Circular DNA expression cassettes for in vivo gene transfer
AssigneeRhone-Poulenc Rorer S.A.
InventorsJoel Crouzet, Daniel Scherman, Beatrice Cameron, Pierre Wils, Anne-Marie Darquet
Life of this patent
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Abstract
Double-stranded DNA molecules characterised in that they are circular and in that they essentially include one or more genes of interest.
Claims43
A double-stranded DNA molecule, comprising an expression cassette containing a gene of interest under control of a transcription promoter and a transcription terminator active in a mammalian cell, wherein said molecule:
- is in circular and supercoiled form,
- lacks an origin of replication,
- lacks a marker gene, and
- comprises a region resulting from site-specific recombination between two sequences, said region being located outside the expression cassette.
A recombinant DNA comprising a polynucleotide comprising an expression cassette positioned between two sequences positioned in direct orientation, which recombine by site-specific recombination in the presence of a recombinase, wherein said expression cassette comprises a gene of interest under control of a transcription promoter and a transcription terminator active in a mammalian cell, and wherein said polynucleotide lacks an origin of replication and a marker gene.
A plasmid comprising:
- a) a bacterial origin of replication and optionally, a marker gene;
- and
- b) a polynucleotide comprising an expression cassette positioned between attP and attB sequences of a bacteriophage lambda, P22, .PHI.80, P1, or HP1, or of plasmid pSAM2, positioned in direct orientation, which recombine by site-specific recombination in the presence of a recombinase, wherein said expression cassette comprises a gene of interest under control of a transcription promoter and a transcription terminator active in a mammalian cell, and wherein said polynucleotide lacks an origin of replication and a marker gene.
A plasmid comprising:
- a) a bacterial origin of replication and optionally, a marker gene;
- and
- b) a polynucleotide comprising an expression cassette positioned between two inverted repeat sequences of bacteriophage P1 (loxP region) positioned in direct orientation, which recombine by site specific recombination in the presence of a recombinase;
- wherein the expression cassette comprises a gene of interest under control of a transcription promoter and a transcription terminator active in a mammalian cell, and wherein the polynucleotide lacks an origin of replication and a marker gene.
A plasmid comprising:
- a) an origin of replication and optionally, a marker gene;
- and
- b) a polynucleotide comprising at least one gene of interest and a sequence which interacts specifically with an oligonucleotide to form a triple helix by hybridization, wherein the at least one gene of interest and the oligonucleotide interacting sequence are positioned between two sequences positioned in direct orientation, which recombine by site-specific recombination in the presence of a recombinase, and wherein the polynucleotide lacks an origin of replication and a marker gene.
A plasmid comprising:
- a) an origin of replication and optionally, a marker gene;
- and
- b) a polynucleotide comprising at least one gene of interest and an mrs sequence originating from a par locus of plasmid RK2, wherein the at least one gene of interest and the mrs sequence are positioned between two sequences positioned in direct orientation, which recombine by site-specific recombination in the presence of a recombinase, and wherein the polynucleotide lacks an origin of replication and a marker gene.
A plasmid comprising:
- a) an origin of replication and optionally, a marker gene;
- and
- b) a polynucleotide comprising:
- 1) a first set of two sequences positioned in direct orientation, which recombine by integrase-dependent site-specific recombination;
- 2) a second set of two sequences positioned in direct orientation, which recombine by resolvase-dependent site-specific recombination;
- 3) at least one gene of interest;
- and,
- 4) optionally, a sequence which interacts specifically with an oligonucleotide to form a triple helix by hybridization,
- wherein each integrase-dependent sequence of 1) is positioned next to a resolvase-dependent sequence of 2) and wherein the at least one gene of 3) and the optional oligonucleotide interacting sequence of 4) are placed between the integrase-dependent/resolvase-dependent sequences, and wherein the polynucleotide lacks an origin of replication and a marker gene.
Dependencies are inferred from the claim text.