Retroviral packaging cassettes amplified in the cytoplasm by autocatalytic togavirus vectors
AssigneeThe Government of the United States as represented by the Secretary of the Department of Human Services
InventorsRICHARD MORGAN, JARMO WAHLFORS, KLEANTHIS XANTHOPOULOS
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Abstract
This invention provides a Togavirus-amplified retrovirus vector and a novel method for packaging a retrovirus cassette that contains a heterologous nucleic acid, which is amplified in a packaging cell cytoplasm by a Togavirus vector. The retroviral cassette is packaged into infectious retrovirus particles by retroviral packaging cells. These retroviral particles carrying the retroviral packaging cassette are then used to infect host cells.
Claims23
A method for packaging a heterologous nucleic acid into a retrovirus vector within a packaging cell which comprises:
- (a) selecting a packaging cell that produces retroviral components including reverse transcriptase, integrase, gag proteins, and envelope proteins;
- (b) transducing the cell with RNA sequences encoding:
- (i) the replicase gene cluster of a togavirus said replicase operably linked to a ribosomal binding site recognized by ribosomes of the packaging cell,
- (ii) a Togavirus replicase target sequence comprising a replicase promoter operably linked to a replicase amplification region said region comprising a subgenomic promoter and a retroviral packaging cassette comprising:
- R and U5 regions at the 5′ end, reverse transcriptase recognition sites, a retroviral packaging sequence, a eukaryote expression cassette comprising a promoter operably linked to a heterologous nucleic acid, and U3 and R regions at the 3′ end of the cassette;
- wherein the R, U5, and U3 regions, a primer binding site and a polypurine tract, and the packaging sequence are compatible with the retroviral components of the packaging cell;
- and,
- (c) culturing the cell under conditions that permit the replicase gene cluster to be translated;
- that permit the replicase to amplify the expression cassette and that permit the retroviral components to package the expression cassette into retrovirus vector particles.
A nucleic acid sequence comprising the following elements:
- (i) a replicase gene cluster of a Togavirus said replicase operably linked to a ribosomal binding region recognized by eukaryotic ribosomes,
- (ii) a replicase target sequence comprising a replicase promoter operably linked to a replicase amplification region said region comprising a subgenomic promoter and a retroviral packaging cassette comprising:
- R and U5 regions at the 5′ end, reverse transcriptase recognition sites, a retroviral packaging sequence, a eukaryote expression cassette comprising a promoter operably linked to a heterologous nucleic acid, and U3 and R regions at the 3′ end of the cassette;
- wherein the R, U5, and U3 regions, a primer binding site and a polypurine tract, and the packaging sequence are able to direct packaging of the replicase amplification region into a retrovirus vector particles and replication in a target cell.
Dependencies are inferred from the claim text.