CN114524811A - LXH0307 and LXH0308 as small molecule inhibitors of gene editing and application thereof - Google Patents

LXH0307 and LXH0308 as small molecule inhibitors of gene editing and application thereof Download PDF

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CN114524811A
CN114524811A CN202210261597.XA CN202210261597A CN114524811A CN 114524811 A CN114524811 A CN 114524811A CN 202210261597 A CN202210261597 A CN 202210261597A CN 114524811 A CN114524811 A CN 114524811A
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crispr
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李慧艳
吴敏
何新华
宋增庆
张学敏
李爱玲
周涛
张宇程
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Abstract

The invention discloses LXH0307 and LXH0308 serving as small molecule inhibitors for gene editing and application thereof, wherein the LXH0307 and the LXH0308 have stronger capability of inhibiting the gene editing activity. The invention also provides a synthesis method of LXH0307, LXH0308, a composition containing LXH0307 and LXH0308, a reagent product, a kit and an application thereof.

Description

LXH0307 and LXH0308 as small molecule inhibitors of gene editing and application thereof
Technical Field
The invention belongs to the field of chemical drugs, and particularly relates to LXH0307 and LXH0308 serving as small molecule inhibitors of SpCas9 and effects of the small molecule inhibitors.
Background
Genome editing technology is a genetic manipulation technology that artificially modifies a DNA sequence at the genome level, including insertion, knock-out, substitution, and point mutation of a specific DNA fragment. Among them, the basic principle of the nuclease-dependent genome editing technology is to generate Double-stranded DNA breaks (DSBs) at specific positions of the genome and then repair them by Non-Homologous end joining (NHEJ) or Homologous Recombination (HR). With the intensive research on nucleases, genome editing techniques have also been rapidly developed, wherein the most commonly used nucleases mainly include meganuclease, zinc finger nuclease, transcription activator-like effector nuclease, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -associated proteins (Cas).
The CRISPR/Cas system is a natural immune system from bacteria and archaea, consisting of CRISPR sequences and highly diverse Cas proteins. CRISPR sequences consist of regions of repeated sequences (Repeats) and Spacers (Spacers). The repeated sequence region contains palindrome sequence and can form hairpin structure, and the spacer region is the captured exogenous DNA sequence. Cas proteins can be divided into 4 modules according to function: the adaptation module mainly participates in the process of obtaining the Spacer and integrating the exogenous gene into the CRISPR sequence; the expression processing module is responsible for the processing of pre-crRNA; the interference and effect sub-module participates in target identification and cutting; the signal transduction and accessory modules are a collection of functionally distinct genes. Based on the sequence, combinatorial differences of Cas proteins, CRISPR systems can be divided into 2 major classes. Class 1 CRISPR/Cas systems are effector submodules consisting of multiple Cas proteins, while class 2 CRISPR/Cas systems are single multi-domain Cas protein effector submodules.
Recent researches show that the CRISPR system can be used for not only cutting viral DNA, but also precisely cutting other DNA sequences, the crRNA-tracrRNA is simplified into Single strand guide RNA (sgRNA), and can be simply designed and synthesized, and the sequence of the sgRNA can be changed to match a target gene, thereby guiding the Cas9 protein to a specified position to cut DNA.
With the development of molecular biology and sequencing technology, people's understanding of diseases enters the molecular level, and the discovery that the occurrence and the progress of many diseases of human beings can be related to gene mutation. For example: mutations in certain proto-oncogenes can contribute to tumorigenesis, and even mutations in certain tumor suppressor genes can contribute to tumorigenesis by causing activation of the proto-oncogenes, etc. (Jiang CY, Lin XH, Zhao ZG. applications of CRISPR/Cas9 technology in the treatment of long cancer. trends Mol Med,2019,25(11): 1039-1049). Therefore, the determination of genes closely related to diseases and functions thereof can help to provide better strategies for clinical treatment. With the rapid development of the CRISPR technology, genome screening based on the CRISPR technology is widely applied in the laboratory level, and the screening method has the advantages of flexible library design, simplicity in operation, wide coverage and the like. Although the CRISPR-Cas9 technology is becoming more sophisticated, there are certain risks associated with its widespread use in clinical gene therapy, which remain to be improved in specificity, safety and in vivo transduction.
The safe application of CRISPR-Cas9 in gene therapy requires the ability to: once the desired use has been achieved, the gene editing activity of the Cas9/sgRNA complex is controlled. Although several engineered systems allow for controlled activation of CRISPR-Cas9 to improve accuracy, all of these systems still lack the ability to provide predictable control and robust inhibition. The development of agents that regulate gene editing has greatly improved efficacy and safety for clinical therapeutic and research applications based on CRISPR systems.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention provides compounds and methods for preparing compounds that modulate gene editing, which compounds have a high ability to modulate gene editing activity.
The invention adopts the following technical scheme:
in a first aspect, the present invention provides a compound for modulating gene editing, said compound having the structure of formula (I):
Figure BDA0003550327940000021
wherein R is an aromatic ring which may be substituted with 0, 1, 2 or 3 substituents R1Substituted, said substituent R1Independently selected from halo, CN, C1-C4Alkyl radical, C1-C4-haloalkyl group, C3-C4-cycloalkyl, OR2、NR2R3(ii) a Each R2And R3Independently is H, C1-C4Alkyl or C1-C4-a haloalkyl group;
in some embodiments, the aromatic ring is a benzene ring or naphthalene.
In some embodiments, the R is2And R3Independently is H, C1-C4An alkyl group.
In a preferred embodiment, the structure of the compound is as follows:
Figure BDA0003550327940000031
in a second aspect, the present invention provides a process for the preparation of a compound according to the first aspect of the invention, said process comprising:
1) reacting bromoaniline, ethyl glyoxylate and BOC-dihydropyrrole to synthesize a compound 2-P;
2) separating by column chromatography to obtain compounds 2-P1 and 2-P2;
3) carrying out suzuki coupling reaction on the compound 2-P1 and 2-fluorobenzeneboronic acid to generate a compound 3-P1;
4) carrying out carboxylic acid reduction reaction on the compound 3-P1 to generate a compound 4-P1;
5) de-Boc the compound 4-P1 under acidic condition to generate a compound 5-P1;
6) compound 5-P1 is sulfonylated to produce LXH0307 and LXH 0308.
In some embodiments, step 1) the method of synthesizing compound 2-P is as follows: ethyl glyoxylate and 4-bromoaniline were added to the toluene solution, followed by MgSO4Stirring to obtain a reaction solution 1; adding BOC-pyrroline into a toluene solution, and then adding scandium trifluoromethanesulfonate to react to obtain a reaction solution 2; adding the reaction solution 1 and the reaction solution 2 together for reaction, and extracting and spin-drying.
In some embodiments, stirring at 0 ℃ provides reaction solution 1.
In some embodiments, scandium triflate is added at 0 ℃.
In some embodiments, the extraction reagent is ethyl acetate/water.
In some embodiments, the eluent in step 2) column chromatography is selected from PE and EA.
In some embodiments, 2-P1 is isolated with 6% EA.
In some embodiments, the catalytic agent for the suzuki coupling reaction of step 3) is Xphos-pd-G3.
In some embodiments, the basic reagent of the suzuki coupling reaction of step 3) is K3PO4。
In some embodiments, the procedure for the step 3) suzuki coupling reaction is as follows:
2-P1, 2-fluorobenzeneboronic acid and K3PO4Dissolved in dioxane, Xphos-pd-G3 was added and the reaction was refluxed at 110 ℃.
In some embodiments, step 3) further comprises isolating 3-P1 using column chromatography.
In some embodiments, the eluent for column chromatography is selected from PE and EA.
In some embodiments, EA of 14% provides 3-P1.
In some embodiments, the process of step 4) carboxylic acid reduction reaction is as follows:
3-P1 was dissolved in THF and a reducing agent solution was slowly added dropwise to carry out the reaction.
In some embodiments, the reducing agent is LiBH4。
In some embodiments, the reducing agent solution is LiBH4The methanol solution of (1).
In some embodiments, the reaction conditions are 0 ℃ for 2h, followed by room temperature for 15 h.
In some embodiments, the acid is added after the reaction is complete.
In some embodiments, the acid is HCl.
In some embodiments, step 4) further comprises extracting the reaction solution.
In some embodiments, the extraction reagent is ethyl acetate/water.
In some embodiments, step 4) further comprises isolating 4-P1 using column chromatography.
In some embodiments, the eluent for column chromatography is selected from DCM and MeOH.
In some embodiments, DCM: MeOH was 4%.
In some embodiments, the Boc removal in step 5) is as follows:
4-P1 was added to HCl/Et2And reacting at normal temperature in O.
In some embodiments, step 5) further comprises spin-drying and washing the reaction solution.
In some embodiments, the washing reagent is a mixed solution of PE, DCM and n-hexane.
In some embodiments, the 5-P1 is a yellow solid.
In some embodiments, step 6) performs the sulfonylation reaction as follows:
5-P1 was added to DCM and Et was slowly added dropwise3N, fully dissolving 5-P1, and then dropwise adding a sulfonylation reagent for reaction;
in some embodiments, the sulfonylating agent is selected from dansyl chloride, 2-methoxybenzenesulfonyl chloride;
in some embodiments, Et is slowly added dropwise at 0 ℃3N。
In some embodiments, step 6) further comprises quenching the reaction solution.
In some embodiments, step 6) further comprises extracting the quenched reaction solution.
In some embodiments, the extraction reagent is dichloromethane/water.
Preferably, step 6) further comprises a column chromatography step.
Preferably, the eluent for column chromatography is selected from PE and EA.
Preferably, the eluents PE and EA are in a proportion of 50%.
In a third aspect, the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof; or a compound or intermediate prepared by the process of the second aspect of the invention;
in some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In a fourth aspect, the present invention provides a method for regulating CRISPR system for gene editing, comprising administering to a subject in need thereof a compound according to the first aspect of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof; or a pharmaceutical composition according to the third aspect of the invention.
In a fifth aspect, the invention provides a method of editing one or more target genomic regions by administering to one or more cells comprising one or more genomic regions
1) A CRISPR editing system; and
2) a compound according to the first aspect of the present invention, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof; or a pharmaceutical composition according to the third aspect of the invention.
In some embodiments, the CRISPR editing system was previously administered into the one or more cells.
In some embodiments, the one or more cells are cultured cells.
In some embodiments, the one or more cells are in vivo cells within an organism.
In some embodiments, the one or more cells are ex vivo cells from an organism.
In some embodiments, the CRISPR editing system is a CRISPR-Cas editing system.
In some embodiments, 1) and 2) are administered via different routes.
In some embodiments, 1) and 2) are administered via the same route.
In some embodiments, the CRISPR editing system is delivered by one or more vectors.
In some embodiments, the one or more vectors are selected from viral vectors, plasmids, or ssDNA.
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.
In some embodiments, the CRISPR editing system is delivered by synthetic RNA.
In some embodiments, the CRISPR editing system is delivered by a nanoformulation.
In a sixth aspect the present invention provides a reagent product comprising:
1) a first agent, which is a gene editing inhibitor, wherein the gene editing inhibitor is the compound according to one aspect of the present invention, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof; and
2) a second agent that performs CRISPR gene editing.
In some embodiments, the second agent comprises one or more agents selected from the group consisting of:
1) cas9 nuclease, a coding sequence for Cas9 nuclease, or a vector expressing Cas9 nuclease, or a combination;
2) a gRNA, a crRNA, or a vector for producing the gRNA or crRNA;
3) template for homogenous directed repair: a single-stranded nucleotide sequence or a plasmid vector.
The invention provides in a seventh aspect a kit for editing one or more target genomic regions, the kit comprising:
1) a first container, and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is the compound of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof; or a composition according to the third aspect of the invention;
2) a second container, and CRISPR editing system reagents located within the second container;
in some embodiments, the CRISPR editing system is a CRISPR-Cas editing system.
In some embodiments, the CRISPR-Cas editing system comprises:
at least one guide RNA element and a Cas protein element.
In some embodiments, the guide RNA element comprises: 1) a targeting RNA comprising a nucleotide sequence 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; 2) and an activating RNA comprising a nucleotide sequence capable of hybridizing to the targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding the activating RNA.
In some embodiments, the Cas protein is a Cas9 protein.
In a preferred embodiment, the Cas9 protein is SpCas9 protein.
In some embodiments, wherein the CRISPR editing system comprises or is packaged in one or more vectors.
In some embodiments, the one or more vectors are selected from viral vectors, plasmids, or ssDNA.
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.
In an eighth aspect, the present invention provides a compound of the first aspect of the present invention, a pharmaceutical composition of the third aspect of the present invention, a reagent product of the sixth aspect of the present invention or a kit of the seventh aspect of the present invention for use in modulating genome editing.
In some embodiments, the genome editing is directed to a disease-causing gene, a tumor-associated gene (e.g., an oncogene), an immune-related gene (e.g., a gene associated with autoimmunity), a vision-related gene, an auditory-related, a metabolic-related, a viral infection-related, a genetic disease-related gene.
In some embodiments, the genome editing employs a CRISPR editing system;
in some embodiments, the CRISPR editing system is selected from a CRISPR-Cas editing system.
In a ninth aspect, the present invention provides the use of a compound according to the first aspect of the present invention, a pharmaceutical composition according to the third aspect of the present invention, a reagent product according to the sixth aspect of the present invention or a kit according to the seventh aspect of the present invention in the manufacture of a medicament for the treatment of a disease.
In some embodiments, the disease is cancer.
In some embodiments, the disease is a genetic disorder.
A tenth aspect of the invention provides a compound of the first aspect of the invention, a pharmaceutical composition of the third aspect of the invention, a reagent product of the sixth aspect of the invention or a kit of the seventh aspect of the invention for use in constructing a disease model.
In some embodiments, the disease model is a cellular disease model or an animal disease model.
In some embodiments, the disease model is used to screen for drugs that treat the disease. If the substance to be screened can treat or relieve the symptoms of the disease, the substance to be screened can be used as a potential drug for treating the disease.
The invention has the beneficial effects that:
the invention provides compounds LXH0307 and LXH0308 for remarkably inhibiting gene editing, which have stronger inhibition effect on CRISPR-Cas mediated shearing efficiency;
the invention provides a simple and efficient strategy for precise gene editing based on LXH0307/LXH0308 and CRISPR editing system.
The preparation and synthesis method of the compound uses a Boc protection strategy, reduces the cost and increases the yield.
Drawings
FIG. 1 is a graph of the effect of SpCas9 inhibitors on in vitro cleavage activity; wherein 1A is inhibitor LXH0307 and 1B is inhibitor LXH 0308.
Detailed Description
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. Co-administration of such Acr inhibitors may provide advantageous assistance in: allows safe and practical biological therapeutics by controlling Cas9 activity spatially or temporally; control of Cas 9-based gene drivers in wild populations to mitigate the ecological consequences of such forced genetic programs; and to contribute to general research into various biotechnological, agricultural and medical applications of gene editing technology.
The CRISPR-Cas system is an adaptive immune system in bacteria and archaea that helps defend against predatory viral attacks. These systems use small RN (CRISPR RNA (crRNA)) as a guide to identify their nucleic acid targets, which are then cleaved and inactivated.
One subset of CRISPR-Cas systems (those called "type II") employ a protein called Cas9, and this protein (along with its RNA guide) has been adapted to be a revolutionary genome editing platform. Variants carrying mutations in their nuclease active sites have also been developed as RNA-guided DNA binding platforms to enable gene labeling and transcriptional control. Cas9 has been widely used as a research tool, and it is also being used for a wide range of biotechnological applications, and in animal husbandry, agriculture and elsewhere.
In addition, the CRISPR-Cas9 platform is being developed as a potentially revolutionary treatment for many diseases, given its potential ability to modify genomic loci that contribute to the disease, disrupt the genome of the pathogen, or introduce therapeutically useful sequences (e.g., chimeric antigen receptors for cancer immunotherapy).
One current limitation of Cas9 application is that its activity can be very difficult to control: it is generally active as long as it is present and is able to access its crRNA guide, usually in the form of a so-called single guide rna (sgrna). This lack of control can introduce difficulties for a number of 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 into undesired sites. This is a concern for many purposes, particularly but not exclusively for clinical therapy.
It is well established that the more functional Cas9/sgRNA present in a cell and the longer Cas9/sgRNA lasts the greater the likelihood of off-target effects. While the mechanism of the present invention is not required to be understood, it is believed that once "on-target" genome editing occurs, the sustained Cas9/sgRNA can no longer recognize and edit the desired locus, thereby accomplishing its intended purpose. Nevertheless, the Cas9/sgRNA complex, which is still functional, can continue to find and edit off-target sites elsewhere.
The present invention contemplates compositions that inhibit Cas9/sgRNA activity. While not necessary to understand the mechanism of the invention, it is believed that these inhibitor compounds interact with active Cas9/sgRNA and act as a "off switch" that minimizes Cas9/sgRNA activity. And/or for a duration necessary for its intended purpose. Further, it is believed that Cas9/sgRNA inhibitor compositions can limit Cas9/sgRNA activity to a particular tissue or organ, or to any given set of user-defined conditions, by "activating a close switch" anywhere, at any time, or under any circumstances when its activity is not desired.
Accordingly, the present invention provides a compound for modulating gene editing, said compound having the structure described by formula (I):
Figure BDA0003550327940000091
wherein R is an aromatic ring which may be substituted with 0, 1, 2 or 3 substituents R1Substituted, said substituent R1Independently selected from halo, CN, C1-C4Alkyl radical, C1-C4-haloalkyl group, C3-C4-cycloalkyl, OR2、NR2R3(ii) a Each R2And R3Independently is H, C1-C4Alkyl or C1-C4-a haloalkyl group;
in some embodiments, the aromatic ring is a benzene ring or naphthalene.
In some embodiments, the R is2And R3Independently is H, C1-C4An alkyl group.
In some embodiments, the derivative has the structure shown in any one of the following:
Figure BDA0003550327940000092
the compounds disclosed herein may be present 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 adduct or derivative that, when administered to a patient in need thereof, is capable of providing, directly or indirectly, a compound as described elsewhere herein, or a metabolite or residue thereof.
Pharmaceutical composition
The pharmaceutical composition comprises the compound, pharmaceutically acceptable salt, optical isomer, racemate or solvate thereof.
The term "pharmaceutically acceptable salts" 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.
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 alginates, ascorbates, aspartates, benzenesulfonates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, caproates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, dodecylsulfates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoate, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, sulfates, tartrates, Thiocyanate, p-toluenesulfonate,Undecanoate salts, pentanoate salts, and the like. Additional exemplary salts include adipate, benzoate, citrate, fumarate, maleate, or succinate. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C)1-4Alkyl radical)4And (3) salt.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, adjuvants or vehicles include any or all of solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like as appropriate for the particular intended dosage form. Various carriers for The formulation of pharmaceutically acceptable compositions and known techniques for their preparation are disclosed in Remington, The Science and Practice of Pharmacy, 21 st edition, 2005, D.B.Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, J.Swarbrick and J.C.Boylan, 1988. Ank 1999, Marcel Dekker, New York (The contents of each of which are incorporated herein by reference). Unless any conventional carrier vehicle is incompatible with the compounds of the present invention (e.g., due to producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of a pharmaceutically acceptable composition), its use is contemplated within the scope of the present invention.
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 silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates (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, ethyl cellulose and cellulose acetate; powdered gum tragacanth; malt; gelatin; talc powder; 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, mold release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir (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 compositions of the present invention may be an aqueous or oleaginous suspension. Such suspensions may be formulated according to the techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Acceptable carriers and solvents that may be used are water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants (such as tweens, spans, and other emulsifiers or bioavailability promoters) commonly used to prepare pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.
The pharmaceutically acceptable compositions of the present invention may be administered orally 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. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in the form of a capsule, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration.
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 compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
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, e.g., sodium citrate or dicalcium phosphate and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerol, d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption promoters, such as quaternary ammonium compounds, g) wetting agents, such as cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay, and i) lubricants, such as talc, calcium stearate, mannitol, sodium chloride, and sodium chloride, Magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Method
The invention provides a method for regulating and controlling a CRISPR editing system to carry out gene editing, which comprises the steps of administering the compound, the pharmaceutically acceptable salt thereof, the optical isomer thereof, the racemate thereof or the solvate thereof to a needed object; or a pharmaceutical composition as hereinbefore described.
As used herein, "administering" refers to contacting, injecting, distributing, delivering, or applying the genome editing system and/or the compounds/compositions described previously to a cell or subject. In some embodiments, administration is by contacting the genome editing system and/or the compounds/compositions described previously with a cell. In some embodiments, the administering is delivering the genome editing system and/or the previously described compounds/compositions to the cell. In some embodiments, administration is to apply the genome editing system and/or the compounds/compositions described previously to the cell. In some embodiments, administration is injection of the genome editing system and/or the compounds/compositions described previously into the cell. Administration can occur in vivo, ex vivo, or in vitro. Administration of the genome editing system and the aforementioned compounds/compositions to the cell can be performed sequentially.
The CRISPR editing system is delivered by one or more vectors.
In some embodiments, the one or more vectors are selected from viral vectors, plasmids, or ssDNA.
A "viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, ex vivo, or in vitro. Examples of the viral vector include a retroviral vector, an adenoviral vector, an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, a herpes simplex viral vector, a chimeric viral vector and the like. In some embodiments, wherein gene transfer is mediated by a retroviral vector, a vector construct refers to a polynucleotide comprising a retroviral genome or portion thereof.
In some embodiments, the CRISPR editing system is delivered by synthetic RNA.
In some embodiments, the CRISPR editing system is delivered by a nanoformulation.
Some embodiments of the present disclosure relate to a vector system comprising one or more vectors or the vectors themselves. The vectors can be designed for expression of 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 e.coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells.
The cells may be primary cells, induced pluripotent stem cells (ipscs), embryonic stem cells (hescs), adult stem cells, progenitor cells, or cell lines. "Primary cells" are cells taken directly from living tissue and grown in vitro. Population doublings of primary cells are rare and population doublings have a limited lifespan in vitro. "stem cells", "embryonic stem cells" and "induced pluripotent stem cells" are non-specialized and undifferentiated cells, capable of self-renewal and having the potential to differentiate into different types of cells with specific functions. A "cell line" includes a cell culture derived from one cell type or a group of cells of the same type that can be immortalized. 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.
In some embodiments, the vector is capable of driving expression of one or more sequences in a mammalian cell using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 and pMT2 PC. When used in mammalian cells, the control functions of the expression vector are typically 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 EF 1a promoter.
The term "editing" or the like refers to any type of engineering, alteration, modification or regulation (in each case, including, but not limited to, by gene knock-out, gene tagging, gene disruption, gene mutation, gene insertion, gene deletion, gene activation, gene silencing or gene knock-in).
CRISPR editing system
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 collectively are referred to as genome editing systems, which comprise one or more guide RNA elements; and one or more RNA-guided endonuclease elements. The guide RNA element comprises a targeting RNA comprising a nucleotide sequence substantially complementary to a nucleotide sequence at one or more target genomic regions, or a nucleic acid comprising a nucleotide sequence encoding a targeting RNA. The RNA-guided endonuclease element comprises an endonuclease that is guided or brought to a target genomic region by a guide RNA element; or a nucleic acid comprising 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.
In a specific embodiment of the invention, the CRISPR editing system is selected from the CRISPR-Cas system.
In some embodiments, the CRISPR-Cas editing system comprises:
at least one guide RNA element and a Cas protein element.
The terms "guide RNA element," "guide RNA," "gRNA molecule," and "synthetic guide RNA" are used interchangeably and refer to a polynucleotide sequence comprising a target RNA that hybridizes to a target nucleic acid sequence or a nucleic acid containing a nucleotide sequence encoding the target RNA. The targeting RNA of the gRNA comprises a targeting domain comprising a nucleotide sequence that is substantially complementary to a nucleotide sequence at a target genomic region. The phrase "substantially complementary" means complementary to a degree of 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.
The guide RNA element may further comprise an activating RNA capable of hybridizing to the targeting RNA or a nucleic acid comprising a nucleotide sequence encoding the activating RNA. The activating RNA and the targeting RNA can be isolated or fused into a single nucleic acid via linker loop sequences to form a single gRNA molecule. gRNA molecules can comprise a number of domains. For example, such grnas comprise, e.g., from 5 'to 3': a targeting domain (which is complementary to the target nucleic acid), a first complementing domain, a linking domain, a second complementing domain (complementary to the first complementing domain), a proximal domain, and optionally a tail domain.
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.
A "linking domain" is used to link a first complementary domain to a second complementary domain of a single gRNA. The linking domain may covalently or non-covalently link the first complementary domain and the second complementary domain.
The "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.
The "tail domain" may be absent or 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.
The guide RNA element can form a complex ("gRNA/nuclease complex") with an endonuclease, such as a Cas endonuclease, of the RNA-guided endonuclease element. An example of a gRNA/nuclease complex is the CRISPR complex described below for CRISR-based systems. 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 to a targeting RNA and an activating RNA.
The targeting domain of the targeting RNA facilitates specific targeting or homing of the gRNA/nuclease complex to the target nucleotide sequence. In some embodiments, the targeting domain may be 10-30bp, such as 15-25bp, 18-22bp, or 20 bp.
Methods for designing grnas are known in the art, including methods for selecting, designing, and validating target domains.
In some embodiments, an RNA-guided endonuclease, such as a Cas enzyme (e.g., type II Cas9 protein), may be used. In some embodiments, modified forms of this Cas enzyme may also be used.
In some embodiments, Cas9 proteins from species other than streptococcus pyogenes (s.pyogenes) and streptococcus thermophilus (s.thermophiles) may be used. Additional Cas9 protein species available and used herein include: acidovorax avenae (Acidovorax avenae), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Actinobacillus succinogenes (Actinobacillus succinogenes), Actinobacillus suis (Actinobacillus suis), Actinomyces sp.sp., cyclophilus denticola, Aminomonas paucivorans (Aminomonas paucivorans), Bacillus cereus (Bacillus cereus); bacillus smithii (Bacillus smithii), Bacillus thuringiensis (Bacillus thuringiensis), Bacteroides sp (Bacteroides sp.), Blastopirella marina, Bradyrhizobium sp (Bradyrhizobium sp.), Brevibacillus laterosporus (Brevibacillus laterosporus), Campylobacter coli (Campylobacter coli), Campylobacter jejuni (Campylobacter jejuni), Campylobacter marini (Campylobacter lari), Candidatus Punica, Clostridium cellulolyticum (Clostridium clostridia), Clostridium perfringens (Clostridium perfringens), Corynebacterium crowding (Corynebacterium acetobacter), Corynebacterium elongatum (Corynebacterium glutamicum), Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium crowding (Corynebacterium glutamicum), Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium (Corynebacterium glutamicum), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus (Corynebacterium glutamicum), Corynebacterium (Corynebacterium glutamicum), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium glutamicum), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium glutamicum), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) strain (Corynebacterium) strain (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) and Bacillus (Corynebacterium) strain (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) strain (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) strain (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) strain (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium (Corynebacterium), Corynebacterium) and Bacillus (Corynebacterium) and Bacillus (Corynebacterium) strain (Corynebacterium), Slime tropheus (llyobacter polytropus), bacillus thuringiensis (Kingella kinggae), lactobacillus crispatus (lactibacillus crispatus), listeria monocytogenes (listeria ivanovii), listeria monocytogenes (listeria monocytogenes), campylobacter sphaericus (mobilus mulitilis), Neisseria bacilli (Neisseria bacteriovoris), Neisseria cinerea (Neisseria cinerea), Neisseria shallot (Neisseria avflns), Neisseria lactis (Neisseria lactia), Neisseria species (Neisseria sp), Neisseria lactis (Neisseria pis), rhodobacter sphaeroides sp), rhodobacter sphaeroides (Neisseria lactis), rhodobacter sphaeroides (bacterium sp), rhodobacter sphaeroides (rhodobacter sphaeroides), rhodobacter sphaeroides sp), rhodobacter sphaeroides, rhodobacter spha, Salmonella miehei (Simnesiella mueller), Sphingomonas sp, Sporolactobacillus vinae, Staphylococcus lugdunensis (Staphyloccocus lugdunensis), Streptococcus sp (Streptococcus sp), Pediococcus sp, Thermus sp, Treeletriparia (Tistrella mobilis), Treponema sp or Phanerochaete gasterensis (Verminthrobacter eisenseniae).
In some embodiments, one or more elements of the CRISPR-based system are derived from a particular organism comprising an endogenous CRISPR system, such as streptococcus pyogenes, Staphylococcus aureus (Staphylococcus aureus), Francisella tularensis (Francisella tularensis), Prevotella species (Prevotella sp.), aminoacidococcus species (Acidaminococcus sp.), and trichospira species (Lachnospiraceae sp.). Generally, CRISPR-based in the present invention, systems are characterized by elements that promote the formation of CRISPR complexes at sites of a target genomic region or target sequence (also referred to as protospacer in the context of endogenous CRISPR systems). In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have substantial complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of the CRISPR complex. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote 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 may be within an organelle of the eukaryotic cell, such as a mitochondrion or chloroplast.
In some embodiments, the CRISPR-based system is a CRISPR-Cas9 system. The targeting RNA of the CRISPR-Cas9 system comprises CRISPR targeting RNA (crrna), and the activating RNA of the CRISPR-Cas9 system comprises trans-activation CRISPR RNA (tracRNA). The Cas protein element of the CRISPR-Cas9 system employs Cas9 protein. The crRNA and tracrRNA may be combined into a single RNA construct separately or via linker loop sequences. Such combined RNA constructs are referred to as single guide RNAs (sgRNAs; or guide RNAs).
Kit for editing one or more target genomic regions
The present invention provides a kit for editing one or more target genomic regions, the kit comprising:
1) a first container and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is the compound, or pharmaceutically acceptable salt thereof, or optical isomer or racemate thereof, or solvate thereof; or a composition as described previously;
2) a second container, and CRISPR editing system reagents located within the second container.
In some embodiments, the CRISPR-based genome editing system comprises a CRISPR sequence, a trans-activating cr (tracr) sequence, a guide sequence, and a Cas endonuclease, or any combination thereof. HDR genome editing efficiency is improved upon administration of SpCas9 inhibitors to cells.
In some embodiments, the CRISPR editing system comprises an rna (crrna) comprising a CRISPR sequence, an rna (tracrrna) comprising a trans-activating cr (tracr) sequence, and a Cas endonuclease, or any combination thereof.
In some embodiments, the CRISPR editing system comprises a CRISPR sequence, a guide sequence, and a Cas endonuclease, or any combination thereof.
Compounds for regulating gene editing and use of genome editing system, kit and composition thereof
Precisely altered genome editing of a particular genomic region has great therapeutic potential.
In some embodiments, provided herein are methods of modifying the expression of one or more genes or proteins, the methods 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 the nucleic acid of the one or more target genomic regions of a target gene resulting in editing the one or more target genomic regions, and wherein the editing modifies the expression of a downstream gene and/or protein associated with the target gene.
Editing one or more target genomic regions includes any kind of genetic manipulation or engineering of the genome of a cell. Editing of one or more target genomic regions may include insertion, deletion, or substitution of genomic regions in the cell by one or more endonucleases. Genomic regions comprise genetic material in a cell, such as DNA, RNA, polynucleotides, and oligonucleotides. The genomic region in a cell also comprises the genome of a mitochondrion or chloroplast contained in the cell.
The Cas9 inhibitor can be any Cas9 inhibitor. The Cas9 inhibitor can be any compound or substance that causes Cas9 inhibition. Cas9 inhibitors can be compounds, small molecules, antibodies, or nucleotide sequences. In some embodiments, the Cas9 inhibitor is a compound represented by structural formulae LXH0307 and LXH 0308.
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 a cell of the subject, the method comprising administering to one or more cells a genome editing system and a Cas9 inhibitor.
In some embodiments, the methods provided herein are used to modify the expression of a gene, RNA molecule, protein, proteome, or downstream protein in a pathway. Such modifications may be useful in the treatment of diseases, dysfunctions, abnormal physiological homeostasis, either acquired or inherited or due to the aging process. As used herein, the term "modifying" includes modulating, enhancing, decreasing, increasing, inserting, deleting, knocking-out, knocking-in, and the like.
It will be understood by those skilled in the art that acquired or genetic or otherwise acquired diseases involve dysregulation of homeostatic mechanisms including those involved in gene or protein function. To this end, the skilled person may use the methods provided herein to modulate, modify, enhance, reduce or provide other aspects of gene function in a subject.
Modifying expression of a gene and subsequent protein expression in a cell can be achieved by the methods provided herein, e.g., by specifically editing (e.g., substituting, inserting, or deleting, any combination thereof) a nucleic acid sequence in any of an exon, an intron, a transcription initiation 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.
In some embodiments, the methods, kits, and compositions provided herein are used to treat a subject having cancer. A method of treating a subject having cancer or a cancer-related disorder includes administering a Cas9 inhibitor and a genome editing system to a cell of the subject. Administration of Cas9 inhibitor and genome editing system can be in vivo or ex vivo.
The cancer may be any type of cancer. Cancers include solid tumors such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreatic, brain, melanoma and other tissue organ tumors, as well as blood cell cancers such as lymphomas and leukemias, including acute myelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia and B-cell lymphoma. The cancer may include melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, hodgkin's lymphoma, chronic lymphocytic leukemia, as well as pancreatic cancer, breast cancer, thyroid cancer, ovarian cancer, uterine cancer, testicular cancer, pituitary cancer, kidney cancer, stomach cancer, esophageal cancer, and rectal cancer.
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 Lymphocytic Leukemia (ALL), acute myelogenous leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal carcinoma, adnexal carcinoma, astrocytoma, childhood cerebellar or basal cell carcinoma of the brain, cholangiocarcinoma, extrahepatic (see cholangiocarcinoma) bladder carcinoma, bone tumor, 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 conduction pathway and hypothalamic glioma brain tumor, breast cancer, bronchial adenoma/carcinoid, Burkitt's lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal primary carcinoma of the central nervous system, childhood cerebellar astrocytoma, primary carcinoma of the central nervous system, primary carcinoma of the brain, primary carcinoma of the periphery of the brain, primary carcinoma of the brain, secondary astrocytoma, secondary carcinoma of the brain, and primary carcinoma of the brain, Pediatric cerebral astrocytoma/glioblastoma, cervical cancer, childhood cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, profibroproliferative small round cell tumors, endometrial cancer, ependymoma, epithelial-like intravascular dermatoma (EHE), esophageal cancer, Ewing's sarcoma of the Ewing tumor family, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, intraocular melanoma (eye cancer), retinoblastoma (eye cancer), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), extracranial, extragonadal or ovarian germ cell tumors, gestational trophoblastic tumors, glioma of the brain stem, glioma, pediatric cerebral astrocytoma glioma, pediatric visual conduction pathway and hypothalamic glioma, Gastric carcinoid, hairy cell leukemia, head and neck cancer, cardiac tumor, hepatocellular (liver) carcinoma, Hodgkin lymphoma, hypopharyngeal carcinoma, childhood hypothalamic and visual conduction pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney carcinoma (renal cell carcinoma), laryngeal carcinoma, leukemia, acute lymphocytic leukemia (also known as acute lymphocytic leukemia), acute myelogenous leukemia (also known as acute myelogenous leukemia), chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), chronic myelogenous leukemia (also known as chronic myelogenous leukemia), hairy cell leukemia, lip and oral cavity carcinoma, liposarcoma, liver cancer (primary), non-small cell lung cancer, lymphoma, AIDS-related lymphoma, cutaneous T cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma, human lymphomas, human immunodeficiency syndrome, human immunodeficiency, non-hodgkin's (old classification of all lymphomas except hodgkin's) lymphoma, primary central nervous system macroglobulinemia, male breast cancer, malignant fibrous histiocytoma/osteosarcoma of bone, medulloblastoma, childhood melanoma, intraocular (ocular) melanoma, Merkel cell carcinoma, mesothelioma, adult malignant mesothelioma, childhood primary focus occult metastatic squamous neck cancer, oral cancer, multiple endocrine tumor syndrome multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disorders, myelogenous leukemia, chronic myelogenous leukemia, adult acute myelogenous leukemia, childhood multiple acute myeloma (myelocarcinoma), myeloproliferative disorders, chronic myxoma, nasal cavity cancer and sinus cancer, nasopharyngeal cancer, neuroblastoma, Non-hodgkin's lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (superficial epithelial-mesenchymal tumor), ovarian germ cell tumor, ovarian low grade malignant potential tumor, pancreatic cancer, pancreatic islet cell pancreatic cancer, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineal blastoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasmacytoma/multiple myeloma, pleuropulmonoblastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (renal carcinoma), renal pelvis and ureteral cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, Ewing 'S tumor family sarcoma, Kaposi' S sarcoma, soft tissue sarcoma, uterine sarcoma, Szary 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-visible skin cancer (non-melanoma), primary focally occult metastatic squamous neck cancer, gastric cancer, supratentorial primary neuroectodermal tumors, cutaneous T-cell lymphoma (mycosis fungoides and Szary syndrome), testicular cancer, laryngeal cancer, thymoma and thymus cancer, thyroid cancer, renal pelvis and ureteral transitional cell carcinoma, gestational trophoblastic tumors, unknown primary site cancer in adults, unknown primary site cancer in children, transitional cell carcinoma of ureters and renal pelvis, urethral carcinoma, uterine carcinoma, endometrial carcinoma, uterine sarcoma, vaginal carcinoma, visual conduction pathway and hypothalamic glioma, neuroblastoma, melanoma, neuroblastoma, carcinoma of the uterine carcinoma of the uterus, carcinoma of the uterine cervix, carcinoma of the rectum, carcinoma of the rectum of the urethra, carcinoma of the rectum of the type, carcinoma of the type, and the type of the type, Vulvar cancer, macroglobulinemia or nephroblastoma (renal cancer).
In some embodiments, the methods, kits, or compositions provided herein are used to treat a subject having a genetic disorder. Methods of treating a subject having a genetic disease or condition or genetic disorder include administering to a cell of the subject a Cas9 inhibitor and a genome editing system Cas9 inhibitor and administration of the genome editing system can be in vivo or ex vivo.
Genetic disorders may result from mutation or replication in a chromosomal region (e.g., by point mutation, deletion, insertion, frameshift, chromosomal replication or deletion). The genetic disorder can be any genetic disorder. The term "genetic disorder" or "genetic disease" includes genetic or acquired mutations in the genome of a subject that cause or may cause a disease.
In some embodiments, the genetic disorder includes, but is not limited to, 22q11.2 deletion syndrome, Angelman syndrome, canavan disease, Charcot-Marie-Tooth disease, achromatopsia, crinis syndrome, down syndrome, duchenne muscular dystrophy, hemochromatosis, hemophilia, Klinefelter syndrome, multiple neurofibromas, phenylketonuria, polycystic kidney disease, Prader-Willi syndrome, sickle cell disease, spinal muscular atrophy, Tay-Sachs disease, turner syndrome, hemoglobinopathy, or any combination thereof.
As used herein, "treating" or "alleviating" or "ameliorating" are used interchangeably. These terms refer to methods for achieving a beneficial or desired result, including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutically beneficial effect is meant any therapeutically relevant improvement or effect on one or more diseases, conditions or symptoms in a treatment. For prophylactic benefit, the compositions can be administered to a subject at risk of 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 been manifested. These terms also mean the treatment of a disease in a mammal, such as a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) remission, i.e., causing regression of the disease state; or (c) cure the disease.
In embodiments, specific post-transcriptional control regulators are targeted to modulate, modify, enhance, or reduce activity by administering a Cas9 inhibitor and a genome editing system.
In some embodiments, the activity of a genetic pathway associated with the cell cycle is modulated, enhanced, or reduced by administering a Cas9 inhibitor and a genome editing system.
In some embodiments, the activity of a gene associated with angiogenesis is modulated, enhanced, or reduced by administering a Cas9 inhibitor and a genome editing system to a cell.
In some embodiments, the activity of a genetic pathway and/or gene associated with mitochondrial function is modulated, enhanced, or reduced by administering a Cas9 inhibitor and a genome editing system to a cell.
In some embodiments, the activity of a genetic pathway and/or gene associated with DNA damage or genomic instability is modulated, enhanced, or reduced.
In some embodiments, a gene encoding a mammalian transcription factor is modulated, enhanced, reduced, or provided to a cell.
In some embodiments, the cell is diseased or carries a mutant. These cells can be manipulated to treat disease, for example to correct mutations or to alter the phenotype of the cell, for example to inhibit the growth of cancer cells. For example, the cell is associated with one or more of the diseases or conditions described herein.
The following detailed description of embodiments of the present application will be made with reference to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present application, are given by way of illustration and explanation only, and are not intended to limit the present application.
Example 1 Synthesis of Small molecule Compounds
Mono, cycloaddition reaction
1. Ethyl glyoxylate (20.4g, 110mmol) and 4-bromoaniline (17g, 100mmol) were added to 200ml of tolueneTo the benzene solution was added 10g of MgSO4Stirring for 30 minutes at 0 ℃;
2. 1-tert-Butoxycarbonyl-2, 3-dihydropyrrole (17g, 100mmol) was added to 60ml of toluene and scandium triflate was added at 0 ℃;
3. adding the reaction liquid obtained in the step 1 and the reaction liquid obtained in the step 2 together, and stirring at room temperature for 6 hours;
4. ethyl acetate/water extraction, spin-drying, weighing 29.75g, yield 68%.
Figure BDA0003550327940000231
Two, chiral separation
1. Column chromatography PE: EA 6% gave 2-P1 as a white solid 17g, 57% yield.
2. Column chromatography PE: EA 8% -15% gave 2-P2 as a colorless oil 8.5g, 29% yield.
Figure BDA0003550327940000241
Three, Suzuki coupling
1. 2-P1(17g, 40mmol), 2-fluorobenzeneboronic acid (6.7g, 48mmol), K3PO4(25.4G, 120mmol) was dissolved in dioxane (70ml) and Xphos-pd-G3(3.2G, 4mmol) was added and the reaction was refluxed at 110 ℃ for 20 h;
2. carrying out suction filtration and spin drying;
3. column chromatography (PE: EA ═ 14%) and spin drying afforded 3-P113 g as a white solid in 73% yield.
Figure BDA0003550327940000242
Tetra, carboxylic acid reduction
1. 3-P1(13g, 28mmol) was dissolved in 100ml THF and LiBH in methanol (1ml) was slowly added dropwise at 0 deg.C4(1.8g, 88mmol), reaction for 2h followed by 15h at room temperature; finally adding 1N HCl;
2. extracting with ethyl acetate/water, and spin-drying;
3. column chromatography (DCM: MeOH ═ 4%) gave 4-p110.5g of white solid in 90% yield.
Figure BDA0003550327940000243
Five, de-Boc protection
1. 10.5g of 4-P1(26mmol) were added to 40ml of HCl/Et2Stirring for 6 hours in O at normal temperature;
2. the reaction mixture was spin-dried and washed twice with a mixture of PE (2X 50mL), DCM (2X 50mL), n-hexane (2X 100 mL);
3. the solution is filtered by suction and dried by spinning to obtain 5-P17.9g yellow solid with the yield of 99 percent.
Figure BDA0003550327940000251
Sixthly, sulfonylation reaction
6.1 preparation of LXH0307
5-P1(280mg, 1mmol) was added to 10ml DCM and Et was slowly added dropwise at 0 ℃ in an ice bath3N, fully dissolving 5-P1, then dropwise adding dansyl chloride (270mg,1mmol), and stirring for reacting for 0.5 h; after the reaction, water (10ml) was added for quenching; DCM/W extraction and spin-drying; and (3) performing column chromatography on the PE, wherein EA is 50 percent, and performing spin drying to obtain a yellow fluorescent solid LXH0307 of 100mg with the yield of 20 percent.
6.2 preparation of LXH0308
5-P1(280mg, 1mmol) was added to 10ml DCM and Et was slowly added dropwise at 0 ℃ in an ice bath3N, fully dissolving 5-P1, then dropwise adding 2-methoxybenzenesulfonyl chloride (270mg,1mmol), and stirring for reacting for 0.5 h; after the reaction, water (10ml) was added for quenching; DCM/W extraction and spin-drying; and (4) performing column chromatography on PE, wherein EA is 50%, and performing spin drying to obtain a light yellow solid LXH 0307180 mg with the yield of 37%.
6.36 preparation of P1-1
1. 5-P1(150mg, 1mmol) was added to 10ml DCM and then Et3N was slowly added dropwise at 0 ℃ to dissolve 5-P1 sufficiently, followed by P-toluenesulfonyl chloride (190mg, 1mmol) dropwise for 30 minutes;
2. after the reaction is finished, adding 10ml of water for quenching;
3. extracting with dichloromethane/water, and spin-drying;
4. column chromatography: petroleum ether: ethyl acetate 10% was spun dry to give 6-P1 as a white solid, 181mg, 80% yield.
Seven, chiral column separation
The chiral preparation of 6-P1 is separated into 6-P1-1.
Example 2 validation of the effectiveness of Small molecule Compounds
Cas9 in vitro enzyme digestion kit of Inovogen company is used for detecting the in vitro enzyme digestion activity of Cas9, and the influence of synthesized LXH0307, LXH0308 and 6-P1-1 small molecule compounds on the shearing efficiency of Cas9 protein is evaluated. The experimental procedures were performed according to the kit instructions.
1. Cas9 cleavage reaction:
preparing cas9 in-vitro cutting reaction buffer solution according to the specification, and uniformly mixing by blowing and sucking to perform reaction;
reaction procedure: 30min at 37 ℃; 10min at 85 ℃.
2. Product detection
1) Mu.l of Cleaner was added to the reaction system and mixed well. Incubating at 55 deg.C for 5 min;
2) and 5. mu.l of the solution was subjected to gel electrophoresis detection and counted.
3. Results
Results as shown in fig. 1, compared with control group DMSO, compounds LXH0307 and LXH0308 significantly inhibited the cleavage efficiency of Cas9 protein, and the inhibition efficiency was significantly higher than 6-P1-1.
The preferred embodiments of the present application have been described in detail with reference to the accompanying drawings, however, the present application is not limited to the details of the above embodiments, and various simple modifications can be made to the technical solution of the present application within the technical idea of the present application, and these simple modifications are all within the protection scope of the present application.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described in the present application.
In addition, any combination of the various embodiments of the present application is also possible, and the same should be considered as disclosed in the present application as long as it does not depart from the idea of the present application.

Claims (10)

1. A compound that modulates gene editing, the compound having the structure of formula (I):
Figure FDA0003550327930000011
wherein R is an aromatic ring which may be substituted with 0, 1, 2 or 3 substituents R1Substituted, said substituent R1Independently selected from halo, CN, C1-C4Alkyl radical, C1-C4-haloalkyl group, C3-C4-cycloalkyl, OR2、NR2R3；
Each R2And R3Independently is H, C1-C4Alkyl or C1-C4-a haloalkyl group;
preferably, the aromatic ring is a benzene ring or naphthalene;
preferably, said R is2And R3Independently is H, C1-C4An alkyl group;
preferably, the structure of the compound is as follows:
Figure FDA0003550327930000012
2. a process for preparing the compound of claim 1, comprising:
1) reacting bromoaniline, ethyl glyoxylate and BOC-dihydropyrrole to synthesize a compound 2-P;
2) separating by column chromatography to obtain compounds 2-P1 and 2-P2;
3) carrying out suzuki coupling reaction on the compound 2-P1 and 2-fluorobenzeneboronic acid to generate a compound 3-P1;
4) carrying out carboxylic acid reduction reaction on the compound 3-P1 to generate a compound 4-P1;
5) de-Boc the compound 4-P1 under acidic condition to generate a compound 5-P1;
6) and performing sulfonylation reaction on the compound 5-P1 to generate LXH0307 and LXH 0308.
Preferably, the method for synthesizing the compound 2-P in step 1) is as follows: ethyl glyoxylate and 4-bromoaniline were added to the toluene solution, followed by MgSO4Stirring to obtain a reaction solution 1; adding BOC-pyrroline into a toluene solution, and then adding scandium trifluoromethanesulfonate to react to obtain a reaction solution 2; adding the reaction solution 1 and the reaction solution 2 together for reaction, and extracting and spin-drying;
preferably, the reaction solution 1 is obtained by stirring at 0 ℃;
preferably, scandium triflate is added at 0 ℃;
preferably, the extraction reagent is ethyl acetate/water;
preferably, the eluent in step 2) column chromatography is selected from PE and EA;
preferably, EA is 6 percent to obtain 2-P1;
preferably, the catalytic agent of the suzuki coupling reaction in the step 3) is Xphos-pd-G3;
preferably, the alkaline reagent for the suzuki coupling reaction in the step 3) is K3PO4；
Preferably, the process of the suzuki coupling reaction of step 3) is as follows:
2-P1, 2-fluorobenzeneboronic acid and K3PO4Dissolving in dioxane, adding Xphos-pd-G3, and refluxing at 110 deg.c;
preferably, the step 3) further comprises the step of separating by using column chromatography to obtain 3-P1;
preferably, the eluent for column chromatography is selected from PE and EA;
preferably, the PE is separated when EA is 14 percent to obtain 3-P1;
preferably, the procedure of the carboxylic acid reduction reaction of step 4) is as follows:
dissolving 3-P1 in THF, and slowly dropwise adding a reducing agent solution for reaction;
preferably, the reducing agent is LiBH4；
Preferably, the reducing agent solution is LiBH4The methanol solution of (4);
preferably, the reaction condition is 0 ℃ for 2h, and then room temperature reaction is carried out for 15 h;
preferably, the acid is added after the reaction is finished;
preferably, the acid is HCl;
preferably, the step 4) further comprises extracting the reaction solution;
preferably, the extraction reagent is ethyl acetate/water;
preferably, the step 4) further comprises the step of separating by using column chromatography to obtain 4-P1;
preferably, the eluent for column chromatography is selected from DCM and MeOH;
preferably, the ratio of DCM: 4% of MeOH;
preferably, the Boc removal process in the step 5) is as follows:
4-P1 was added to HCl/Et2Reacting at normal temperature in O;
preferably, the step 5) further comprises spin-drying and washing the reaction solution;
preferably, the washing reagent is a mixed solution of PE, DCM and n-hexane;
preferably, the 5-P1 is a yellow solid;
preferably, the step of conducting the sulfonylation reaction of step 6) is as follows:
5-P1 was added to DCM and Et was slowly added dropwise3N, fully dissolving 5-P1, and then dropwise adding a sulfonylation reagent for reaction;
preferably, the sulfonylating agent is selected from dansyl chloride, 2-methoxybenzenesulfonyl chloride;
preferably, Et is slowly added dropwise at 0 deg.C3N；
Preferably, the step 6) further comprises quenching the reaction solution;
preferably, the step 6) further comprises extracting the quenched reaction solution;
preferably, the extraction reagent is dichloromethane/water;
preferably, step 6) further comprises a column chromatography step;
preferably, the eluent for column chromatography is selected from PE and EA;
preferably, the eluents PE and EA are in a proportion of 50%.
3. A pharmaceutical composition comprising a compound of claim 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof; or a compound or intermediate prepared by the process of claim 2;
preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
4. A method for modulating a CRISPR editing system for gene editing, comprising administering a compound of claim 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof to a subject in need thereof; or a pharmaceutical composition according to claim 3.
5. A method of editing one or more target genomic regions comprising administering to one or more cells comprising one or more genomic regions
1) A CRISPR editing system; and
2) the compound of claim 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a racemate thereof, or a solvate thereof; or the pharmaceutical composition of claim 3;
preferably, the CRISPR editing system was previously administered into the one or more cells;
preferably, the one or more cells are cultured cells;
preferably, the one or more cells are in vivo cells within an organism;
preferably, the one or more cells are ex vivo cells from an organism;
preferably, the CRISPR editing system is a CRISPR-Cas editing system;
preferably, 1) and 2) are administered via different routes;
preferably, 1) and 2) are administered via the same route;
preferably, the CRISPR editing system is delivered by one or more vectors;
preferably, the one or more vectors are selected from viral vectors, plasmids or ssDNA;
preferably, the viral vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector and/or a herpes simplex viral vector;
preferably, the CRISPR editing system is delivered by synthetic RNA;
preferably, the CRISPR editing system is delivered by a nanoformulation.
6. A reagent product, comprising:
1) a first agent which is a gene editing inhibitor, wherein the gene editing inhibitor is the compound of claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof; and
2) a second agent that performs CRISPR gene editing;
preferably, the second agent comprises one or more agents selected from the group consisting of:
1) cas9 nuclease, a coding sequence for Cas9 nuclease, or a vector expressing Cas9 nuclease, or a combination;
2) a gRNA, a crRNA, or a vector for producing the gRNA or crRNA;
3) template for homogenous directed repair: a single-stranded nucleotide sequence or a plasmid vector.
7. A kit for editing one or more target genomic regions, the kit comprising:
1) a first container, and a gene editing inhibitor in the first container, wherein the gene editing inhibitor is the compound of claim 1, or a pharmaceutically acceptable salt thereof, or an optical isomer or racemate thereof, or a solvate thereof; or the composition of claim 3;
2) a second container, and CRISPR editing system reagents located within the second container;
preferably, the CRISPR editing system is a CRISPR-Cas editing system;
preferably, the CRISPR-Cas editing system comprises:
at least one guide RNA element and a Cas protein element;
preferably, the guide RNA element comprises: 1) a targeting RNA comprising a nucleotide sequence 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; 2) and an activating RNA comprising a nucleotide sequence capable of hybridizing to the targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding the activating RNA;
preferably, the Cas protein is a Cas9 protein;
preferably, the Cas9 protein is SpCas9 protein;
preferably, wherein said CRISPR editing system comprises or is packaged in one or more vectors;
preferably, the one or more vectors are selected from viral vectors, plasmids or ssDNA;
preferably, 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.
8. Use of a compound of claim 1, a pharmaceutical composition of claim 3, a reagent product of claim 6 or a kit of claim 7 for regulating genome editing;
preferably, the genome editing is directed to a disease-causing gene, a tumor-associated gene (e.g., an oncogene), an immune-related gene (e.g., a gene associated with autoimmunity), a vision-related gene, an auditory-related, a metabolic-related, a viral infection-related, a genetic disease-related gene;
preferably, the genome editing employs a CRISPR editing system;
preferably, the CRISPR editing system is selected from a CRISPR-Cas editing system.
9. Use of a compound according to claim 1, a pharmaceutical composition according to claim 3, a reagent product according to claim 6 or a kit according to claim 7 for the manufacture of a medicament for the treatment of a disease;
preferably, the disease is selected from cancer or a genetic disorder.
10. Use of a compound according to claim 1, a pharmaceutical composition according to claim 3, a reagent product according to claim 6 or a kit according to claim 7 for the construction of a disease model;
preferably, the disease model is a cellular disease model or an animal disease model;
preferably, the disease model is used to screen drugs for treatment of disease.
CN202210261597.XA 2022-03-16 2022-03-16 LXH0307 and LXH0308 as small molecule inhibitors of gene editing and application thereof Pending CN114524811A (en)

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CN109863143A (en) * 2016-07-13 2019-06-07 威泰克斯制药公司 Improve method, composition and the kit of genome editorial efficiency
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