Small molecules for increasing precise genome editing efficiency
View Patent ↗The present invention relates to compounds suitable to increase precise genome editing efficiency in a eukaryotic target cell or target organism. Thus, the present invention can be applied in gene therapy.
1 . A method for editing a genome of a eukaryotic genome editing target cell or a eukaryotic genome editing target organism, the method comprising introducing Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof into the eukaryotic genome editing target cell or the eukaryotic genome editing target organism, wherein the eukaryotic genome editing target cell or the eukaryotic target genome editing organism comprises a DNA cleavage enzyme selected from the group consisting of a CRISPR/Cas9 enzyme, a mutated nickase version of CRISPR/Cas9, a CRISPR/Cpf1 enzyme, or a split-fusio version of any of the foregoing, wherein introducing the Nedisertib (M3814) or the physiologically acceptable salt or solvate thereof results in an increase in homologous recombination (HDR)-mediated genome-editing efficiency that is greater than that achieved by treatment with known DNA-PKcs inhibitors under equivalent conditions.
2 . The method according to claim 1 , wherein the genome editing target cell is a vertebrate genome editing target cell.
3 . The method according to claim 1 , wherein the genome editing target cell is a mammalian genome editing target cell.
4 . The method according to claim 3 , wherein said mammalian cell is a rodent genome editing target cell or a human genome editing target cell.
5 . The method according to claim 1 , wherein the genome editing target cell is a stem cell including an induced or embryonic pluripotent stem cell of a eukaryotic genome editing target organism.
6 . The method according to claim 5 , wherein the induced or embryonic pluripotent stem cell of a eukaryotic genome editing target organism is a human induced or embryonic pluripotent stem cell.
7 . The method according to claim 1 , wherein the genome editing target organism is a mammalian genome editing target organism.
8 . The method according to claim 1 , further comprising introducing at least one further compound different from Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof, into said eukaryotic genome editing target cell or said eukaryotic genome editing target organism.
9 . The method according to claim 8 , wherein the at least one further compound is selected from the group consisting of:
(a) a HDAC inhibitor,
(b) a NAE inhibitor,
(c) a RPA inhibitor, and
(d) a combination of at least 2 of compounds (a), (b) and/or (c).
10 . The method according to claim 9 , wherein the compound (a) is Trichostatin A, the compound (b) is MLN4924, and/or the compound (c) is NSC15520.
11 . The method according to claim 8 , wherein the at least one further compound is selected from the group consisting of an inhibitor of the microhomology mediated end-joining (MMEJ) pathway and an inhibitor of the single strand annealing (SSA) pathway.
12 . The method according to claim 11 , wherein the inhibitor of the MMEJ pathway is selected from the group consisting of an inhibitory RNA molecule directed against the PolQ mRNA, a DNA cleavage enzyme adapted for nicking the coding strand of a PolQ gene and a combination thereof.
13 . The method according to claim 12 , wherein the inhibitory RNA molecule directed against the PolQ mRNA is an inhibitory RNA molecule which binds to the PolQ mRNA upstream a sequence encoding a first RAD51 binding domain.
14 . The method according to claim 11 , wherein the inhibitor of the SSA pathway is selected from 6-hydroxy-dopa, and 5-aminoimidazol-4-carboxamide (AICA).
15 . The method according to claim 14 , wherein the 5-aminoimidazol- 4-carboxamide (AICA) is AICA ribonucleotide 5′-monophosphate (AICAR).
16 . The method according to claim 1 , wherein the genome editing comprises introducing a staggered cut or a blunt-ended cut into the double-stranded genome of the eukaryotic genome editing target cell or the eukaryotic genome editing target organism.
17 . The method according to claim 16 , wherein the staggered cut is a staggered cut with 5′ overhangs.
18 . The method according to claim 1 , wherein the DNA cleavage enzyme is a CRISPR/Cas9D10A enzyme.
19 . The method according to claim 1 , wherein the genome editing further comprises introducing a donor DNA molecule carrying a desired mutation into the genome editing target cell or the genome editing target organism, wherein said donor DNA molecule is a single-stranded or double-stranded DNA molecule.
20 . The method according to claim 19 , wherein said donor DNA molecule is a single-stranded DNA molecule.
21 . The method according to claim 1 , wherein said Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof is introduced in combination with a knock-down or inhibition of endogenous Polymerase Theta in the genome editing target cell or the genome editing target organism.
22 . The method according to claim 1 , wherein the genome of a eukaryotic genome editing target cell or the eukaryotic genome editing target organism is edited in vivo or ex vivo and used in gene therapy.
23 . The method of claim 1 , wherein introducing the Nedisertib (M3814) or the physiologically acceptable salt or solvate thereof produces HDR-mediated genome-editing efficiency greater than that obtained with NU7026 and NU7441 under equivalent conditions.
24 . A method for editing a genome of a eukaryotic genome editing target cell in vitro, the method comprising introducing Nedisertib (M 3814 ) or a physiologically acceptable salt or solvate thereof into the eukaryotic genome editing target cell, wherein the eukaryotic genome editing target cell comprises a DNA cleavage enzyme selected from the group consisting of a CRISPR/Cas9 enzyme, a mutated nickase version of CRISPR/Cas9, a CRISPR/Cpf 1 enzyme, or a split-fusion version of any of the foregoing, wherein introducing the Nedisertib (M3814) or the physiologically acceptable salt or solvate thereof produces a homologous recombination (HDR)-mediated genome-editing efficiency greater than that obtained with known DNA-PKcs inhibitors under equivalent conditions.
25 . The method according to claim 24 , wherein said eukaryotic genome editing target cell is a mammalian genome editing target cell.
26 . The method according to claim 24 , wherein said mammalian genome editing target cell is a human genome editing target cell.
27 . The method of claim 24 , wherein introducing the Nedisertib (M3814) or the physiologically acceptable salt or solvate thereof produces HDR-mediated genome-editing efficiency greater than that obtained with NU7026 and NU7441 under equivalent conditions.