Use of aminoquinoline compounds for higher gene integration
OwnerAlexandre JUILLERAT, Philippe DUCHATEAU +1inventors
InventorsAlexandre JUILLERAT, Philippe DUCHATEAU, Ming YANG
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Abstract
The invention provides aminoquinoline compounds as powerful enhancers of genetic recombination in living cells, especially to perform site-directed gene integration of exogenous DNA template by homologous recombination. In particular, disclosed are methods by which cells are treated with chloroquine and/or hydroxychloroquine prior to, or concomitantly with, the introduction of exogenous DNA templates, and optionally in presence of rare-cutting endonucleases, to obtain higher rates of gene integration or correction.
Claims51
1) Use of aminoquinoline compound(s) to increase the frequency of targeted genome modification by a sequence-specific gene editing reagent at a selected locus in the genome of a cell.
4) Method to increase the frequency of targeted modification into the genome of a cell, characterized in that said method comprises the step of treating the cell with a sequence-specific endonuclease or nickase reagent and at least one aminoquinoline compound(s).
6) Method for targeted integration of an exogenous nucleic acid template at a selected locus in the genome of cells, said method comprising the steps of:
- i) contacting the cells with aminoquinoline compound(s);
- ii) introducing into said cells at least one sequence-specific endonuclease or nickase reagent that specifically targets said selected locus,
- iii) introducing into said cells an exogenous nucleic acid template to be integrated at said locus,
- iv) cultivating the cells to induce DNA repair and integration of the exogenous nucleic acid template at said selected locus targeted by said sequence-specific endonuclease or nickase;
- v) optionally, selecting the cells which have integrated the exogenous nucleic acid template at the selected locus in their genome.
39) An oligo capture assay (OCA), characterized in that cells are treated with an aminoquinoline compound(s) to increase oligonucleotide markers integration into the genome of said cells.
40) A composition, therapeutic composition, kit, or nanoparticle comprising:
- (a) an aminoquinoline compound, and
- (b) an exogenous nucleic acid template to be integrated into the genome of a cell at a selected locus.
45) A cell culture medium comprising at least 0.005 mM of an aminoquinoline compound (as claimed before), preferably between 0.01 and 0.5 mM.
46) A cell culture medium comprising between 0.005 and 1 mM, preferably between 0.01 and 0.5 mM, and more preferably between 0.01 and 0.1 mM chloroquine or hydroxychloroquine.
47) An ex-vivo gene therapy method comprising the step of contacting a cell sequentially or concomitantly with (1) an aminoquinoline compound, and (2) an exogenous nucleic acid template, and optionally (3) sequence-specific gene editing reagent, preferably a nuclease or nickase reagent.
48) A gene therapy method comprising the step of administrating, sequentially or in combination:
- (a) an aminoquinoline compound, and (b) an exogenous nucleic acid template, and optionally (c) a sequence-specific gene editing nuclease reagent.
Dependencies are inferred from the claim text.