one or more alphavirus structural proteins.
Dependencies are inferred from the claim text.