LXH0307 and LXH0308 as small molecule inhibitors of gene editing and their applications

Abstract

The present invention discloses LXH0307 and LXH0308 as small molecule inhibitors of gene editing and their applications. LXH0307 and LXH0308 have stronger ability to inhibit gene editing activity. The present invention also provides a synthesis method of LXH0307 and LXH0308, and a composition, reagent product and kit containing LXH0307 and LXH0308 and their applications.

Description

Technical Field

[0001] The present invention belongs to the field of chemical drugs, and specifically relates to LXH0307 and LXH0308 as small molecule inhibitors of SpCas9 and their effects. Background Art

[0002] Genome editing technology is a genetic manipulation technology that uses artificial means to modify DNA sequences at the genome level, including the insertion, knockout, replacement and point mutation of specific DNA fragments. Among them, the basic principle of nuclease-dependent genome editing technology is to generate double-stranded DNA breaks (DSBs) at specific locations in the genome and then repair them by non-homologous end joining (NHEJ) or homologous recombination (HR). With the more in-depth study of nucleases, genome editing technology has also developed rapidly. The most commonly used nucleases mainly include giant nucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas).

[0003] The CRISPR / Cas system is a natural immune system from bacteria and archaea, consisting of CRISPR sequences and highly diverse Cas proteins. The CRISPR sequence consists of repeat regions and spacers. The repeat region contains palindromic sequences that can form a hairpin structure, and the spacer is a captured exogenous DNA sequence. Cas proteins can be divided into four modules according to their functions: the adaptation module is mainly involved in the acquisition of Spacer and the integration of exogenous genes into the CRISPR sequence; the expression processing module is responsible for the processing of pre-crRNA; the interference and effector modules are involved in target recognition and cutting; the signal transduction and auxiliary modules are a collection of genes with different functions. Based on the sequence and combination differences of Cas proteins, CRISPR systems can be divided into two major categories. Class 1 CRISPR / Cas systems are effector modules composed of multiple Cas proteins, while Class 2 CRISPR / Cas systems are single multi-domain Cas protein effector modules.

[0004] Recent studies have found that the CRISPR system can not only be used to cut viral DNA, but also can accurately cut other DNA sequences. crRNA-tracrRNA is simplified to single-stranded guide RNA (sgRNA), which can be easily designed and synthesized. By changing the sequence of sgRNA, the target gene can be matched, thereby guiding the Cas9 protein to the designated location to cut DNA.

[0005] With the development of molecular biology and sequencing technology, people's understanding of diseases has entered the molecular level, and it has been found that the occurrence and progression of many human diseases may be related to gene mutations. For example, mutations in certain oncogenes can promote the occurrence of tumors, and even mutations in certain tumor suppressor genes can cause tumors by activating oncogenes (Jiang CY, Lin XH, Zhao ZG. Applications of CRISPR / Cas9 technology in the treatment of lung cancer. Trends Mol Med, 2019, 25(11): 1039-1049). Therefore, determining genes closely related to diseases and their functions will help provide better strategies for clinical treatment. With the rapid development of CRISPR technology, genome screening based on CRISPR technology has been widely used at the laboratory level. This screening method has the advantages of flexible library design, simple operation, and wide coverage. Although CRISPR-Cas9 technology is becoming more and more sophisticated, there are still certain risks in its widespread application in clinical gene therapy, and its specificity, safety, and in vivo transduction still need to be improved.

[0006] The safe application of CRISPR-Cas9 in gene therapy requires the ability to control the gene editing activity of the Cas9 / sgRNA complex once the desired use has been achieved. Although several modified systems allow controlled activation of CRISPR-Cas9 to improve precision, all of these systems still lack the ability to provide predictable control and robust inhibition. The development of agents that regulate gene editing will greatly improve the efficacy and safety of clinical therapeutics and research applications based on CRISPR systems. Summary of the invention

[0007] In order to make up for the deficiencies of the prior art, the present invention provides compounds for regulating gene editing and methods for preparing the compounds. The compounds of the present invention can have a higher ability to regulate gene editing activity.

[0008] The present invention adopts the following technical solutions:

[0009] The first aspect of the present invention provides a compound for regulating gene editing, wherein the compound has a structure described by formula (I):

[0010]

[0011] Wherein, R is an aromatic ring, and the aromatic ring may be substituted by 0, 1, 2 or 3 substituents R 1 Substituted, the substituent R 1 independently selected from halo, CN, C1-C4-alkyl, C1-C4-haloalkyl, C3-C4-cycloalkyl, OR 2 NR 2 R 3 ; Each R 2 and R 3 are independently H, C1-C4 alkyl or C1-C4-haloalkyl;

[0012] In some embodiments, the aromatic ring is a benzene ring or naphthalene.

[0013] In some embodiments, the R 2 and R 3 Independently H, C1-C4 alkyl.

[0014] In a preferred embodiment, the structure of the compound is as follows:

[0015]

[0016] The second aspect of the present invention provides a method for preparing the compound according to the first aspect of the present invention, the method comprising:

[0017] 1) using bromoaniline, ethyl glyoxylate, and BOC-dihydropyrrole to react and synthesize compound 2-P;

[0018] 2) using column chromatography to separate and obtain compounds 2-P1 and 2-P2;

[0019] 3) subjecting compound 2-P1 to a Suzuki coupling reaction with 2-fluorophenylboronic acid to generate compound 3-P1;

[0020] 4) subjecting compound 3-P1 to carboxylic acid reduction to generate compound 4-P1;

[0021] 5) Compound 4-P1 is de-Bocated under acidic conditions to generate compound 5-P1;

[0022] 6) Compound 5-P1 is subjected to sulfonylation reaction to generate LXH0307 and LXH0308.

[0023] In some embodiments, the method for synthesizing compound 2-P in step 1) is as follows: ethyl glyoxylate and 4-bromoaniline are added to a toluene solution, and then MgSO4 is added and stirred to obtain a reaction solution 1; BOC-dihydropyrrole is added to a toluene solution, and then scandium trifluoromethanesulfonate is added to react to obtain a reaction solution 2; reaction solution 1 and reaction solution 2 are added together to react, and the mixture is extracted and spin-dried.

[0024] In some embodiments, the reaction solution 1 is obtained by stirring at 0°C.

[0025] In some embodiments, scandium triflate is added at 0 °C.

[0026] In some embodiments, the extraction reagent is ethyl acetate / water.

[0027] In some embodiments, the eluent in step 2) column chromatography is selected from PE and EA.

[0028] In some embodiments, 2-P1 is isolated at 6% PE:EA.

[0029] In some embodiments, the catalytic reagent of step 3) the Suzuki coupling reaction is Xphos-pd-G3.

[0030] In some embodiments, the alkaline reagent in step 3) the suzuki coupling reaction is K3PO4.

[0031] In some embodiments, the process of step 3) Suzuki coupling reaction is as follows:

[0032] Dissolve 2-P1, 2-fluorophenylboronic acid and K3PO4 in dioxane, then add Xphos-pd-G3 and reflux at 110°C for reaction.

[0033] In some embodiments, step 3) further comprises separating by column chromatography to obtain 3-P1.

[0034] In some embodiments, the eluent for column chromatography is selected from PE and EA.

[0035] In some embodiments, 3-P1 is isolated at 14% PE:EA.

[0036] In some embodiments, the process of step 4) carboxylic acid reduction reaction is as follows:

[0037] 3-P1 was dissolved in THF, and the reducing agent solution was slowly added dropwise to react.

[0038] In some embodiments, the reducing agent is LiBH4.

[0039] In some embodiments, the reducing agent solution is a solution of LiBH4 in methanol.

[0040] In some embodiments, the reaction conditions are 0° C. for 2 h, followed by room temperature for 15 h.

[0041] In some embodiments, the acid is added after the reaction is complete.

[0042] In some embodiments, the acid is HCl.

[0043] In some embodiments, step 4) further comprises extracting the reaction solution.

[0044] In some embodiments, the extraction reagent is ethyl acetate / water.

[0045] In some embodiments, step 4) further comprises separating by column chromatography to obtain 4-P1.

[0046] In some embodiments, the eluent for column chromatography is selected from DCM and MeOH.

[0047] In some embodiments, the DCM:MeOH is 4%.

[0048] In some embodiments, the process of step 5) removing Boc is as follows:

[0049] 4-P1 was added to HCl / Et2O and reacted at room temperature.

[0050] In some embodiments, step 5) further comprises spinning and washing the reaction solution.

[0051] In some embodiments, the washing reagent is a mixed solution of PE, DCM and n-hexane.

[0052] In some embodiments, the 5-P1 is a yellow solid.

[0053] In some embodiments, the step of performing the sulfonylation reaction in step 6) is as follows:

[0054] 5-P1 was added to DCM, Et3N was slowly added dropwise to fully dissolve 5-P1, and then the sulfonylating agent was added dropwise to react;

[0055] In some embodiments, the sulfonylating agent is selected from dansyl chloride, 2-methoxybenzenesulfonyl chloride;

[0056] In some embodiments, Et3N is added slowly dropwise at 0°C.

[0057] In some embodiments, the step 6) further comprises quenching the reaction solution.

[0058] In some embodiments, the step 6) further comprises extracting the quenched reaction solution.

[0059] In some embodiments, the extraction reagent is dichloromethane / water.

[0060] Preferably, step 6) further comprises a column chromatography step.

[0061] Preferably, the eluent for column chromatography is selected from PE and EA.

[0062] Preferably, the ratio of the eluents PE and EA is 50%.

[0063] The third aspect of the present invention provides a pharmaceutical composition, which comprises the compound described in the first aspect of the present invention, its pharmaceutically acceptable salt, its optical isomer, its racemate, or its solvate; or the compound or intermediate prepared by the method described in the second aspect of the present invention;

[0064] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0065] The fourth aspect of the present invention provides a method for regulating the CRISPR system for gene editing, administering to a subject in need thereof the compound described in the first aspect of the present invention, its pharmaceutically acceptable salt, its optical isomer, its racemate, or its solvate; or the pharmaceutical composition described in the third aspect of the present invention.

[0066] A fifth aspect of the present invention provides a method for editing one or more target genomic regions, comprising administering to one or more cells comprising the one or more genomic regions

[0067] 1) CRISPR editing system; and

[0068] 2) The compound described in the first aspect of the present invention, its pharmaceutically acceptable salt, its optical isomer, its racemate, or its solvate; or the pharmaceutical composition described in the third aspect of the present invention.

[0069] In some embodiments, the CRISPR editing system is previously administered to the one or more cells.

[0070] In some embodiments, the one or more cells are cultured cells.

[0071] In some embodiments, the one or more cells are in vivo cells within an organism.

[0072] In some embodiments, the one or more cells are ex vivo cells from an organism.

[0073] In some embodiments, the CRISPR editing system is a CRISPR-Cas editing system.

[0074] In some embodiments, 1) and 2) are administered via different routes.

[0075] In some embodiments, 1) and 2) are administered via the same route.

[0076] In some embodiments, the CRISPR editing system is delivered by one or more vectors.

[0077] In some embodiments, the one or more vectors are selected from a viral vector, a plasmid, or ssDNA.

[0078] In some embodiments, the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and / or a herpes simplex viral vector.

[0079] In some embodiments, the CRISPR editing system is delivered via synthetic RNA.

[0080] In some embodiments, the CRISPR editing system is delivered via nanoformulation.

[0081] A sixth aspect of the present invention provides a reagent product, comprising:

[0082] 1) a first agent, wherein the first agent is a gene editing inhibitor, wherein the gene editing inhibitor is a compound according to one aspect of the present invention, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; and

[0083] 2) A second reagent, wherein the second reagent is a reagent for CRISPR gene editing.

[0084] In some embodiments, the second reagent includes one or more reagents selected from the group consisting of:

[0085] 1) Cas9 nuclease, a coding sequence of Cas9 nuclease, or a vector expressing Cas9 nuclease, or a combination thereof;

[0086] 2) gRNA, crRNA, or a vector for producing the gRNA or crRNA;

[0087] 3) Template for homology-directed repair: single-stranded nucleotide sequence or plasmid vector.

[0088] A seventh aspect of the present invention provides a kit for editing one or more target genomic regions, the kit comprising:

[0089] 1) a first container, and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; or the composition described in the third aspect of the present invention;

[0090] 2) a second container, and a CRISPR editing system reagent located in the second container;

[0091] In some embodiments, the CRISPR editing system is a CRISPR-Cas editing system.

[0092] In some embodiments, the CRISPR-Cas editing system comprises:

[0093] At least one guide RNA element and a Cas protein element.

[0094] In some embodiments, the guide RNA element comprises: 1) a targeting RNA comprising a nucleotide sequence that is substantially complementary to a nucleotide sequence at the one or more target genomic regions, or a nucleic acid comprising a nucleotide sequence encoding the targeting RNA; and 2) an activating RNA comprising a nucleotide sequence capable of hybridizing with the targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding the activating RNA.

[0095] In some embodiments, the Cas protein is a Cas9 protein.

[0096] In a preferred embodiment, the Cas9 protein is SpCas9 protein.

[0097] In some embodiments, the CRISPR editing system is contained or packaged in one or more vectors.

[0098] In some embodiments, the one or more vectors are selected from a viral vector, a plasmid, or ssDNA.

[0099] In some embodiments, the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and / or a herpes simplex viral vector.

[0100] The eighth aspect of the present invention provides the use of the compound described in the first aspect of the present invention, the pharmaceutical composition described in the third aspect of the present invention, the reagent product described in the sixth aspect of the present invention or the kit described in the seventh aspect of the present invention in regulating genome editing.

[0101] In some embodiments, the genome editing targets pathogenic genes, tumor-related genes (such as oncogenes), immune-related genes (such as genes related to autoimmunity), vision-related genes, hearing-related genes, metabolism-related genes, viral infection-related genes, and genetic disease-related genes.

[0102] In some embodiments, the genome editing uses the CRISPR editing system;

[0103] In some embodiments, the CRISPR editing system is selected from a CRISPR-Cas editing system.

[0104] The ninth aspect of the present invention provides the use of the compound described in the first aspect of the present invention, the pharmaceutical composition described in the third aspect of the present invention, the reagent product described in the sixth aspect of the present invention or the kit described in the seventh aspect of the present invention in the preparation of a drug for treating a disease.

[0105] In some embodiments, the disease is cancer.

[0106] In some embodiments, the disease is a genetic disorder.

[0107] The tenth aspect of the present invention provides the use of the compound described in the first aspect of the present invention, the pharmaceutical composition described in the third aspect of the present invention, the reagent product described in the sixth aspect of the present invention or the kit described in the seventh aspect of the present invention in constructing a disease model.

[0108] In some embodiments, the disease model is a cellular disease model or an animal disease model.

[0109] In some embodiments, the disease model is used to screen drugs for treating diseases. If the substance to be screened can treat or alleviate the symptoms of the disease, it means that the substance to be screened can be used as a potential drug for treating the disease.

[0110] Beneficial effects of the present invention:

[0111] The present invention provides compounds LXH0307 and LXH0308 that significantly inhibit gene editing, and the compounds have a strong inhibitory effect on CRISPR-Cas-mediated shearing efficiency;

[0112] The present invention is based on LXH0307 / LXH0308 and the CRISPR editing system, providing a simple and efficient strategy for precise gene editing.

[0113] The preparation and synthesis method of the compound of the present invention uses a Boc protection strategy to reduce costs and increase yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] Figure 1This is a diagram showing the effect of SpCas9 inhibitors on in vitro enzymatic cleavage activity; among them, 1A is the inhibitor LXH0307, ​​and 1B is the inhibitor LXH0308. DETAILED DESCRIPTION

[0115] The present invention relates to the field of CRISPR-Cas9 gene editing platforms. In particular, the present invention identifies type II-C Cas9 anti-CRISPR (Acr) inhibitors that control Cas9 gene editing activity. The co-application of such Acr inhibitors can provide advantageous assistance in the following aspects: allowing safe and practical biological treatments to be carried out by controlling Cas9 activity in space or time; controlling Cas9-based gene drives in wild populations to mitigate the ecological consequences of such forced genetic programs; and contributing to general research in various biotechnology, agricultural and medical applications entering gene editing technology.

[0116] CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that help defend against attacks by predatory viruses. These systems use small RNAs (CRISPR RNAs (crRNAs)) as guides to identify their nucleic acid targets, which are then cleaved and inactivated.

[0117] A subset of CRISPR-Cas systems (those known as "Type II") employ a protein known as Cas9, and this protein (along with its RNA guide) has been adapted as a revolutionary genome editing platform. Variants carrying mutations in its nuclease active site have also been developed as RNA-guided DNA binding platforms to enable gene tagging and transcriptional control. Cas9 has been widely used as a research tool, and it is also being used in a wide range of biotechnology applications, and in animal husbandry, agriculture, and elsewhere.

[0118] Additionally, the CRISPR-Cas9 platform is being developed as a potentially revolutionary therapeutic approach for many diseases, given its potential ability to modify genomic loci that contribute to disease, disrupt the genome of a pathogen, or introduce therapeutically useful sequences (e.g., chimeric antigen receptors for cancer immunotherapy).

[0119] One current limitation of Cas9 applications is that its activity can be very difficult to control: it is generally active as long as it is present and accessible to its crRNA guide, usually in the form of a so-called single guide RNA (sgRNA). This lack of control can introduce difficulties for many reasons. Most notably, although Cas9 is often highly accurate in editing only the genomic locus to which it is directed, off-target effects are not uncommon. Off-target Cas9 activity has the undesirable consequence of introducing mutations to undesirable sites. This is a matter of concern for many purposes, especially but not exclusively for clinical therapeutics.

[0120] It is well established that the more functional Cas9 / sgRNA there is in a cell, and the longer the Cas9 / sgRNA persists, the greater the likelihood of off-target effects. Although it is not necessary to understand the mechanism of the present invention, it is believed that once "on-target" genome editing has occurred, the persistent Cas9 / sgRNA may no longer recognize and edit the desired locus, thereby accomplishing its intended purpose. Nevertheless, the still functional Cas9 / sgRNA complex may continue to find and edit off-target sites elsewhere.

[0121] The present invention contemplates compositions that inhibit Cas9 / sgRNA activity. Although it is not necessary to understand the mechanism of the present invention, it is believed that these inhibitor compounds interact with active Cas9 / sgRNA and act as a "shutdown switch" that reduces Cas9 / sgRNA activity to a minimum amount. And / or maintains the duration necessary for its intended purpose. Further, it is believed that the Cas9 / sgRNA inhibitor composition can limit Cas9 / sgRNA activity to specific tissues or organs, or to any given set of user-defined conditions, by "touching the shutoff switch" anywhere, at any time, or under any circumstances when its activity is not desired.

[0122] Therefore, the present invention provides a compound for regulating gene editing, wherein the compound has a structure described by formula (I):

[0123]

[0124] Wherein, R is an aromatic ring, and the aromatic ring may be substituted by 0, 1, 2 or 3 substituents R 1 Substituted, the substituent R 1 independently selected from halo, CN, C1-C4-alkyl, C1-C4-haloalkyl, C3-C4-cycloalkyl, OR 2 NR 2 R 3 ; Each R 2 and R 3are independently H, C1-C4 alkyl or C1-C4-haloalkyl;

[0125] In some embodiments, the aromatic ring is a benzene ring or naphthalene.

[0126] In some embodiments, the R 2 and R 3 Independently H, C1-C4 alkyl.

[0127] In some embodiments, the derivative has a structure as shown in any of the following:

[0128]

[0129] The compounds disclosed herein may exist in free form or, where appropriate, as pharmaceutically acceptable derivatives thereof. Pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable prodrugs, salts, esters, salts of such esters, or any other adducts or derivatives that, when administered to a patient in need thereof, are capable of providing, directly or indirectly, a compound as described elsewhere herein, or a metabolite or residue thereof.

[0130] Pharmaceutical composition

[0131] The pharmaceutical composition of the present invention comprises the aforementioned compound, its pharmaceutically acceptable salt, its optical isomer, its racemate, or its solvate.

[0132] The term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like.

[0133] Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds disclosed herein include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed using inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or using organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include alginate, ascorbate, aspartate, benzenesulfonate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, dodecylsulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Additional exemplary salts include adipate, benzoate, citrate, fumarate, maleate, or succinate. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N+(C 1-4 Alkyl)4 salt.

[0134] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, adjuvants or vehicles include any or all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid adhesives, lubricants, etc. suitable for a specific expected dosage form. In Remington: The Science and Practice of Pharmacy, 21st edition, 2005, DB Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, J. Swarbrick and JC Boylan, 1988-1999, Marcel Dekker, New York (the contents of each of them are incorporated herein by reference) discloses various carriers for preparing pharmaceutically acceptable compositions and known techniques for their preparation. Unless any conventional carrier vehicle is incompatible with the compounds of the invention (for example, by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other ingredients of the pharmaceutically acceptable composition), its use is contemplated as being within the scope of the present invention.

[0135] Some examples of substances that can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, lanolin; sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose , ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol, and phosphate buffered solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0136] The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraocular, intrahepatic, intralesional, epidural, intraspinal and intracranial injection or infusion techniques. Preferably, the composition is administered orally, intraperitoneally or intravenously. The sterile injectable form of the composition of the present invention may be an aqueous or oily suspension. These suspensions may be prepared using suitable dispersants or wetting agents and suspending agents according to techniques known in the art. Sterile injection preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Available acceptable carriers and solvents are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are typically used as solvents or suspending media.

[0137] For this purpose, any gentle fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) can be used to prepare injections, as can natural pharmaceutically acceptable oils (such as olive oil or castor oil), particularly in polyoxyethylated forms. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, such as carboxymethyl cellulose or similar dispersants commonly used in the preparation of pharmaceutically acceptable dosage forms (including emulsions and suspensions). Other conventional surfactants (such as tweens, spans and other emulsifiers or bioavailability enhancers) commonly used in the preparation of pharmaceutically acceptable solids, liquids or other dosage forms can also be used to achieve the purpose of the preparation.

[0138] The pharmaceutically acceptable compositions of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in the form of capsules, available diluents include lactose and dry corn starch. When an aqueous suspension is required for oral use, the active ingredient is combined with an emulsifier and a suspending agent. If necessary, certain sweeteners, flavorings or coloring agents may also be added. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be matched to the mode of administration.

[0139] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (specifically, cottonseed oil, peanut oil, corn oil germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, the oral composition may also include adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and aromatics.

[0140] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia, c) humectants such as glycerol, d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retardants such as paraffin, f) absorption promoters such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glyceryl monostearate, h) absorbents such as kaolin and bentonite, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

[0141] method

[0142] The present invention provides a method for regulating the CRISPR editing system to perform gene editing, administering the aforementioned compound, its pharmaceutically acceptable salt, its optical isomer, its racemate, or its solvate; or the aforementioned pharmaceutical composition to a subject in need.

[0143] As used herein, "administering" refers to contacting, injecting, distributing, delivering, or applying a genome editing system and / or the aforementioned compound / composition to a cell or subject. In some embodiments, administration is contacting a genome editing system and / or the aforementioned compound / composition with a cell. In some embodiments, administration is delivering a genome editing system and / or the aforementioned compound / composition to a cell. In some embodiments, administration is applying a genome editing system and / or the aforementioned compound / composition to a cell. In some embodiments, administration is injecting a genome editing system and / or the aforementioned compound / composition into a cell. Administration can occur in vivo, ex vivo, or in vitro. Administration of a genome editing system and the aforementioned compound / composition to a cell can be performed sequentially.

[0144] The CRISPR editing system is delivered by one or more vectors.

[0145] In some embodiments, the one or more vectors are selected from a viral vector, a plasmid, or ssDNA.

[0146] "Viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide to be delivered into a host cell in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, herpes simplex viral vectors, and chimeric viral vectors, among others. In some embodiments in which gene transfer is mediated by a retroviral vector, a vector construct refers to a polynucleotide comprising a retroviral genome or a portion thereof.

[0147] In some embodiments, the CRISPR editing system is delivered via synthetic RNA.

[0148] In some embodiments, the CRISPR editing system is delivered via nanoformulation.

[0149] Some embodiments of the present disclosure relate to vector systems comprising one or more vectors or the vectors themselves. The vectors can be designed to express CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells.

[0150] Cell can be primary cell, induced pluripotent stem cell (iPSC), embryonic stem cell (hESC), adult stem cell, progenitor cell or cell line."Primary cell" is a cell taken directly from living tissue and placed in vitro for growth. The population doubling of primary cells is seldom, and the population doubling in vitro has a limited life span."Stem cell", "embryonic stem cell" and "induced pluripotent stem cell" are non-specialized and undifferentiated cells, which can self-renew and have the potential to be differentiated into different types of cells with special functions."Cell line" includes a cell culture derived from a cell type or a group of cells of the same type that can be infinitely propagated.Non-limiting examples of mammalian cell lines can include CD34 cells, 293 cells, HEK cells, CHO cells, BHK cells, CV-1 cells, Jurkat cells, HeLa cells or any variants thereof.

[0151] In some embodiments, the vector can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 and pMT2PC. When used in mammalian cells, the control function of the expression vector is generally provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters disclosed herein and known in the art. Other promoters may include, for example, the EF1 promoter or the EF1α promoter.

[0152] The terms "editing" and the like refer to any type of engineering, alteration, modification or regulation (in each case including but not limited to by gene knockout, gene marking, gene disruption, gene mutation, gene insertion, gene deletion, gene activation, gene silencing or gene knock-in).

[0153] CRISPR editing system

[0154] In the present invention, "CRISPR-based system", "CRISPR-based gene editing system", "CRISPR-genome editing", "CRISPR editing system", "CRISPR-gene editing", "CRISPR-endonuclease-based genome editing" and the like are used interchangeably herein and are collectively referred to as genome editing systems, which include one or more guide RNA elements; and one or more RNA-guided endonuclease elements. The guide RNA element comprises a targeting RNA containing a nucleotide sequence that is substantially complementary to a nucleotide sequence at one or more target genomic regions, or a nucleic acid containing a nucleotide sequence encoding the targeting RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or brought to the target genomic region by the guide RNA element; or a nucleic acid containing a nucleotide sequence encoding such an endonuclease. Examples of such CRISPR-based gene editing systems include CRISPR-based systems, i.e., CRISPR-Cas systems or CRISPR-Cpf systems.

[0155] In a specific embodiment of the invention, the CRISPR editing system is selected from the CRISPR-Cas system.

[0156] In some embodiments, the CRISPR-Cas editing system comprises:

[0157] At least one guide RNA element and a Cas protein element.

[0158] The terms "guide RNA element", "guide RNA", "gRNA", "gRNA molecule" and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a targeting RNA that hybridizes to a target nucleic acid sequence or a nucleic acid containing a nucleotide sequence encoding a targeting RNA. The targeting RNA of the gRNA comprises a targeting domain that comprises a nucleotide sequence that is substantially complementary to a nucleotide sequence at a target genomic region. The phrase "substantially complementary" means that the degree of complementarity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or that two nucleic acids hybridize under stringent conditions.

[0159] The guide RNA element may also include an activation RNA capable of hybridizing with the targeting RNA or a nucleic acid containing a nucleotide sequence encoding the activation RNA. The activation RNA and the targeting RNA may be separated or fused into a single nucleic acid via a linker loop sequence to form a single gRNA molecule. The gRNA molecule may include many domains. For example, such gRNAs may include, for example, from 5' to 3': a targeting domain (which is complementary to the target nucleic acid), a first complementary domain, a connecting domain, a second complementary domain (complementary to the first complementary domain), a proximal domain, and an optional tail domain.

[0160] The "first complementary domain" has substantial complementarity with the second complementary domain and can form a double-stranded region under at least some physiological conditions.

[0161] The "connection domain" is used to connect the first complementary domain of the single-molecule gRNA to the second complementary domain. The connection domain can covalently or non-covalently connect the first complementary domain and the second complementary domain.

[0162] A "proximal domain" may be 3-25 nucleotides in length, or may be 5-20 nucleotides in length. The proximal domain may share homology with or be derived from a naturally occurring proximal domain.

[0163] The "tail domain" may be absent or be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. The tail domain may comprise sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions.

[0164] The guide RNA element can form a complex with an endonuclease of an RNA-guided endonuclease element, such as a Cas endonuclease ("gRNA / nuclease complex"). An example of a gRNA / nuclease complex is a CRISPR complex described below for a CRISR-based system. In some embodiments, the CRISPR complex comprises an endonuclease of an RNA-guided endonuclease system complexed with a targeting RNA. In some embodiments, the CRISPR complex comprises an endonuclease of an RNA-guided endonuclease system complexed with a targeting RNA and an activating RNA.

[0165] The targeting domain of the targeting RNA promotes the gRNA / nuclease complex to specifically target or home to the target nucleotide sequence. In some embodiments, the targeting domain may be 10-30bp, such as 15-25bp, 18-22bp or 20bp.

[0166] Methods for designing gRNAs are known in the art, including methods for selecting, designing, and validating target domains.

[0167] In some embodiments, an RNA-guided endonuclease, such as a Cas enzyme (e.g., a type II Cas9 protein) can be used. In some embodiments, a modified form of such a Cas enzyme can also be used.

[0168] In some embodiments, Cas9 proteins from species other than S. pyogenes and S. thermophiles can be used. Additional Cas9 protein strains available and used herein include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus; Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp.), Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium perfingens, Corynebacterium accolens, Corynebacterium dolichum, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemoplzilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicohacter cinaedi, Helicobacter mustelae, llyobacter polytropus, Kingellakingae, lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria griseus cinerea), Neisseria flavescens, Neisseria lactamica, Neisseria sp.), Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutells, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis), Treponema sp., or Verminephrobacter eiseniae.

[0169] In some embodiments, one or more elements of a CRISPR-based system are derived from a specific organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes, Staphylococcus aureus, Francisella tularensis, Prevotella sp., Acidaminococcus sp., and Lachnospiraceae sp. Typically, CRISPR-based In the present invention, the system is characterized by promoting the formation of elements of a CRISPR complex at the site of a target genomic region or a target sequence (also referred to as a protospacer sequence in the context of an endogenous CRISPR system). In the context of forming a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence is designed to have substantial complementarity, wherein hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence can be in an organelle of a eukaryotic cell such as a mitochondria or a chloroplast.

[0170] In some embodiments, the CRISPR-based system is a CRISPR-Cas9 system. The targeting RNA of the CRISPR-Cas9 system includes a CRISPR targeting RNA (crRNA), and the activation RNA of the CRISPR-Cas 9 system includes a trans-activating CRISPR RNA (tracRNA). The Cas protein element of the CRISPR-Cas9 system uses Cas9 protein. CrRNA and tracrRNA can be separated or combined into a single RNA construct via a linker loop sequence. This combined RNA construct is referred to as a single guide RNA (sgRNA; or guide RNA).

[0171] Kits for editing one or more targeted genomic regions

[0172] The present invention provides a kit for editing one or more target genomic regions, the kit comprising:

[0173] 1) a first container, and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is the aforementioned compound, or a pharmaceutically acceptable salt thereof, or an optical isomer thereof, or a racemate thereof, or a solvate thereof; or the aforementioned composition;

[0174] 2) a second container, and a CRISPR editing system reagent located in the second container.

[0175] In some embodiments, the CRISPR-based genome editing system includes a CRISPR sequence, a transactivating cr (tracr) sequence, a guide sequence, and a Cas endonuclease, or any combination thereof. After administering a SpCas9 inhibitor to the cell, the HDR genome editing efficiency is improved.

[0176] In some embodiments, the CRISPR editing system comprises an RNA containing a CRISPR sequence (crRNA), an RNA containing a trans-activating cr (tracr) sequence (tracrRNA), and a Cas endonuclease, or any combination thereof.

[0177] In some embodiments, the CRISPR editing system comprises a CRISPR sequence, a guide sequence, and a Cas endonuclease, or any combination thereof.

[0178] Compounds for regulating gene editing and genome editing systems, kits and uses of their compositions

[0179] Genome editing for precise alterations of specific genomic regions has great therapeutic potential.

[0180] In some embodiments, provided herein is a method for modifying the expression of one or more genes or proteins, the method comprising administering a genome editing system and a Cas9 inhibitor described herein to one or more cells comprising one or more target genomic regions, wherein the genome editing system interacts with nucleic acids of the one or more target genomic regions of the target genes, resulting in editing of the one or more target genomic regions, and wherein the editing modifies the expression of downstream genes and / or proteins associated with the target genes.

[0181] Editing one or more target genomic regions includes any kind of genetic manipulation or engineering of the cell genome. Editing of one or more target genomic regions may include insertion, deletion or replacement of a genomic region in a cell by one or more endonucleases. The genomic region includes genetic material in the cell, such as DNA, RNA, polynucleotides and oligonucleotides. The genomic region in the cell also includes the genome of mitochondria or chloroplasts contained in the cell.

[0182] The Cas9 inhibitor can be any Cas9 inhibitor. The Cas9 inhibitor can be any compound or substance that causes Cas9 inhibition. The Cas9 inhibitor can be a compound, a small molecule, an antibody, or a nucleotide sequence. In some embodiments, the Cas9 inhibitor is a compound represented by the structural formula LXH0307 and LXH0308.

[0183] In some embodiments, provided herein are methods of treating a subject having a disease or condition requiring editing of one or more target genomic regions in cells of the subject, the method comprising administering a genome editing system and a Cas9 inhibitor to the one or more cells.

[0184] In some embodiments, the methods provided herein are used to modify the expression of genes, RNA molecules, proteins, proteomes, or downstream proteins in a pathway. Such modifications can be used to treat acquired or hereditary diseases, dysfunctions, abnormal physiological homeostasis, or diseases caused by the aging process. As used herein, the term "modification" includes regulating, enhancing, reducing, increasing, inserting, deleting, knocking out, knocking in, etc.

[0185] Those skilled in the art will appreciate that acquired or hereditary or otherwise acquired diseases involve disorders of homeostatic mechanisms including those involved in gene or protein function. To this end, the skilled person can use the methods provided herein to regulate, modify, enhance, reduce or provide other aspects of gene function in a subject.

[0186] Modifying the expression of a gene in a cell and subsequent protein expression can be achieved by the methods provided herein, for example, by specifically editing (e.g., replacing, inserting or deleting, any combination thereof) a nucleic acid sequence in any of an exon, an intron, a transcription start site, a promoter region, an enhancer region, a silencer region, an insulator region, an anti-repressor, a post-translational regulatory element, a polyadenylation signal (e.g., minimal poly A), a conserved region, a transcription factor binding site, or any combination thereof.

[0187] In some embodiments, the methods, kits, and compositions provided herein are used to treat subjects with cancer. The method of treating a subject with cancer or a cancer-related condition includes administering a Cas9 inhibitor and a genome editing system to the subject's cells. The administration of the Cas9 inhibitor and the genome editing system can be in vivo or ex vivo.

[0188] Cancer can be any type of cancer.Cancer includes solid tumors, such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma and other tissue and organ tumors, and blood cell cancers, such as lymphoma and leukemia, including acute myeloid leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia and B cell lymphoma.Cancer can include melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin lymphoma, chronic lymphocytic leukemia, and pancreatic cancer, breast cancer, thyroid cancer, ovarian cancer, uterine cancer, testicular cancer, pituitary cancer, kidney cancer, gastric cancer, esophageal cancer and rectal cancer.

[0189] In some embodiments, the kits and compositions provided herein are used to treat a subject having any one or more of the following cancers: acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma of the cerebellum or brain in children, bile duct cancer, extrahepatic (see bile duct cancer) bladder cancer, bone tumors, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain cancer, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumor brain tumor, visual pathway and hypothalamic glioma brain tumor, breast cancer, bronchial Ductal adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal cancer of unknown primary, primary central nervous system lymphoma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma / glioblastoma, cervical cancer, childhood cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma of the Ewing family of tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma (eye cancer), retinoblastoma (eye cancer), gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor tumors, gastrointestinal stromal tumors (GIST), extracranial, extragonadal or ovarian germ cell tumors, gestational trophoblastic tumors, brainstem gliomas, gliomas, astrocytoma gliomas in children, gliomas of the visual pathway and hypothalamus in children, gastric carcinoids, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, gliomas of the hypothalamus and visual pathway in children, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphocytic leukemia (also known as acute lymphocytic leukemia), acute myeloid leukemia (also known as acute myeloid leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), chronic myeloid Chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, cutaneous T-cell lymphoma Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin (old classification of all lymphomas except Hodgkin) lymphoma, primary central nervous system macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, childhood melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, childhood squamous neck cancer with occult metastasis from primary lesion, oral cancer, multiple endocrine neoplasia syndrome multiple myeloma / plasmacytoma, mycosis fungoides,Myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myeloid leukemia, chronic myeloid leukemia, acute myeloid leukemia in adults, multiple acute myeloma (bone marrow cancer) in children, myeloproliferative disorders, chronic myxoma, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-mesenchymal ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, islet cell pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasmacytoma / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter Carcinoma, Transitional Cell Carcinoma, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Carcinoma, Ewing Tumor Family Sarcomas, Kaposi Sarcoma, Soft Tissue Sarcoma, Uterine Sarcoma, Sézary Syndrome, Skin Cancer (Non-Melanoma), Skin Cancer (Melanoma), Merkel Cell Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma - See Skin Cancer (Non-Melanoma), Squamous Neck Cancer with Occult Metastatic Primary, Gastric Cancer, Supratentorial Primitive Neuroectodermal Tumor, Cutaneous T-cell Lymphoma (mycosis fungoides and Sézary syndrome), testicular cancer, laryngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, cancer of unknown primary site in adults, cancer of unknown primary site in children, transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, vulvar cancer, macroglobulinemia, or Wilms tumor (kidney cancer).

[0190] In some embodiments, the methods, kits, or compositions provided herein are used to treat a subject with a genetic disorder. The method of treating a subject with a genetic disease or condition or a genetic disorder comprises administering a Cas9 inhibitor and a genome editing system to the subject's cells. The administration of the Cas9 inhibitor and the genome editing system can be in vivo or ex vivo.

[0191] A hereditary disorder can be caused by a mutation or duplication in a chromosomal region (e.g., by a point mutation, deletion, insertion, frameshift, chromosomal duplication or deletion). A hereditary disorder can be any hereditary disorder. The term "hereditary disorder" or "hereditary disease" includes an inherited or acquired mutation in the subject's genome that causes or may cause a disease.

[0192] In some embodiments, the genetic disorders include but are not limited to 22q11.2 deletion syndrome, Angelman syndrome, Canavan disease, Charcot-Marie-Tooth disease, color blindness, Cry-a-cat syndrome, Down syndrome, Duchenne muscular dystrophy, hemochromatosis, hemophilia, Klinefelter syndrome, multiple neurofibromatosis, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, sickle cell disease, spinal muscular atrophy, spinal muscular atrophy, Tay-Sachs disease, Turner syndrome, hemoglobinopathy, or any combination thereof.

[0193] As used herein, "treat" or "alleviate" or "improve" are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefits and / or prophylactic benefits. By therapeutic benefit is meant any treatment-related improvement or effect on one or more diseases, conditions or symptoms in treatment. For prophylactic benefits, the composition can be administered to a subject at risk for developing a particular disease, condition or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition or symptom has not yet manifested. These terms also refer to treatment of a disease in a mammal, such as a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) alleviating the disease, i.e., causing regression of the disease state; or (c) curing the disease.

[0194] In embodiments, specific post-transcriptional control regulators are targeted by administering a Cas9 inhibitor and a genome editing system to modulate, modify, enhance or decrease activity.

[0195] In some embodiments, the activity of a genetic pathway associated with the cell cycle is modulated, enhanced, or decreased by administering a Cas9 inhibitor and a genome editing system.

[0196] In some embodiments, the activity of genes associated with angiogenesis is modulated, enhanced, or decreased by administering a Cas9 inhibitor and a genome editing system to cells.

[0197] In some embodiments, the activity of genetic pathways and / or genes associated with mitochondrial function is modulated, enhanced, or decreased by administering a Cas9 inhibitor and a genome editing system to a cell.

[0198] In some embodiments, the activity of genetic pathways and / or genes associated with DNA damage or genomic instability is modulated, enhanced, or decreased.

[0199] In some embodiments, a gene encoding a mammalian transcription factor is modulated, enhanced, decreased, or provided to a cell.

[0200] In some embodiments, the cell is a diseased or mutant cell. These cells can be manipulated to treat a disease, such as to correct a mutation or to change the phenotype of the cell, such as to inhibit the growth of a cancer cell. For example, the cell is associated with one or more diseases or conditions described herein.

[0201] The specific implementation of the present application is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present application, and is not used to limit the present application.

[0202] Example 1 Synthesis of small molecule compounds

[0203] 1. Cycloaddition reaction

[0204] 1. Add ethyl glyoxylate (20.4 g, 110 mmol) and 4-bromoaniline (17 g, 100 mmol) to 200 ml of toluene solution, then add 10 g of MgSO4, and stir at 0°C for 30 minutes;

[0205] 2. Add 1-tert-butyloxycarbonyl-2,3-dihydropyrrole (17 g, 100 mmol) to 60 ml of toluene, and add scandium trifluoromethanesulfonate at 0°C;

[0206] 3. Add the reaction solutions of step 1 and step 2 together and stir at room temperature for 6 hours;

[0207] 4. Extract with ethyl acetate / water, spin dry, weigh 29.75 g, yield 68%.

[0208]

[0209] 2. Chiral Separation

[0210] 1. Column chromatography PE:EA=6% to obtain 2-P1 as a white solid (17 g). Yield: 57%.

[0211] 2. Column chromatography PE:EA=8%-15% to obtain 2-P2 as a colorless oil (8.5 g). Yield: 29%.

[0212]

[0213] 3. Suzuki coupling

[0214] 1. Dissolve 2-P1 (17 g, 40 mmol), 2-fluorophenylboric acid (6.7 g, 48 mmol), and K3PO4 (25.4 g, 120 mmol) in dioxane (70 ml), then add Xphos-pd-G3 (3.2 g, 4 mmol), and reflux at 110°C for 20 h;

[0215] 2. Filter and spin dry;

[0216] 3. Column chromatography (PE:EA=14%), spin-drying to obtain 13 g of white solid 3-P1 with a yield of 73%.

[0217]

[0218] 4. Carboxylic Acid Reduction

[0219] 1. Dissolve 3-P1 (13 g, 28 mmol) in 100 ml THF, then slowly add LiBH4 (1.8 g, 88 mmol) dissolved in methanol (1 ml) at 0°C, react for 2 h, then react at room temperature for 15 h; finally, add 1N HCl;

[0220] 2. Extract with ethyl acetate / water and spin dry;

[0221] 3. Column chromatography (DCM:MeOH=4%), spin-drying to obtain 10.5 g of white solid 4-P1 with a yield of 90%.

[0222]

[0223] 5. Removal of Boc protection

[0224] 1. Add 10.5 g 4-P1 (26 mmol) to 40 ml HCl / Et2O and stir at room temperature for 6 h;

[0225] 2. The reaction solution was spin dried and washed twice with a mixture of PE (2×50 mL), DCM (2×50 mL), and n-hexane (2×100 mL);

[0226] 3. Filter and spin dry to obtain 7.9 g of yellow solid 5-P1 with a yield of 99%.

[0227]

[0228] 6. Sulfonylation reaction

[0229] 6.1 Preparation of LXH0307

[0230] 5-P1 (280 mg, 1 mmol) was added to 10 ml of DCM. Et3N was then slowly added dropwise in an ice bath at 0°C to fully dissolve 5-P1. Dansyl chloride (270 mg, 1 mmol) was then added dropwise and the reaction was stirred for 0.5 h. After the reaction was completed, water (10 ml) was added to quench the reaction. The mixture was extracted with DCM / W and dried by spin-drying. Column chromatography with PE:EA=50% was performed and the yellow fluorescent solid LXH0307 was obtained by spin-drying. The yield was 20%.

[0231] 6.2 Preparation of LXH0308

[0232] 5-P1 (280 mg, 1 mmol) was added to 10 ml of DCM. Then, Et3N was slowly added dropwise in an ice bath at 0°C to fully dissolve 5-P1. Subsequently, 2-methoxybenzenesulfonyl chloride (270 mg, 1 mmol) was added dropwise and the reaction was stirred for 0.5 h. After the reaction was completed, water (10 ml) was added to quench the reaction. The reaction was extracted with DCM / W and dried by spin-drying. Column chromatography with PE:EA=50% was performed and the mixture was dried by spin-drying to obtain 180 mg of light yellow solid LXH0307 with a yield of 37%.

[0233] 6.3 Preparation of 6-P1-1

[0234] 1. Add 5-P1 (150 mg, 1 mmol) to 10 ml DCM, then slowly add Et3N at 0°C to fully dissolve 5-P1, then add p-toluenesulfonyl chloride (190 mg, 1 mmol) and react for 30 minutes;

[0235] 2. After the reaction is complete, add 10 ml of water to quench;

[0236] 3. Extract with dichloromethane / water and spin dry;

[0237] 4. Column chromatography: petroleum ether:ethyl acetate = 10%, spin-dried to obtain white solid 6-P1, 181 mg, yield 80%.

[0238] 7. Chiral column separation

[0239] Chiral preparative liquid phase separation of 6-P1 gave 6-P1-1.

[0240] Example 2: Validation of the effectiveness of small molecule compounds

[0241] The Cas9 in vitro cleavage kit from Inovogen was used to detect the in vitro cleavage activity of Cas9 and evaluate the effects of the synthesized small molecule compounds LXH0307, ​​LXH0308 and 6-P1-1 on the cleavage efficiency of Cas9 protein. The experimental steps were carried out according to the instructions of the kit.

[0242] 1. Cas9 cleavage reaction:

[0243] Prepare Cas9 in vitro cleavage reaction buffer according to the instructions, mix evenly by pipetting, and perform the reaction;

[0244] Reaction procedure: 37°C for 30 min; 85°C for 10 min.

[0245] 2. Product testing

[0246] 1) Add 5 μl Cleaner to the reaction system and mix well. Incubate at 55°C for 5 min;

[0247] 2) Take 5 μl for gel electrophoresis and perform statistics.

[0248] 3. Results

[0249] The results are as follows Figure 1 As shown, compared with the control group DMSO, compounds LXH0307 and LXH0308 significantly inhibited the cleavage efficiency of Cas9 protein, and the inhibition efficiency was significantly higher than that of 6-P1-1.

[0250] The preferred embodiments of the present application are described in detail above in conjunction with the accompanying drawings; however, the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, a variety of simple modifications can be made to the technical solution of the present application, and these simple modifications all fall within the protection scope of the present application.

[0251] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not further describe various possible combinations.

[0252] In addition, the various implementation modes of the present application may be arbitrarily combined, and as long as they do not violate the concept of the present application, they should also be regarded as the contents disclosed in the present application.

Claims

1. A compound for regulating gene editing or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown below: 。 2. A method for preparing a compound that inhibits SpCas9, characterized in that: The method comprises: 1) Compound 2-P was synthesized by reacting bromoaniline, ethyl glyoxylate, and BOC-dihydropyrrole; 2) Compounds 2-P1 and 2-P2 were separated by column chromatography; 3) Compound 2-P1 is subjected to a Suzuki coupling reaction with 2-fluorophenylboronic acid to generate compound 3-P1; 4) subjecting compound 3-P1 to carboxylic acid reduction to generate compound 4-P1; 5) Compound 4-P1 is de-Bocated under acidic conditions to generate compound 5-P1; 6) Compound 5-P1 is subjected to sulfonylation reaction to generate LXH0307, The structure of the LXH0307 is shown below: 。 3. The method according to claim 2, characterized in that Step 1) The method for synthesizing compound 2-P is as follows: ethyl glyoxylate and 4-bromoaniline are added to a toluene solution, and then MgSO4 is added and stirred to obtain a reaction solution 1; BOC-dihydropyrrole is added to a toluene solution, and then scandium trifluoromethanesulfonate is added to react to obtain a reaction solution 2; reaction solution 1 and reaction solution 2 are added together to react, and the mixture is extracted and spin-dried.

4. The method according to claim 3, characterized in that The reaction mixture was stirred at 0°C to obtain reaction solution 1.

5. The method according to claim 3, characterized in that: Scandium trifluoromethanesulfonate was added at 0°C.

6. The method according to claim 3, characterized in that The extraction reagent was ethyl acetate / water.

7. The method according to claim 2, characterized in that Step 2) The eluent in the column chromatography is selected from PE and EA.

8. The method according to claim 7, characterized in that 2-P1 was isolated when PE:EA was 6%.

9. The method according to claim 2, characterized in that: Step 3) The catalytic reagent for the Suzuki coupling reaction is Xphos-pd-G3.

10. The method according to claim 2, characterized in that Step 3) The alkaline reagent for the Suzuki coupling reaction is K3PO4.

11. The method according to claim 9 or 10, characterized in that Step 3) The process of the Suzuki coupling reaction is as follows: Dissolve 2-P1, 2-fluorophenylboronic acid and K3PO4 in dioxane, then add Xphos-pd-G3 and reflux at 110°C for reaction.

12. The method according to claim 11, characterized in that Step 3) further comprises separating by column chromatography to obtain 3-P1.

13. The method according to claim 12, characterized in that The eluent for column chromatography is selected from PE and EA.

14. The method according to claim 13, characterized in that 3-P1 was isolated when the PE:EA ratio was 14%.

15. The method according to claim 2, characterized in that Step 4) The process of carboxylic acid reduction reaction is as follows: 3-P1 was dissolved in THF, and the reducing agent solution was slowly added dropwise to react.

16. The method according to claim 15, characterized in that The reducing agent is LiBH4.

17. The method according to claim 15, characterized in that The reducing agent solution is a methanol solution of LiBH4.

18. The method according to claim 15, characterized in that The reaction conditions were 0°C for 2 h and then room temperature for 15 h.

19. The method according to claim 18, characterized in that After the reaction is complete, the acid is added.

20. The method according to claim 19, characterized in that The acid is HCl.

21. The method according to claim 15, characterized in that Step 4) also includes extracting the reaction solution.

22. The method according to claim 21, characterized in that The extraction reagent was ethyl acetate / water.

23. The method according to claim 2, characterized in that Step 4) further comprises separating 4-P1 using column chromatography.

24. The method according to claim 23, characterized in that The eluent for column chromatography was selected from DCM and MeOH.

25. The method according to claim 24, characterized in that DCM:MeOH 4%.

26. The method according to claim 2, characterized in that Step 5) The process of removing Boc is as follows: 4-P1 was added to HCl / Et2O and reacted at room temperature.

27. The method according to claim 2, characterized in that Step 5) also includes spinning and washing the reaction solution.

28. The method according to claim 27, characterized in that The washing reagent is a mixed solution of PE, DCM and n-hexane.

29. The method according to claim 2, characterized in that The 5-P1 is a yellow solid.

30. The method according to claim 2, characterized in that Step 6) The steps for carrying out the sulfonylation reaction are as follows: 5-P1 was added to DCM, and Et3N was slowly added dropwise to allow 5-P1 to fully dissolve, and then the sulfonylating agent was added dropwise to react.

31. The method according to claim 30, characterized in that The sulfonylating agent is selected from dansyl chloride and 2-methoxybenzenesulfonyl chloride.

32. The method according to claim 31, characterized in that Et3N was slowly added dropwise at 0°C.

33. The method according to claim 2, characterized in that The step 6) further comprises quenching the reaction solution.

34. The method according to claim 33, characterized in that The step 6) further comprises extracting the quenched reaction solution.

35. The method according to claim 34, characterized in that The extraction reagent was dichloromethane / water.

36. The method according to claim 2, characterized in that Step 6) also includes a column chromatography step.

37. The method according to claim 36, characterized in that The eluent for column chromatography is selected from PE and EA.

38. The method according to claim 37, characterized in that The ratio of eluent PE to EA is 50%.

39. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound according to claim 1 or a pharmaceutically acceptable salt thereof; or the compound prepared by the method according to claim 2.

40. The pharmaceutical composition according to claim 39, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

41. A method for regulating a CRISPR editing system for gene editing for non-therapeutic purposes, characterized in that: Administering compound LXH0307 or LXH0308, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 39 to 40 to a subject in need thereof, wherein the structural formula of compound LXH0307 or LXH0308 is as follows: or .

42. A method for editing one or more target genomic regions for non-therapeutic purposes, characterized in that administering to one or more cells comprising one or more genomic regions 1) CRISPR editing system; and 2) Compound LXH0307 or LXH0308, or a pharmaceutically acceptable salt thereof; or a pharmaceutical composition according to any one of claims 39 to 40, wherein the structural formula of compound LXH0307 or LXH0308 is as follows: or .

43. The method according to claim 42, characterized in that The CRISPR editing system is previously administered to the one or more cells.

44. The method according to claim 43, characterized in that The one or more cells are cultured cells.

45. The method according to claim 43, characterized in that The one or more cells are in vivo cells within an organism.

46. ​​The method according to claim 43, characterized in that The one or more cells are ex vivo cells from an organism.

47. The method according to claim 42, characterized in that The CRISPR editing system is a CRISPR-Cas editing system.

48. The method according to claim 42, characterized in that 1) and 2) are administered via different routes.

49. The method according to claim 42, characterized in that 1) and 2) are administered via the same route.

50. The method of claim 42, wherein: The CRISPR editing system is delivered by one or more vectors.

51. The method according to claim 50, characterized in that The one or more vectors are selected from viral vectors, plasmids or ssDNA.

52. The method according to claim 51, characterized in that The viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector and / or a herpes simplex viral vector.

53. The method according to claim 42, characterized in that The CRISPR editing system is delivered via synthetic RNA.

54. The method according to claim 42, characterized in that The CRISPR editing system is delivered via nanoformulation.

55. A reagent product, characterized in that include: 1) A first reagent, wherein the first reagent is a gene editing inhibitor, wherein the gene editing inhibitor compound is LXH0307 or LXH0308, or a pharmaceutically acceptable salt thereof; and 2) A second reagent, wherein the second reagent is a reagent for CRISPR gene editing; The structural formula of compound LXH0307 or LXH0308 is as follows: or .

56. The product according to claim 55, characterized in that The second reagent comprises one or more reagents selected from the group consisting of: 1) Cas9 nuclease, a coding sequence of Cas9 nuclease, or a vector expressing Cas9 nuclease, or a combination thereof; 2) gRNA, crRNA, or a vector for producing the gRNA or crRNA; 3) Template for homology-directed repair: single-stranded nucleotide sequence or plasmid vector.

57. A kit for editing one or more target genomic regions, characterized in that The kit comprises: 1) A first container, and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is a compound LXH0307 or LXH0308, or a pharmaceutically acceptable salt thereof; or a composition according to any one of claims 39 to 40; 2) a second container, and a CRISPR editing system reagent located in the second container, The structural formula of compound LXH0307 or LXH0308 is as follows: or .

58. The kit according to claim 57, characterized in that The CRISPR editing system is a CRISPR-Cas editing system.

59. The kit according to claim 58, characterized in that The CRISPR-Cas editing system includes: At least one guide RNA element and a Cas protein element.

60. The kit according to claim 59, characterized in that The guide RNA element comprises: 1) a targeting RNA comprising a nucleotide sequence that is substantially complementary to a nucleotide sequence at the one or more target genomic regions, or a nucleic acid comprising a nucleotide sequence encoding the targeting RNA; and 2) an activating RNA comprising a nucleotide sequence capable of hybridizing with the targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding the activating RNA.

61. The kit according to claim 59, characterized in that The Cas protein is Cas9 protein.

62. The kit according to claim 61, characterized in that The Cas9 protein is SpCas9 protein.

63. The kit according to claim 57, characterized in that The CRISPR editing system is contained or packaged in one or more vectors.

64. The kit according to claim 63, characterized in that The one or more vectors are selected from viral vectors, plasmids or ssDNA.

65. The kit according to claim 64, characterized in that The viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector and / or a herpes simplex viral vector.

66. Use of compound LXH0307 or LXH0308, the pharmaceutical composition of any one of claims 39 to 40, the reagent product of any one of claims 55 to 56, or the kit of any one of claims 57 to 65 in the preparation of a product for regulating genome editing, wherein: The structural formula of compound LXH0307 or LXH0308 is shown below: or .

67. The use according to claim 66, characterized in that The genome editing targets pathogenic genes, tumor-related genes, immune-related genes, vision-related genes, hearing-related genes, metabolism-related genes, viral infection-related genes, and genetic disease-related genes.

68. The use according to claim 67, characterized in that The tumor gene is selected from oncogenes, and the immune-related gene is selected from genes related to autoimmunity.

69. The use according to claim 66, characterized in that The genome editing uses the CRISPR editing system.

70. The use according to claim 69, characterized in that The CRISPR editing system is selected from the CRISPR-Cas editing system.

71. Use of the compound LXH0307 or LXH0308, the pharmaceutical composition according to any one of claims 39 to 40, the reagent product according to any one of claims 55 to 56, or the kit according to any one of claims 57 to 65 in the preparation of a drug for treating a disease, wherein: The structural formula of compound LXH0307 or LXH0308 is shown below: or .

72. The use according to claim 71, characterized in that The disease is selected from cancer or a genetic disorder.

73. Use of the compound LXH0307 or LXH0308, the pharmaceutical composition of any one of claims 39 to 40, the reagent product of any one of claims 55 to 56, or the kit of any one of claims 57 to 65 in constructing a disease model, wherein: The structural formula of compound LXH0307 or LXH0308 is shown below: or .

74. The use according to claim 73, characterized in that The disease model is a cell disease model or an animal disease model.

75. The use according to claim 73, characterized in that The disease model is used to screen drugs for treating diseases.

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