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.
Claims25
A recombinant DNA molecule for transiently expressing alphavirus structural proteins comprising a constitutive promoter for directing the transcription of RNA from a DNA sequence operably linked to a DNA sequence comprising 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 method of producing infectious, replication-defective alphavirus replicon particles, comprising introducing into a population of cells,
- (i) a first recombinant DNA molecule for transiently expressing alphavirus structural proteins comprising a constitutive promoter for directing the transcription of RNA from a DNA sequence operably linked to a DNA sequence encoding at least one alphavirus structural protein-coding sequence, with the proviso that the DNA sequence does not encode alphaviral 5′ or 3′ replication recognition sequences or an alphavirus subgenomic promoter,
- (ii) a second recombinant DNA molecule for transiently expressing alphavirus structural proteins comprising a constitutive promoter for directing the transcription of RNA from a DNA sequence operably linked to a DNA sequence encoding at least one alphavirus structural protein-coding sequence not present in the at least one alphavirus structural protein-coding sequence of the first recombinant DNA molecule, with the proviso that the DNA sequence does not encode alphaviral 5′ or 3′ replication recognition sequences or an alphavirus subgenomic promoter, wherein the first and second recombinant DNA molecules together encode all alphavirus structural proteins;
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- (iii) an alphavirus replicon RNA encoding at least one heterologous RNA, under conditions wherein the first and second recombinant DNA molecules are expressed from autonomous plasmids, whereby the alphavirus structural protein coding sequences are transiently expressed from the first and second recombinant DNA molecules to produce alphavirus structural proteins and infectious, replication-defective alphavirus replicon particles are produced and wherein the first and second recombinant DNA molecules are introduced into the population of cells by electroporation.
Dependencies are inferred from the claim text.