Patent Description:
CRISPR is a bacterial nuclease immune system against viral DNA, which has been adopted to accurately cut chromosomal DNA sequences in eukaryotic cells. Such DNA breaks are repaired by two competing pathways: Non-homologous-End-Joining (NHEJ) or Homology directed Repair (HDR).

In NHEJ, the first proteins to bind to the DNA ends are Ku70/Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) (Shrivastav et al. The kinase phosphorylates itself and other downstream effectors at the repair site. Recruitment and phosphorylation of several proteins like Artemis result in end-processing ligation by ligase IV (LIG4), X-ray repair cross-complementing protein <NUM> (XRCC4) and Non-homologous end-joining factor <NUM> (XLF) (Dueva, Iliakis <NUM>).

If this canonical NHEJ pathway is repressed, an alternative NHEJ pathway (A-NHEJ) also referred to as microhomology mediated end-joining (MMEJ) becomes active (Nussenzweig & Nussenzweig <NUM>). It requires Polymerase theta (POLQ), Poly(ADP-ribose)-Polymerase <NUM> (PARP-<NUM>), Werner syndrome ATP-dependent (WRN) helicase and DNA ligase <NUM> (LIG3) or DNA ligase I (LIG1) amongst other proteins. Binding of the MRN-complex (Mre11, Rad50 and Nbs1) complex to the double strand break (DSB) initiates HDR (Shrivastav et al. Along with other proteins like DNA endonuclease RBBP8 (CtIP), Bloom helicase (BLM) and Exonuclease <NUM> (EXO1), terminal nucleotides in the <NUM>' ends are removed, generating long <NUM>' single-stranded DNA (ssDNA) overhangs on both sides of the break of the DNA. These tails are then coated and stabilized by the Replication protein A (RPA) complex, followed by breast cancer <NUM> (BRCA2) assisted generation of a Rad51 nucleoprotein filament (Shrivastav et al. Rad52 facilitates replacement of RPA bound to ssDNA with Rad51 and promotes ssDNA annealing (Grimme et al. Strand invasion with the donor DNA and subsequent DNA synthesis by a polymerase finally results in precisely repaired DNA. The protein kinase ataxia-telangiectasia mutated (ATM) plays a major role in HDR, as it phosphorylates at least <NUM> repair proteins (Shrivastav et al.

NHEJ of CRISPR Cas9-induced DSBs is error prone and frequently introduces insertions and deletions (indels) at the cut site. It is therefore useful for knocking out a targeted gene. In contrast, HDR allows precise repair of a DSB by using a homologous donor DNA sequence. If this donor sequence is provided in the experiment and carries mutations, these will be introduced into the genome.

A requirement for a DSB introduced by Cas9 is an NGG sequence (PAM site) in DNA. Targeting of Cas9 is determined by a bound guide RNA (gRNA) which is complementary to <NUM> nucleotides adjacent to the PAM site. However, the Cas9 nuclease may also cut the genome at sites that carry sequence similarity to those targeted by the gRNA (Fu et al. Those off-target double stranded cuts mean that unwanted mutations can appear elsewhere in the genome together with the desired mutation.

One strategy to reduce such off-target cuts is to use a mutated Cas9 that introduces single-stranded nicks instead of DSBs such as Cas9 D10A (Shen et al. Using two gRNAs to introduce two nicks on opposite DNA strands in close proximity to each other will result in a DSB at the desired locus while reducing the risk of two off-target nicks occurring elsewhere in the genome close enough to cause a DSB. Another strategy is to use Cpf1 (Zetsche et al. This nuclease introduces a staggered cut near a T-rich PAM site and has been shown to produce less off-target effects (Kim et al. <NUM>) (Kleinstiver et al.

In current approaches, precise genome editing (PGE) efficiencies, especially for targeted nucleotide substitutions in stem cells, are usually low, ranging from <NUM>-<NUM>% (Yu et al. <NUM>) (Gonzalez et al. Several researchers addressed the low rate of precise genome editing by trying to promote HDR or decrease NHEJ.

Cell cycle synchronization to G2/M phase was shown to increase PGE with single stranded oligodeoxynucleotide (ssODN) donors in HEK293T cells (from <NUM>% to <NUM>%), human primary neonatal fibroblasts (from undetectable to <NUM>%) and human embryonic stem cells (hESCs) (from undetectable to <NUM>%) (Lin et al. <NUM>) and with double stranded oligodeoxynucleotide (dsODN) donors in hESCs (from <NUM> to <NUM>% after sorting) (Yang et al. <NUM>), since homologous recombination is restricted to this phase and its proteins are upregulated.

Also, improved efficiency was achieved by suppressing key proteins like Ku70/<NUM> and ligase IV with siRNA (from <NUM> to <NUM>%) or co-expression of adenovirus type <NUM> proteins 4E1 B55K and E4orf6 (from <NUM> to <NUM>%) in HEK293/TLR cells using dsODN donors (Chu et al. E1B55K and E4orf6 proteins mediate the ubiquitination and proteosomal degradation of LIG4 among other targets.

A common strategy to increase genome editing has been the use of small molecules. The small molecule ligase IV inhibitor SCR7 has been claimed to block NHEJ and to increase the efficiency of PGE (from <NUM> to <NUM>%) in mouse embryos (Maruyama et al. Other researchers described similar increase in HEK293/TLR cells, a marginal but significant increase in HEK293A, or found no significant effect in mouse embryos, rabbit embryos and human stem cells (Chu et al. <NUM>) (Pinder et al. <NUM>) (Song et al. <NUM>) ( Yang et al. <NUM>) (Zhang et al. Recently, Greco et al. reanalysed the structure and inhibitory properties of SCR7 (Greco et al. They conclude that SCR7 and its derivates are neither selective nor potent inhibitors of human LIG4.

Pharmacological inhibition of DNA-PK, a key protein complex in the NHEJ-pathway, by the small molecules NU7441, KU-<NUM> and NU7026 was shown to moderately reduce the frequency of NHEJ and to increase PGE in HEK293/TLR cells (from <NUM> to <NUM>%), HEK293 (<NUM> to <NUM>%) and human induced pluripotent stem cells (hiPSCs) (from <NUM> to <NUM>%) with dsODN donors and in mouse embryonic fibroblasts (from <NUM> to <NUM>%) with ssODN donors (Robert et al. <NUM>) (Suzuki et al. <NUM>) (Zhang et al.

Also, a single small molecule enhancing homologous recombination with CRISPR-Cas9 has been described. The RAD51 stimulatory compound RS-<NUM> increased PGE in rabbit embryos (from <NUM> to <NUM>%), HEK293A cells (from <NUM> to <NUM>%) and U2OS cells (from <NUM> to <NUM>%)(Song et al. <NUM>) (Pinder et al. <NUM>), but not in hiPSCs (Zhang et al. , <NUM>), all with dsODN donors. No effect of RS-<NUM> on PGE efficiency was found in porcine fetal fibroblasts using ssODN donors (Wang et al.

Furthermore, using a library screen of around <NUM> small molecules, Yu et al. found the β3-adrenergic receptor agonist L755507 to increase PGE in hiPSCs (from <NUM> to <NUM>%) using ssODN and using dsODN donors in mouse ESCs (from <NUM> to <NUM>%), while the repair pathway target of that molecule is not known (Yu et al. Others did not find significant stimulation of PGE by L755507 in HEK293A cells or hiPSCs (Pinder et al. <NUM>) (Zhang et al. Pinder et al. compared SCR7, RS-<NUM> and L755507 singly and together and found no additive effect when adding SCR7 and L755507 together with RS-<NUM> compared to RS-<NUM> alone.

<CIT> describes that certain compounds when applied as a combination of two or more different compounds selected from inhibitors of histone deacetylase (HDAC) inhibitors of NEDD8 activating enzyme (NAE), inhibitors of DNA-dependent Protein Kinase (DNA-PK) in particular of its catalytic subunit (DNA-PKcs), and inhibitors of replication protein A (RPA) and combinations of compounds selected from these different classes of inhibitors, are capable of increasing genome editing efficiency.

Further, <CIT> describes that a DNA-PKcs which is catalytically inactive, but structurally intact, increases precise genome editing efficacy, independently from the presence of compounds as indicated above.

The present inventors have found that certain small molecules known as anticancer agents are capable of increasing precise genome editing demonstrate a surprisingly strong increase in homology-directed repair (HDR) efficiency while only exhibiting moderate toxicity. Further, these small molecules were found to be effective under conditions where previously tested small molecules did not exhibit any effect. Thus, these compounds are suitable both in non-medical applications, e.g. as research tool or in medical applications, e.g. for in vivo or ex vivo use.

Nedisertib is a compound having the structure
<CHM>.

The present inventors have found that Nedisertib (M3814) which is an inhibitor of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an extremely high potency in increasing HDR efficiency in contrast to other known DNA-PKc inhibitors such as NU7026 and NU7441. In particular, the inventors found that administration of M3814 to K562 tumor cells expressing wild-type DNA-PKcs shows a very strong increase in precise genome editing from <NUM>% to <NUM>% while exhibiting only moderate toxicity. In contrast thereto, NU7026 and NU7441 show significantly less precise genome editing efficiency in K562 cells. Corresponding results also were found in human induced pluripotent stem cells. Further, the inventors found that administration of M3814 combination with at least one inhibitor of the microhomology mediated end-joining (MMEJ) pathway and/or at least one inhibitor of the single strand annealing (SSA) pathway may even lead to a synergistic increase in precise genome editing.

Nedisertib is described in <CIT>. It was found to be an inhibitor of serine threonine protein kinases which is suitable for the sensitization of cancer cells to anticancer agents and/or ionizing radiation. It is stated that this effect is caused through specific inhibition of the repair of DNA double strand breaks (non-homologous end-joining). A use of the compound in genome editing is not described in <CIT>.

<NPL>) discloses that Nedisertib (M3814) is a highly potent and selective inhibitor of DNA-PK for use in the treatment of cancer however a use of this compound in genome editing is not described.

<CIT> relates to methods and compositions for inhibiting repair via classic and alternative NHEJ mechanisms in cells to increase gene disruption mediated by inhibiting the critical enzymatic activities of these NHEJ DNA repair pathways, for example using DNA-PKcs such as NU7026 and NU7441.

<CIT> relates to DNA-PKcs capable of increasing genome editing efficiency such as NU7026.

A first aspect of the present invention relates to Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof, for use in medicine in the treatment of disorders associated with an undesired genotype of a patient in need of the treatment, the disorder being a metabolic dysfunction or cancer, comprising genome editing in a eukaryotic target cell or in a eukaryotic target organism, wherein processes for cloning human beings, processes for modifying the germ line genetic identity of human beings, and uses of human embryos for industrial and commercial purposes are excluded.

A further aspect of the present invention relates to an in vitro method for editing the genome of a eukaryotic target cell comprising introducing Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof into the target cell, wherein processes for cloning human beings, processes for modifying the germ line genetic identity of human beings, and uses of human embryos for industrial and commercial purposes are excluded.

Still a further aspect of the present invention relates to the in vitro use of Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof for genome editing in a eukaryotic target cell, particularly in a mammalian target cell, more particularly in a human target cell, wherein processes for cloning human beings, processes for modifying the germ line genetic identity of human beings, and uses of human embryos for industrial and commercial purposes are excluded.

In certain embodiments, Nedisertib (M3814) may be used alone, i.e. as the only active agent in a method comprising genome editing in a eukaryotic target cell or in a eukaryotic target organism, wherein the target organism is a mammalian target organism, particularly a human. In certain further embodiments, Nedisertib (M3814) may be used in combination with other active agents in a method comprising genome editing or in gene therapy in a eukaryotic target cell or target cell organism.

In certain embodiments, Nedisertib may be used in combination with an inhibitor of histone deacetylase (HDAC), an inhibitor of NEDD8 activating enzyme (NAE) and/or inhibitor of replication protein A (RPA).

HDAC inhibitors are known as cytostatic agents for inhibiting tumor cell proliferation by inducing cell cycle arrest, differentiation and/or apoptosis. HDAC inhibitors usually act by binding to the zinc-containing catalytic domain of HDACs. They may be classified according to the chemical moiety that binds to the zinc ion. Examples of suitable classes of HDAC inhibitors are:.

HDAC inhibitors are reviewed e.g. by <NPL>) and <NPL>).

According to the present invention, HDAC inhibitors are preferably selected from synthetic non-nucleosidic compounds, e.g. small molecules having a molecular mass of <NUM> Da or less or <NUM> Da or less. Specific examples of HDAC inhibitors are selected from Trichostatin A, Vorinostat, Entinostat, Panobinostat, Mocetinostat, Belinostat, Romidepsin, MC1568, Tubastatin A HCl, Givinostat, LAQ824, CUDC-<NUM>, Quisinostat 2HCl, Pracinostat, PCl-<NUM>, Droxinostat, PCI-<NUM>, RGFP966, AR-<NUM>, Rocilinostat, Valproic acid, Cl994, CUDC-<NUM>, Tubacin, M344, Resminostat, RG2833, Divalproex Sodium, Scriptaid, Phenylbutyrate, Tubastatin A, CAY <NUM>, NexturastatA, BG45, LMK-<NUM>, Santacruzamate A, BRD73954, HPOB, TMP269, Tasquinimod and 4SC-<NUM> as well as salts or solvates thereof, in particular pharmaceutically acceptable salts or solvates thereof. According to the present invention a preferred HDAC inhibitor is Trichostatin A including salts and solvates thereof.

NAE inhibitors are known as anti-tumor agents as reviewed e.g. by <NPL>) or as antiviral agents as reviewed e.g. by Le-<NPL>).

According to the present invention, NAE inhibitors are preferably selected from synthetic non-nucleosidic compounds, e.g. small molecules having a molecular mass of <NUM> Da or less or <NUM> Da or less. A preferred NAE inhibitor is MLN4924 (Pevonedistat) or any salt or solvate thereof, in particular any pharmaceutically acceptable salt or solvate thereof.

RPA inhibitors are known as anti-tumor agents as reviewed e.g. by <NPL>).

According to the present invention, RPA inhibitors are preferably selected from synthetic non-nucleosidic compounds, e.g. small molecules having a molecular mass of <NUM> Da or less or <NUM> Da or less. Specific examples of RPA inhibitors are NSC15520, TDRL-<NUM> and NSC111847, as well as salts or solvates thereof, in particular pharmaceutically acceptable salts and solvates thereof. According to the present invention a preferred of a RPA inhibitor is NSC15520 including salts and solvates thereof.

Nedisertib (M3814) may further be used in combination with a compound for synchronizing cells in the G2/M phase such as Nocodazole and ABT-<NUM> (Yang et al. , <NUM>), paclitaxel (<NPL>), or colchicine or vincristine (<NPL>), or salts or solvates thereof. In a further embodiment, the combination may include an Alt-NHEJ inhibitor such as NSC19630 or a salt or solvate thereof.

In further embodiments, Nedisertib (M3814) may be used in combination with at least one inhibitor of the microhomology mediated end-joining (MMEJ) pathway and/or at least one inhibitor of the single strand annealing (SSA) pathway. Especially preferred is the use of Nedisertib (M3814), with both at least one inhibitor of the MMEJ pathway and at least one inhibitor of the SSA pathway.

In further embodiments, Nedisertib (M3814) may be used in combination with an inhibitor of the MMEJ pathway, particularly with a knock-down or inhibition of any endogenous polymerase theta (PolQ) in the target cell or target organism. PolQ is needed for alternative NHEJ or MMEJ (<NPL>) and has two RAD51 binding domains that inhibit homologous recombination (<NPL>). A knock-down or inhibition of the endogenous polymerase theta gene in the target cell may be effected, e.g. by CRISPR genome editing, by targeted homologous recombination, by use of RNA interference, e.g. by administering inhibitory RNA molecules such as small interfering RNA molecules (siRNAs), by administering antisense molecules, by transient DNA nicking with a CRISPR enzyme, by administering antibodies against PolQ and/or by administering small molecule inhibitors (<NPL>).

In particular embodiments, inhibition of PolQ is carried out by use of RNA interference, e.g. by administering at least one inhibitory RNA molecule such as an siRNA molecule, more particularly by administering at least one inhibitory RNA molecule such as an siRNA molecule which binds to the PolQ mRNA before the sequence encoding the first RAD51 binding domain and/or a DNA cleavage enzyme adapted for nicking the coding strand of a PolQ gene or any combination thereof.

In further embodiments, Nedisertib (M3814) may be used in combination with an inhibitor of the RAD52 dependent SSA pathway. A knock-down or inhibition of the endogenous RAD52 gene in the target cell may be effected, e.g. by CRISPR genome editing, by targeted homologous recombination, by use of RNA interference, e.g. by administering inhibitory RNA molecules such as small interfering RNA molecules (siRNAs), by administering antisense molecules, by transient DNA nicking with a CRISPR enzyme, by administering antibodies against PolQ and/or by administering small molecule inhibitors (<NPL>; <NPL>).

In particular embodiments, inhibition of RAD52 is carried out by administering at least one small molecule inhibitor such as <NUM>-hydroxy-dopa or a related compound and/or by administering <NUM>-aminoimidazol-<NUM>-carboxamide (AICA) or a related compound, e.g. a nucleoside or nucleotide derivative thereof such as AICA ribonucleotide <NUM>'-monophosphate (AlCAR).

According to certain embodiments Nedisertib (M3814), is used alone, i.e. without concomitant use of other active agents, e.g. without a HDAC inhibitor, a NAE inhibitor and a RPA inhibitor. In further embodiments Nedisertib (M3814) , is used without a further DNA-PKcs inhibitor which is different from a compound of formula (I) or (II).

As indicated above, Nedisertib (M3814) may be used in combination with further active agents. The term "combination" in the context of the present invention encompasses compositions comprising at least two compounds as indicated above together in admixture optionally together with a suitable carrier, e.g. a pharmaceutically acceptable carrier. The term "combination" also encompasses kits comprising at least two compounds as indicated above in separate forms, each optionally together with a suitable carrier, e.g. a pharmaceutically acceptable carrier.

Nedisertib (M3814) is suitable for use in genome editing in a eukaryotic target cell, particularly in a eukaryotic target cell as described in the following, including a vertebrate target cell, e.g. an animal target cell such as a mammalian target cell, e.g. a human target cell, but also target cell from non-human animals such as rodents, e.g. mice or zebrafish including a stem cell, e.g. human stem cell, for example an embryonic stem cell or a pluripotent stem cell. In some embodiments, the target cell is a stem cell of a eukaryotic target organism, including an induced or embryonic pluripotent stem cell such as a human induced or embryonic pluripotent stem cell but also an induced or embryonic pluripotent stem cell from non-human animals. In other embodiments, the target cell is a hematopoietic cell or a hematopoietic progenitor cell. In still other embodiments, the target cell is an immortalized cell such as a cancer cell.

In certain embodiments Nedisertib (M3814) is used in a method wherein the genome editing comprises introducing a staggered cut, into the doubled-stranded genome of the target cell or target organism. In certain further embodiments, Nedisertib (M3814) is used in a method comprising introducing a blunt-ended cut into the double-stranded genome of the target organism.

The compound is intended for use in any type of genome editing including multiplexed genome editing on both chromosomes both in non-medical applications and in medical applications.

Nedisertib (M3814) may be used in a genome editing procedure which comprises introducing a staggered cut, or a blunt-ended cut into the genome of the target cell. In order to achieve this result, the target cell may comprise CRISPR/Cas9 enzyme, or a mutated nickase version of CRISPR/Cas9 such as a CRISPR/Cas9 D10A or CRISPR/Cas9 H840A enzyme or a CRISPR/Cpf1 enzyme. Alternatively, other genome editing enzymes, e.g. CRISPRs, transcription activator-like effector-based nucleases (TALENs), zinc finger nuclease proteins, Argonaute of the bacterium Thermus thermophiles (TtAgo), recombinases, or meganucleases or other enzymes may be present which provide staggered cuts or blunt-ended cuts in a double stranded target DNA. The present invention is also suitable together with split-fusion versions of the above enzymes, e.g. split-fusion versions of Cas9 or Cas9 D10A (Zetsche et al.

The enzyme(s) may be introduced into the target cell as such, e.g. as protein or ribonucleoprotein or as nucleic acid molecule encoding the respective enzyme(s). The nucleic acid molecule may be introduced as an expression vector such as a plasmid in operative linkage with appropriate expression control elements for transient or stable expression in the target cell. Suitable transfection techniques for introducing proteins or nucleic acids into the eukaryotic target cells are well known in the art and include lipofection, electroporation, e.g. nucleofection, Ca-phosphate or virus-based methods.

Nedisertib (M3814) is suitable for use with all kinds of donor nucleic acid molecules including but not limited to single stranded molecules or double stranded DNA molecules whether amplified in vivo or in vitro or chemically synthesized. The length of the donor nucleic acid molecules is usually in the range of about <NUM> to <NUM> nt or more, e.g. about <NUM> to <NUM> nt, <NUM> to <NUM> nt or <NUM> to <NUM> nt. The donor nucleic acid molecules are designed to include at least one desired mutation in view of the wild type sequence which is to be introduced into the genome of the target cell by genome editing. The mutation may be a single nucleotide mutation or a mutation encompassing a plurality of nucleotides. In this context, the term mutation refers to a substitution, deletion, or insertion of single nucleotides or of a plurality of nucleotides.

The above aspects comprise a use in vivo, e.g. in isolated cells or cell clusters, but also in vitro, in cells of a target organism. The combinations can be applied in cell types and with genome editing procedures as indicated above, including the use of DNA cleavage enzyme systems capable of introducing a staggered cut, or a blunt-ended cut in a DNA double strand. This aspect also includes a use in medicine including human or veterinary medicine.

Still a further aspect of the present invention is the use of Nedisertib (M3814) or a combination comprising Nedisertib (M3814) and at least one further active agent in medicine including human or veterinary medicine. An effective dose of the compounds according to the invention, or their salts, solvates or prodrugs thereof is used, in addition to physiologically acceptable carriers, diluents and/or adjuvants for producing a pharmaceutical composition. The dose of the active compounds can vary depending on the route of administration, the age and weight of the patient, the nature and severity of the diseases to be treated, and similar factors. The daily dose can be given as a single dose, which is to be administered once, or be subdivided into two or more daily doses, and is as a rule <NUM>-<NUM>. Particular preference is given to administering daily doses of <NUM>-<NUM>, e.g. <NUM>-<NUM>.

Suitable administration forms are oral, parenteral, intravenous, transdermal, topical, inhalative, intranasal and sublingual preparations. Particular preference is given to using oral, parenteral, e.g. intravenous or intramuscular, intranasal preparations, e.g. dry powder or sublingual, of the compounds according to the invention. The customary galenic preparation forms, such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, can be used.

Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid), gelatine, agar agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol); preparations which are suitable for oral administration can comprise additional flavourings and/or sweetening agents, if desired.

Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.

Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials. Use is preferably made of solutions of the active compound, preferably aqueous solution and, in particular, isotonic solutions and also suspensions. These injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the active compound, for example the lyophilisate, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.

Intranasal preparations can be present as aqueous or oily solutions or as aqueous or oily suspensions. They can also be present as lyophilisates which are prepared before use using the suitable solvent or suspending agent.

Inhalable preparations can present as powders, solutions or suspensions. Preferably, inhalable preparations are in the form of powders, e.g. as a mixture of the active ingredient with a suitable formulation aid such as lactose.

The preparations are produced, aliquoted and sealed under the customary antimicrobial and aseptic conditions.

The compounds of the invention may be administered alone or as a combination therapy with further active agents.

The medical use of Nedisertib (M3814) particularly encompasses target gene therapy, e.g. the treatment of disorders associated with an undesired genotype of a patient in need of the treatment. For example, the disorder is a metabolic dysfunction or cancer. By means of the invention, cells from the patient may be subjected to a genome editing procedure in the presence of a combination as described above, thereby increasing the precise genome editing efficiency. This procedure may be carried out in vivo, i.e. by administering the combination to the patient or ex vivo with cells isolated from the patients, which are - after successful genome editing - reimplanted into the patient. The patient may be a vertebrate animal such as a mammal, preferably a human patient. Finally, the compound of formula (I) or (II) is also suitable for genome editing in plant cells or plants.

Further, the invention shall be explained in more detail by the following Figures and Examples.

Methods involving H9 cells are for reference only and do not form part of the invention.

We recently created an iCRISPR-Cas9n line from human induced pluripotent stem cells (hiPSCs) (<NUM>-B2, female, Riken BioResource Center) and human embryonic stem cells (hESCs) (H9) as described by Gonzalez et al. Stem cells were grown on Matrigel Matrix (Corning, <NUM>) in mTeSR1 medium (StemCell Technologies, <NUM>) with supplement (StemCell Technologies, <NUM>) that was replaced daily. K562 cells (ECACC, <NUM>) were grown with IMDM (ThermoFisher, <NUM>) supplemented with <NUM>% FBS. Cells were grown at <NUM> in a humidified incubator gassed with <NUM>% CO<NUM>. Media was replaced every second day for non-pluripotent cell lines. Cell cultures were maintained <NUM>-<NUM> days until ~ <NUM>% confluency, and subcultured at a <NUM>:<NUM> to <NUM>:<NUM> dilution. Adherent cells were dissociated using EDTA (VWR, 437012C). The media was supplemented with <NUM> Rho-associated protein kinase (ROCK) inhibitor Y-<NUM> (Calbiochem, <NUM>) after cell splitting for one day in order to increase cell survival.

A commercially available small molecule used in this study was M3814 (MedChemExpress, HY-<NUM>). A stock of <NUM> was made using dimethylsulfoxide (DMSO) (Thermo Scientific, D12345). Suitable working solutions for different concentrations were made so that addition of M3814 accounts for a final concentration of <NUM>% DMSO in the media.

We designed gRNAs and donors for two nicks per editing site and single-stranded oligodeoxynucleotide DNA donors (ssODNs) carrying the desired amino acid changing mutations. When necessary, the ssODNs carried additional silent non-coding mutations to prevent repeated cutting of the DNA once the targeted substitutions have been introduced (see Table <NUM>).

The recombinant A. Cpf1 and S. Cas9 protein and electroporation enhancer was ordered from IDT (Coralville, USA) and nucleofection was done using the manufacturer's protocol, except for the following alterations. Nucleofection was done using the B-<NUM> program of the Nucleofector 2b Device (Lonza) in cuvettes for <NUM>µl Human Stem Cell nucleofection buffer (Lonza, VVPH-<NUM>) containing <NUM> million cells of the respective lines, <NUM> pmol electroporation enhancer, 160pmol of each gRNA (crRNA/tracR duplex for Cas9 and crRNA for Cpf1) (320pmol for double nicking with both gRNAs for one gene), 200pmol ssODN donor, 252pmol CRISPR protein. For editing with the iCRISPR-Cas9n lines only gRNAs and single stranded DNA donors were electroporated. Cells were counted using the Countess Automated Cell Counter (Invitrogen).

Three days after editing cells were dissociated using Accutase (SIGMA, A6964), pelleted, and resuspended in <NUM>µl QuickExtract (Epicentre, QE0905T). Incubation at <NUM> for <NUM>, <NUM> for <NUM> and finally <NUM> for <NUM> was performed to yield ssDNA as a PCR template. Primers for the targeted loci of FRMD7 containing adapters for Illumina sequencing were ordered from IDT (Coralville, USA). PCR was done in a T100 Thermal Cycler (Bio-Rad) using the KAPA2G Robust PCR Kit (Peqlab, <NUM>-KK5532-<NUM>) with supplied buffer B and <NUM>µl of cell extract in a total volume of <NUM>µl. The thermal cycling profile of the PCR was: <NUM> <NUM>; 34x (<NUM>° <NUM> sec, <NUM> <NUM> sec, <NUM> <NUM> sec); <NUM> <NUM> sec. P5 and P7 Illumina adapters with sample specific indices were added in a second PCR reaction (Kircher et al. <NUM>) using Phusion HF MasterMix (Thermo Scientific, F-<NUM>) and <NUM>µl of the first PCR product. The thermal cycling profile of the PCR was: <NUM> <NUM> sec; 25x (<NUM>° <NUM> sec, <NUM> <NUM> sec, <NUM> <NUM> sec); <NUM> <NUM>. Amplifications were verified by size separating agarose gel electrophoresis using EX gels (Invitrogen, G4010-<NUM>). The indexed amplicons were purified using Solid Phase Reversible Immobilization (SPRI) beads (Meyer, Kircher <NUM>). Double-indexed libraries were sequenced on a MiSeq (Illumina) giving paired-end sequences of <NUM> x <NUM> bp. After base calling using Bustard (Illumina) adapters were trimmed using leeHom (Renaud et al.

CRISPResso (Pinello et al. <NUM>) was used to analyse sequencing data from CRISPR genome editing experiments for percentage of wildtype, targeted nucleotide substitutions (TNS), indels and mix of TNS and indels. Parameters used for analysis were '-w <NUM>', '-min_identity_score <NUM>' and '--ignore_substitutions' (analysis was restricted to amplicons with a minimum of <NUM>% similarity to the wildtype sequence and to a window of <NUM> bp from each gRNA; substitutions were ignored, as sequencing errors would be falsly characterized as NHEJ-events). Sequence homology for an HDR occurrence was set to <NUM>%. Unexpected substitutions were ignored as sequencing putative errors. Since CRISPResso cannot distinguish reads with indels to be from NHEJ or microhomology-mediated end joining (MMEJ), we wrote a python script to call MMEJ events.

Cells were either seeded with or without editing reagents. The media was supplemented with or without M3814 and each condition was carried out in duplicate. After <NUM> media was aspirated and <NUM>µl fresh media together with <NUM>µl resazurin solution (Cell Signaling, <NUM>) was added. Resazurin is converted into fluorescent resorfin by cellular dehydrogenases and resulting fluorescence (Excitation: <NUM>-<NUM>, Emission: <NUM>-<NUM>) is considered as a linear marker for cell viability (O'Brien et al. Cells were incubated with resazurin at <NUM>. The redox reaction was measured every hour by fluorescence readings using a Typhoon <NUM> imager (Amersham Biosciences). After <NUM> the fluorescence scan showed a good contrast without being saturated, and was used to quantify the fluorescence using ImageJ and the 'ReadPlate' plugin. Duplicate wells with media and resazurin, but without cells, were used a blank.

We aimed to test the precise genome editing efficiency of the small molecule M3814 in K562 and H9 hES cells.

We tested the potency of the DNA-PKcs small molecule inhibitor M3814 to increase HDR after a Cas9 or Cas9n induced DSB, even though several small molecule inhibitors of DNA-PK have been described to moderately increase HDR. We show that transient treatment of K562 cells expressing wild-type DNA-PKcs with M3814 has a strong HDR-increasing effect (<NUM>% to <NUM>%) (<FIG>) while only exhibiting moderate toxicity (<FIG>). We furthermore show that increased HDR efficiencies by M3814 are comparable to what is achievable by total inactivation of the DNA-PKcs catalytic active site (K3753R mutation) (<FIG>). Also, residual indels due to MMEJ after NHEJ inactivation can be avoided by inactivation of POLQ leading to quantitative HDR (<FIG>).

We further compared the potency of M3814 and other DNA-PKcs small molecule inhibitors NU7026 and NU7441 to increase HDR after a Cas9 induced DSB. We show that transient treatment of K562 cells expressing wild-type DNA-PKcs with <NUM> and <NUM> M3814 has a stronger HDR-increasing effect than treatment with NU7026 and NU7441 at the same concentrations (<FIG>). We furthermore show that strongly increased HDR efficiencies by M3814 are also obtained in human induced pluripotent stem cells (hiPCs) 409B2 at a concentration of <NUM> whereas treatment with NU7026 and NU7441 at the same concentration resulted in much lower efficiencies (<FIG>).

Further increasing HDR by inhibition of MMEJ and/or SSA together with inhibition of NHEJ by M3814.

For many targets NHEJ inhibition by the surprisingly potent small molecule M3814 results in drastically increased HDR. For some targets HDR is increased but a substantial portion of genome editing events however still consists of indels. These are due to the microhomology mediated end-joining (MMEJ) pathway (also referred to as alternative NHEJ) which can compete with NHEJ and serves as a back-up pathway which relies on short stretches of microhomology at the cleavage site. MMEJ is dependent on Polymerase Theta (PoIQ) (Mateos-Gomez et al. , Nature, <NUM>, supra). PolQ has two RAD51 binding domains that inhibit homologous recombination (Ceccaldi et al. , Nature, <NUM>, supra). We found that siRNAs against PolQ decrease indels with MMEJ signature but do not necessarily always increase HDR (<FIG>). The PolQ siRNA SMART pool (Dharmacon ON-TARGET plus Human POLQ (<NUM>)) contains four siRNAs that bind the mRNA downstream of the first RAD51 binding domain. We speculated that the PolQ mRNA is partially translated into a truncated protein containing the RAD51 binding domain that prevents an HDR increase. We also tested transient nicking of the first exon of PolQ (before the first RAD51 binding domain) to prevent mRNA expression and there is a tendency for increased HDR when the coding strand is nicked as expected. DNA nick repair has very high fidelity so no permanent PolQ editing is expected. Combining SMART pool siRNA and coding strand nicking resulted in a strong increase in HDR with almost no indels, which is comparable to a cell line with DNA-PKcs KR and PolQ knockout. This high HDR can also be achieved by using siRNA aa765 (hs. <NUM>, IDT DNA Technologies) that binds mRNA before the sequence corresponding to the first RAD51 binding domain.

Claim 1:
Nedisertib (M3814) or a physiologically acceptable salt or solvate thereof, for use in medicine in the treatment of disorders associated with an undesired genotype of a patient in need of the treatment, the disorder being a metabolic dysfunction or cancer, comprising genome editing in a eukaryotic target cell or in a eukaryotic target organism, wherein processes for cloning human beings, processes for modifying the germ line genetic identity of human beings, and uses of human embryos for industrial and commercial purposes are excluded.