Transient expression vectors, preparation and uses thereof
AssigneeNewvectys
InventorsChamsy Sarkis, Dorothee Altemir, Nicolas Grandchamp, Stephanie Philippe
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Abstract
The present invention describes a recombinant retroviral vector which cannot by itself achieve complete reverse transcription as well as its uses, in particular for transiently transferring in vitro, ex vivo or in vivo at least one ribonucleic acid sequence of interest in a cell. Such a transient transgene expression is of interest in the context of research, therapy and more generally in the field of biotechnology.
Claims14
A recombinant HIV-1 vector which cannot on its own achieve complete reverse transcription wherein the vector is a recombinant ribonucleic vector genome comprising (i) an intact 5′ LTR retroviral sequence and a 3′ LTR retroviral sequence flanking:
- an intact primer binding site (PBS) sequence, a retroviral psi encapsidation sequence and at least one transgene, and (ii) a coding sequence for a reverse transcriptase which is non-functional for completing reverse transcription, wherein the non-functional reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at position 110 of SEQ ID NO:
- 1 and/or a mutation affecting the RNAseH domain at position 478 of SEQ ID NO:
- 1, and wherein formation of DNA from the vector is prevented due to the presence of the non-functional reverse transcriptase.
A method for producing an HIV-1 vector comprising expressing within a cell the following transcomplementation elements:
- i) a transcomplementation capsid cassette, optionally split into several cassettes, comprising sequences derived from an HIV-1 genome encoding a retroviral gag sequence, said transcomplementation capsid cassette lacking any functional psi encapsidation signal, wherein said transcomplementation capsid cassette comprises a retroviral pol sequence encoding a reverse transcriptase which is non-functional for complete reverse transcription wherein the non-functional reverse transcriptase coding sequence contains a mutation affecting the DNA polymerase domain at position 110 of SEQ ID NO:
- 1 and/or a mutation affecting the RNAseH domain at position 478 of SEQ ID NO:
- 1, and wherein said transcomplementation capsid cassette comprises an intact primer binding site (PBS) sequence,
- ii) a transcomplementation envelope cassette encoding for an envelope glycoprotein, and
- iii) a transcomplementation vector cassette encoding for an HIV-1 recombinant ribonucleic vector genome comprising an intact 5′ LTR retroviral sequence and a 3′ LTR retroviral sequence flanking a retroviral psi encapsidation sequence, at least one transgene and, optionally, at least one post-transcriptional regulatory sequence, and
- recovering the HIV-1 vectors produced from the expression of the elements encoded by each cassette.
Dependencies are inferred from the claim text.