Nucleic acid molecule for inhibiting d2HGDH, inhibitor and use thereof

Abstract

A nucleic acid molecule for specifically inhibiting the transcription or translation of a D2HGDH gene or an inhibitor for inhibiting the activity of a protein encoded by the D2HGDH gene, and the use thereof in preparing a drug for treating tumors. The nucleic acid molecule for specifically inhibiting the transcription or translation of the D2HGDH gene is a shRNA, a siRNA and / or a sgRNA. The inhibitor for inhibiting the activity of the protein encoded by the D2HGDH gene comprises at least one of ginkgolic acid C15:1 and NSC87877.

Description

A nucleic acid molecule and inhibitor for inhibiting D2HGDH and its use

[0001] This application claims priority to and the benefits of Chinese Patent Application No. 202311809864.3 filed with the State Intellectual Property Office of China on December 26, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a nucleic acid molecule that specifically inhibits the transcription or translation of a D2HGDH gene, an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene, and uses thereof. Background Art

[0003] The function of isocitrate dehydrogenase (IDH) is to catalyze the dehydrogenation of isocitrate to produce α-ketoglutarate (α-KG) and convert it into NAD + or NADP + It generates NADH or NADPH for coenzymes. There are three IDH subtypes in human cells: IDH1, IDH2, and IDH3. In 2008, a study published in Science found the IDH1R132H mutation in gliomas. Subsequent studies have shown that this mutation primarily occurs in WHO grade II or III gliomas, with a mutation frequency of an astonishing 85-90%. In addition to gliomas, numerous IDH1 and IDH2 mutations have been found in a variety of malignant tumors, including acute myeloid leukemia (10-20%), angioimmunoblastic T-cell lymphoma (10-40%), chondrosarcoma (56%), cholangiocarcinoma (20%), and sinus cancer (80%), indicating that these mutations are closely linked to the development of these tumors.

[0004] In 2009, Dang et al. reported a landmark discovery in Nature: mutant IDH1 had acquired a new function, capable of reducing the wild-type IDH1 product α-KG to D-2-hydroxyglutarate (D-2HG). Subsequent studies have shown that tumor-associated IDH1 or IDH2 mutations share the common characteristic of producing D-2HG. D-2HG is structurally very similar to α-KG and can competitively inhibit the activity of various dioxygenases that use α-KG as a substrate, such as TET2 and KDMs. TET2 and KDMs are important genes in DNA and histone demethylation. D-2HG inhibits their function, leading to DNA and histone hypermethylation, which reshapes the cellular epigenetic landscape, affects the normal expression of numerous genes, and promotes tumor development and progression. Therefore, D-2HG is considered an "oncometabolite."

[0005] Notably, IDH1 or IDH2 mutations occur early in tumorigenesis and are present in all cells within a tumor. Therefore, IDH1 or IDH2 mutants are considered ideal therapeutic targets. In recent years, pharmaceutical companies such as Agios have developed a variety of IDH1 or IDH2 mutant inhibitors, such as ivosidenib, enasidenib, vorasidenib, and BAY1436032. Currently, these IDH1 or IDH2 mutant inhibitors are clinically used to treat a variety of IDH1 or IDH2 mutant tumors. Among them, ivosidenib and enasidenib have been approved for marketing in the United States. These two approved IDH1 or IDH2 mutant inhibitors have achieved complete remission rates of approximately 20% for refractory or relapsed acute myeloid leukemia (AML) harboring IDH1 mutations. However, clinical trials targeting solid tumors harboring IDH1 or IDH2 mutations have shown that while IDH1 or IDH2 inhibitors can effectively inhibit D-2HG production, they have no significant therapeutic effect on solid tumors, including gliomas harboring IDH1 or IDH2 mutations. Therefore, there is an urgent need to develop alternative treatment strategies for tumors harboring IDH1 or IDH2 mutations. Summary of the Invention

[0006] SUMMARY OF THE INVENTION

[0007] To provide an effective treatment strategy for IDH1 mutation or IDH2 mutation tumors, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a nucleic acid molecule that specifically inhibits the transcription or translation of the D2HGDH gene, or an inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene, for use in the preparation of a medicament for treating tumors. By specifically inhibiting the transcription or translation of the D2HGDH gene, or inhibiting the activity of the protein encoded by the D2HGDH gene, the present invention can effectively inhibit tumor growth.

[0009] In a second aspect, the present invention provides a nucleic acid molecule, which is shRNA, siRNA and / or sgRNA.

[0010] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect and a viral vector.

[0011] In a fourth aspect, the present invention provides a nucleic acid molecule delivery system, which comprises the nucleic acid molecule described in the second aspect.

[0012] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect or the recombinant vector described in the third aspect.

[0013] In a sixth aspect, the present invention provides a composition comprising the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, the nucleic acid molecule delivery system described in the fourth aspect, the host cell described in the fifth aspect, and at least one of an antibody to a protein encoded by D2HGDH or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene.

[0014] In a seventh aspect, the present invention provides a method for delivering nucleic acid molecules to cells for non-therapeutic purposes.

[0015] Detailed Description of the Invention

[0016] In order to solve the aforementioned problems, the present invention provides the following technical solutions.

[0017] In a first aspect, the present invention provides a use.

[0018] A use of a nucleic acid molecule that specifically inhibits the transcription or translation of the D2HGDH gene, or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene, in the preparation of a drug for treating tumors, wherein the nucleic acid molecule is shRNA, siRNA and / or sgRNA.

[0019] The gene D2HGDH (Gene ID: 728294, Ensembl: ENSG00000180902) described in the present invention is derived from humans and is located on human chromosome 2. Its mRNA contains 16 splice variants, of which the longest splice variant is NM_152783.5, with the nucleotide sequence: Seq ID NO. 46.

[0020] The amino acid sequence of the D2HGDH protein, namely UniProtKB-Q8N465, is: Seq ID NO.47.

[0021] In some embodiments, the target sequence of the nucleic acid molecule includes Seq ID NO.1, Seq ID NO.4, Seq ID NO.7, Seq ID NO.13, Seq ID NO.16 or Seq ID NO.19.

[0022] In some embodiments, the shRNA comprises shRNA-1, shRNA-2, or shRNA-3;

[0023] The shRNA-1 includes a sense strand and an antisense strand, the sense strand of the shRNA-1 contains Seq ID NO.2 (Seq ID NO.2: 5′-TGGAAGGACAACACGGGCTATTCAAGAGATAGCCCGTGTTGTCCTTCCTTTTTTC-3′), and the antisense strand of the shRNA-1 contains Seq ID NO.3 (Seq ID NO.3: 5′-TCGAGAAAAAAGGAAGGACAACACGGGCTATCTCTTGAATAGCCCGTGTTGTCCTTCCA-3′) (the target sequence of the shRNA-1 is Seq ID NO.1: 5′-GGAAGGACAACACGGGCTA-3′);

[0024] The shRNA-2 includes a sense strand and an antisense strand, the sense strand of the shRNA-2 contains Seq ID NO.5 (Seq ID NO.5: 5′-TGGGGAAACGTGGCAACCAATTCAAGAGATTGGTTGCCACGTTTCCCCTTTTTTC-3′), and the antisense strand of the shRNA-2 contains Seq ID NO.6 (Seq ID NO.6: 5′-TCGAGAAAAAAGGGGAAACGTGGCAACCAATCTCTTGAATTGGTTGCCACGTTTCCCC-3′) (the target sequence of the shRNA-2 is Seq ID NO.4: 5′-GGGGAAACGTGGCAACCAA-3′);

[0025] The shRNA-3 includes a sense chain and an antisense chain, the sense chain of the shRNA-3 contains Seq ID NO.8 (Seq ID NO.8: 5′-TGGGCTGTGAACGTGGCTTTTTCAAGAGAAAAGCCACGTTCACAGCCCTTTTTTC-3′), and the antisense chain of the shRNA-3 contains Seq ID NO.9 (Seq ID NO.9: 5′-TCGAGAAAAAAGGGCTGTGAACGTGGCTTTTCTCTTGAAAAAGCCACGTTCACAGCCCA-3′) (the target sequence of the shRNA-3 is Seq ID NO.7: 5′-GGGCTGTGAACGTGGCTTT-3′).

[0026] In some embodiments, the siRNA comprises siRNA-1, siRNA-2, or siRNA-3;

[0027] The siRNA is an RNA sequence containing 19 bases, the siRNA-1 sequence is Seq ID NO.10: 5′-GGAAGGACAACACGGGCUA-3′; the siRNA-2 sequence is Seq ID NO.11: 5′-GGGGAAACGUGGCAACCAA-3′; and the siRNA-3 sequence is Seq ID NO.12: 5′-GGGCUGUGAACGUGGCUUU-3′.

[0028] The siRNA-1, siRNA-2 and siRNA-3 sequences are the RNA forms of the target sequence of shRNA-1, shRNA-2 and shRNA-3 respectively, and their biological effects are equivalent to those of the corresponding shRNA.

[0029] In some embodiments, the sgRNA comprises sgRNA-1, sgRNA-2, or sgRNA-3;

[0030] The sgRNA-1 includes a sense strand and an antisense strand, the sense strand of the sgRNA-1 contains Seq ID NO.14 (Seq ID NO.14: 5′-CACCGACGGGACTTCATCATGCCGC-3′), and the antisense strand of the sgRNA-1 contains Seq ID NO.15 (Seq ID NO.15: 5′-AAACGCGGCATGATGAAGTCCCGTC-3′) (the target sequence of the sgRNA-1 is Seq ID NO.13: 5′-ACGGGACTTCATCATGCCGC-3′);

[0031] The sgRNA-2 includes a sense strand and an antisense strand, the sense strand of the sgRNA-2 contains Seq ID NO.17 (Seq ID NO.17: 5′-CACCGGAGAACGGCAAGCGCCGCAC-3′), and the antisense strand of the sgRNA-2 contains Seq ID NO.18 (Seq ID NO.18: 5′-AAACGTGCGGCGCTTGCCGTTCTCC-3′) (the target sequence of the sgRNA-2 is Seq ID NO.16: 5′-GAGAACGGCAAGCGCCGCAC-3′);

[0032] The sgRNA-3 includes a sense chain and an antisense chain, the sense chain of the sgRNA-3 contains Seq ID NO.20 (Seq ID NO.20: 5′-CACCGGCCGGGCACGATGCGCTCAA-3′), and the antisense chain of the sgRNA-3 contains Seq ID NO.21 (Seq ID NO.21: 5′-AAACTTGAGCGCATCGTGCCCGGCC-3′) (the target sequence of the sgRNA-3 is Seq ID NO.19: 5′-GCCGGGCACGATGCGCTCAA-3′).

[0033] In some embodiments, the inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene includes but is not limited to at least one of ginkgolic acid (C15:1) and NSC87877.

[0034] In some embodiments, the tumor is selected from a tumor carrying an IDH1 mutation or an IDH2 mutation.

[0035] IDH1 mutation refers to a mutation in which the mutated IDH1 protein can produce D-2HG, including: IDH1 R132H, IDH1 R132C, IDH1R132G, IDH1 R132L, IDH1 R132S, IDH1 R132Q, IDH1 R132V, IDH1 R132P, IDH1 R100Q, etc.

[0036] IDH2 mutation refers to a mutation in which the mutated IDH2 protein can produce D-2HG, including: IDH2 R140Q, IDH2 R140C, IDH2R172K, IDH2 R172S, IDH2 R172G, IDH2 R172M, IDH2 R172S, IDH2 R172T, IDH2 R172W, etc.

[0037] The amino acid sequence of IDH1 is: Seq ID NO.48:

[0038] The amino acid sequence of IDH2 is: Seq ID NO.49:

[0039] Each mutation site of the IDH1 mutant is mutated at the corresponding position in the above IDH1 amino acid sequence Seq ID NO.48. For example, the amino acid sequence of IDH1 R132H is: Seq ID NO.50: MSKKISGGSVVEMQGDEMTRIIWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAAEAIKKHNVGVKCATITPDEKRVEEFKLKQMWKSPNGTIRNILGGTVFREAIICKNIPRLVSGWVKPIIIGHHAYGDQYRATDFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGMYNQDKSIEDFAHSSFQMALSKG WPLYLSTKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKIWYEHRLIDDMVAQAMKSEGGFIWACKNYDGDVQSDSVAQGYGSLGMMTSVLVCPDGKTVEAEAAHGTVTRHYRMYQKGQETSTNPIASIFAWTRGLAHRAKLDNNKELAFFANALEEVSIETIEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKIKLAQAKL. The mutation patterns of other IDH1 mutations (IDH1 R132C, IDH1 R132G, IDH1 R132L, IDH1 R132S, IDH1 R132Q, IDH1 R132V, IDH1 R132P, IDH1 R100Q, etc.) are similar.

[0040] Each mutation site of the IDH2 mutant is mutated at the corresponding position in the above IDH2 amino acid sequence Seq ID NO.49. For example, the amino acid sequence of IDH2 R140Q is: Seq ID NO.51: MAGYLRVVRSLCRASGSRPAWAPAALTAPTSQEQPRRHYADKRIKVAKPVVEMDGDEMTRIIWQFIKEKLILPHVDIQLKYFDLGLPNRDQTDDQVTIDSALATQKYSVAVKCATITPDEARVEEFKLKKMWKSPNGTIQNILGGTVFREPIICKNIPRLVPGWTKPITIGRHAHGDQYKATDFVADRAGTFKMVFTPKDGSGVKEWEVYNFPAGGVGMGMYN TDESISGFAHSCFQYAIQKKWPLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKIWYEHRLIDDMVAQVLKSSGGFVWACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAAHGTVTRHYREHQKGRPTSTNPIASIFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKLNEHFLNTTDFLDTIKSNLDRALGRQ. The mutation patterns of other IDH2 mutations (IDH2 R140C, IDH2 R172K, IDH2 R172S, IDH2 R172G, IDH2 R172M, IDH2 R172S, IDH2 R172T, IDH2 R172W, etc.) are similar.

[0041] In some embodiments, the tumor comprises glioma, acute myeloid leukemia, angioimmunoblastic T-cell lymphoma, chondrosarcoma, cholangiocarcinoma, or sinonasal cancer.

[0042] In the present invention, the structure of Ginkgolic acid (C15:1) is:

[0043] In the present invention, the structure of NSC87877 is:

[0044] In a second aspect, the present invention provides a nucleic acid molecule.

[0045] A nucleic acid molecule, as shown in the nucleic acid molecule of the first aspect.

[0046] In a third aspect, the present invention provides a recombinant vector.

[0047] A recombinant vector comprising the nucleic acid molecule described in the second aspect and a viral vector.

[0048] In some embodiments, the viral vector comprises a retroviral vector, a lentiviral vector, or an adenoviral vector.

[0049] In a fourth aspect, the present invention provides a nucleic acid molecule delivery system.

[0050] A nucleic acid molecule delivery system, comprising the nucleic acid molecule according to the second aspect.

[0051] In some embodiments, the nucleic acid molecule delivery system includes at least one of N-acetylated galactosamine coupling modification, lipid nanoparticles, polymer nanoparticles (such as polyethyleneimine and chitosan), and exosomes.

[0052] In a fifth aspect, the present invention provides a host cell.

[0053] A host cell comprising the nucleic acid molecule of the second aspect or the recombinant vector of the third aspect.

[0054] In a sixth aspect, the present invention provides a composition.

[0055] A composition comprising the nucleic acid molecule of the second aspect, the recombinant vector of the third aspect, the nucleic acid molecule delivery system of the fourth aspect, the host cell of the fifth aspect, and at least one of an antibody against a protein encoded by D2HGDH or an inhibitor that inhibits the activity of a protein encoded by the D2HGDH gene.

[0056] In some embodiments, the inhibitor comprises at least one of ginkgolic acid C15:1 (CAS number of ginkgolic acid C15:1 is 22910-60-7) and NSC87877 (CAS number of NSC87877 is 56932-43-5).

[0057] In some embodiments, the composition further comprises a pharmaceutically acceptable adjuvant.

[0058] In some embodiments, the pharmaceutically acceptable adjuvant includes dimethyldioctadecyl ammonium (DDA) and / or monophosphoryl lipid A (MPL) and / or saline or other adjuvants.

[0059] In some embodiments, the other adjuvant comprises aluminum adjuvant or Freund's adjuvant.

[0060] In some embodiments, the composition can be in various forms such as injection, tablet, capsule, powder, ointment, nanoformulation, etc. The above-mentioned various dosage forms of drugs can be prepared according to the methods in the field of gene drugs / oligonucleotide drugs.

[0061] In a seventh aspect, the present invention provides a method for delivering nucleic acid molecules to cells for non-therapeutic purposes.

[0062] A method for delivering a nucleic acid molecule to a cell for non-therapeutic purposes, comprising contacting the cell with at least one of the nucleic acid molecule of the second aspect, the recombinant vector of the third aspect, the nucleic acid molecule delivery system of the fourth aspect, the host cell of the fifth aspect, or the composition of the sixth aspect. Beneficial effects

[0063] Compared with the prior art, an embodiment of the present invention has at least one of the following beneficial technical effects:

[0064] (1) The present invention specifically inhibits the transcription or translation of the D2HGDH gene through nucleic acid molecules, or inhibits the activity of the protein encoded by the D2HGDH gene through inhibitors (preferably at least one of ginkgolic acid C15:1 (CAS number of ginkgolic acid C15:1 is 22910-60-7) and NSC87877 (CAS number of NSC87877 is 56932-43-5)) or antibodies, which is beneficial for inhibiting the growth of tumors with IDH1 mutation or IDH2 mutation (such tumors include glioma, acute myeloid leukemia, angioimmunoblastic T-cell lymphoma, chondrosarcoma, cholangiocarcinoma or sinus cancer, etc.).

[0065] (2) The nucleic acid molecule of the present invention is shRNA, siRNA and / or sgRNA, wherein the shRNA is preferably selected from shRNA-1, shRNA-2 or shRNA-3; the siRNA is preferably selected from siRNA-1, siRNA-2 or siRNA-3; the sgRNA is preferably selected from sgRNA-1, sgRNA-2 or sgRNA-3, which can effectively inhibit the expression of D2HGDH protein in tumor cells.

[0066] (3) The inhibitor of the present invention is an inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene and can effectively inhibit the activity of the D2HGDH protein in tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 shows Western blot images of effective shRNA and sgRNA target sequence screening for D2HGDH knockdown or knockout in Example 1. Figure A shows D2HGDH expression knockdown in U251 cells using six independent shRNA pairs, and D2HGDH expression was detected by Western blot; Figure B shows D2HGDH expression knockdown in U251 cells using six independent siRNA pairs, and D2HGDH expression was detected by Western blot; Figure C shows D2HGDH expression knockdown in U251 cells using six independent sgRNA pairs, and D2HGDH expression was detected by Western blot.

[0068] Figure 2 is a diagram showing the construction results of cell lines overexpressing wild-type IDH1 or wild-type IDH2 and overexpressing mutant IDH1 or mutant IDH2 in Example 2. Figure A shows the D-2HG concentration in the culture medium of U251 cells overexpressing wild-type IDH1 cell line (Flag IDH1 WT) or mutant IDH1 cell line (Flag IDH1 R132H) or U118 cells overexpressing wild-type IDH1 cell line (Flag IDH1 WT) or mutant IDH1 cell line (Flag IDH1 R132H) (bar graph in the figure), and Western blot detection of intracellular IDH1 (Flag) or IDH1 R132H and D2HGDH protein expression (Western blot graph in the figure). Figure B shows the D-2HG concentration in the culture medium of JURKAT cells overexpressing IDH2 WT or IDH2 R140Q (bar graph in the figure), and the expression of IDH2 (Flag) or IDH2 R140Q and D2HGDH proteins in the cells detected by Western blot (Western blot graph in the figure).

[0069] Figure 3 shows the expression of D2HGDH knocked down in U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080 and SW1353 cells using three pairs of independent shRNAs in Example 2, a cell proliferation curve plotted by cell counting (the curve graph in the figure), and a Western blot diagram of D2HGDH protein expression in cells detected by Western blot (the Western blot graph in the figure).

[0070] Figure 4 shows the results of the decreased proliferation ability of IDH1 mutant tumor cells or IDH2 mutant tumor cells caused by knockdown of D2HGDH in Example 3. Panels A, B, C, and D show the results of knocking down D2HGDH expression in U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080, and SW1353 cells using shRNA, and complementing wild-type D2HGDH (rWT) and the G477R mutant (rG477R) lacking D2HGDH enzyme activity; the graph shows the cell proliferation curve drawn by cell counting, and the Western blot shows the expression of D2HGDH protein in the cells.

[0071] Figure 5 shows the results of knocking down D2HGDH to inhibit the in vivo tumorigenicity of IDH1 mutant tumor cells in Example 4. D2HGDH expression was knocked down in HT1080 cells, and wild-type D2HGDH (rWT) and its enzymatically inactive G477R mutant (rG477R) were complemented. These cells were then injected subcutaneously into nude mice for tumor formation experiments; Figure A shows a representative image of tumor-bearing nude mice, Figure B shows the statistical results of subcutaneous tumor volume, and Figure C shows the statistical results of subcutaneous tumor weight.

[0072] Figure 6 shows the results of the construction and optimization of the D2HGDH inhibitor screening system in Example 5. By adjusting the concentrations of D2HGDH protein, D-2HG, FAD+, and INT in the D2HGDH enzyme activity reaction system, the enzyme activity reaction was optimized to meet the requirements of drug screening. Panel A shows the combined information of different component concentrations in the reaction system and the corresponding signal-to-noise ratio; Panel B shows the enzyme activity reaction curves of various reaction systems in Panel A.

[0073] Figure 7 illustrates the process and results of the high-throughput screening of D2HGDH small molecule inhibitors in Example 5. Figure A illustrates the high-throughput drug screening process; Figure B shows the inhibition curves of the screened candidate D2HGDH small molecule inhibitors against D2HGDH enzyme activity; and Figure C shows the half-inhibitory concentrations (IC50s) of Ginkgolic acid (C15:1) and NSC87877 against D2HGDH.

[0074] Figure 8 shows the results of Example 5, demonstrating the decreased proliferation of IDH1 or IDH2 mutant tumor cells induced by Ginkgolic acid (C15:1). Panel A shows the statistical results of cell proliferation rates in U251 IDH1 WT and U251 IDH1 R132H cells after treatment with a gradient of concentrations of Ginkgolic acid (C15:1) (GA). Panels B, C, D, and E show the corresponding experimental results in Panel A for U118 IDH1 WT and U118 IDH1 R132H, JURKAT IDH2 WT and JURKAT IDH2 R140Q, HuCCT1 IDH1 WT and HuCCT1 IDH1 R132H, HT1080, and SW1353 cells, respectively. Panel F shows the statistical results of the half-inhibitory concentration of Ginkgolic acid (C15:1) on the proliferation rates of the aforementioned cells.

[0075] Figure 9 shows the results of the decreased proliferation of IDH1 or IDH2 mutant tumor cells induced by NSC87877 in Example 5. Panel A shows the statistical results of cell proliferation rates in U251 IDH1 WT and U251 IDH1 R132H cells after treatment with a series of gradient concentrations of NSC87877 (NSC). Panels B, C, D, and E show the corresponding experimental results in Panel A for U118 IDH1 WT and U118 IDH1 R132H, JURKAT IDH2 WT and JURKAT IDH2 R140Q, HuCCT1 IDH1 WT and HuCCT1 IDH1 R132H, HT1080, and SW1353 cells, respectively. Panel F shows the statistical results of the half-inhibitory concentration of NSC87877 on the proliferation rates of the aforementioned cells.

[0076] Definition of terms:

[0077] In the present invention, "room temperature" refers to ambient temperature, which may be 20°C-30°C; in some embodiments, 22°C-28°C; in some embodiments, 24°C-26°C; in some embodiments, 25°C.

[0078] In the foregoing text of the present invention, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. Based on the disclosed numbers, the value of each numerical value may vary by less than ±10% or by a reasonable difference deemed by a person skilled in the art, such as ±1%, ±2%, ±3%, ±4%, or ±5%.

[0079] The terms "optional", "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur. For example, "optional surfactant" means that a surfactant may or may not be present.

[0080] The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

[0081] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction. DETAILED DESCRIPTION

[0082] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0083] The reagents used in the present invention can be purchased from the market or prepared by the method described in the present invention.

[0084] The experimental methods without specific conditions specified in the examples are generally based on well-known methods in the art.

[0085] Unless otherwise specified, "%" in each solution of the present invention represents mass percentage when indicating the content of the solid after dissolving the solid solute in the solvent, and represents volume percentage when indicating the content of the liquid solute after dissolving the liquid solute in the solvent.

[0086] Main materials and their sources

[0087] The amino acid sequence of the wild-type IDH1 of the present invention is as described in Seq ID NO.48, and the amino acid sequence of the wild-type IDH2 is as described in Seq ID NO.49.

[0088] Example 1: Screening of shRNA and sgRNA target sequences that inhibit D2HGDH expression

[0089] 1. Construction of recombinant plasmid

[0090] 1.1 Construction of shRNA recombinant plasmid

[0091] 1.1.1 shRNA primer design

[0092] There are many websites for designing shRNA targeting sequences. The primary method is to use the BLAST function on the NCBI website for sequence alignment and scoring of the interference effect of the targeting sequence. In this paper, we used the siDESIGN Center (https: / / horizondiscovery.com / en / ordering-and-calculation-tools / sidesign-center) for targeting sequence design. The shRNA primers consist of the selected targeting sequence, linker, and neck-loop structure. The final primer sequences are shown in Table 1.

[0093] Table 1: shRNA primer sequences and their target sequences

[0094] 1.1.2 Primer annealing

[0095] Before connecting to the vector, the primers need to be annealed to form double-stranded DNA fragments from the single-stranded DNA primers. The annealing system is shown in the following table:

[0096] 1.1.3 Preparation of vector

[0097] The pLentiLox3.7 vector was double-digested with XhoⅠ and HpaⅠ. The enzyme digestion reaction system is shown in the following table:

[0098] After the enzyme digestion is completed, the enzyme-digested vector is separated by agarose gel electrophoresis and recovered and purified using the commercial product glass milk.

[0099] 1.1.4 Annealed primers and vector ligation and transformation

[0100] In the present invention, T4 ligase is used to connect the vector and the annealed product, and the ligation reaction system is as follows:

[0101] After ligation with T4 ligase, add 200 μL of competent cells to the ligation product. Place on ice for 20 minutes, then heat shock in a 42°C water bath for 45 seconds. Place the heat-shocked competent cells on ice for 2 minutes, then spread them evenly on solid LB medium. Place the plate upside down in a 37°C incubator and incubate for approximately 12 hours. Once clearly visible colonies appear on the plate, they can be selected for identification.

[0102] 1.1.5 Screening of positive clones

[0103] When screening for correctly linked recombinant plasmid monoclonal colonies, colony PCR can be used for detection. This detection method primarily utilizes primers specific for the target fragment or specific primers on the vector, using the selected monoclonal colony as a template for PCR amplification. Positive clones can amplify DNA fragments of the same size as the set fragment, and candidate positive clones can be identified by agarose gel electrophoresis. Specifically, colonies on the LB plate are picked with a 0.5-10μL pipette tip and transferred to 50μL of liquid LB with the corresponding resistance for a brief expansion (37°C, 4 hours), and PCR amplification is performed using the bacterial solution as a template.

[0104] The colony PCR reaction system is shown in the following table:

[0105] The PCR reaction program is shown in the following table:

[0106] After the PCR reaction is completed, perform agarose gel electrophoresis to identify the correct clones based on the molecular weight of the PCR products. Select 2-3 candidate positive clones and amplify the briefly expanded culture solution again (in 50 mL of liquid LB for 12 hours) to extract sufficient plasmid DNA for later experiments.

[0107] 1.1.6 Plasmid extraction

[0108] Plasmids in Escherichia coli can be extracted and purified using alkaline lysis. The principle is: alkaline solution can lyse Escherichia coli, and the nucleic acid substances (genomic DNA and plasmid DNA) released after lysis are in a denatured state. At this time, an acidic solution is added to adjust the solution to neutral, and the nucleic acid substances will renature. Since genomic DNA is linear DNA and its length is much longer than plasmid DNA, it cannot maintain a normal folding state during the rapid denaturation and renaturation process, resulting in renaturation failure and precipitation. Compared with genomic DNA, plasmid DNA has a small molecular weight and is circular. It can still return to its original soluble state during the rapid denaturation and renaturation process, so it can be dissolved in the solution. Based on this difference between genomic and plasmid DNA, plasmid DNA can be separated and purified. The specific steps are as follows:

[0109] ① Transfer the expanded culture solution to a 50 mL centrifuge tube, centrifuge at 10,000 g for 5 min, and discard the supernatant;

[0110] ② Add 2.0 mL of pH 8.0 P1 aqueous solution (containing a final concentration of 50 mM Tris-HCl (pH 8.0), a final concentration of 10 mM EDTA, and a final concentration of 100 μg / mL RNase) and resuspend until there are no lumps;

[0111] ③ Add 2.0 mL of P2 aqueous solution (containing a final concentration of 0.2 M NaOH and a final concentration of 10% SDS) and gently shake to allow the bacteria to fully lyse in an alkaline environment, releasing nucleic acid substances and causing them to denature;

[0112] ④ Add 2.0 mL of P3 aqueous solution (containing KAc with a final concentration of 3 M and glacial acetic acid with a final concentration of 11.5%) and mix well to neutralize the alkaline substances in the P2 aqueous solution, restore the solution to a neutral environment, and allow the nucleic acid to renature;

[0113] ⑤ After standing for 10 minutes, centrifuge at 10,000 g for 10 minutes and transfer the supernatant to a new centrifuge tube; add 6.0 mL of isopropanol to the supernatant and mix thoroughly to precipitate the plasmid. Let it stand for 10 minutes to allow the plasmid to fully precipitate, centrifuge at 10,000 g for 10 minutes, remove the supernatant, and invert to dry;

[0114] ⑥ Add 500 μL of TE aqueous solution (containing a final concentration of (10 mM Tris-HCl pH 8.0) and a final concentration of 1 mM EDTA) to dissolve the precipitated plasmid. After the plasmid precipitate is completely dissolved, add 1.5 mL of guanidine hydrochloride and let it stand for 10 minutes to remove protein impurities in the solution;

[0115] ⑦ Purify the plasmid using a DNA adsorption column. Before use, add 200 μL of TE solution to the DNA adsorption column for 5-10 minutes to pre-condition the column. Centrifuge at 10,000 g for 2 minutes.

[0116] ⑧ Pass the liquid in the centrifuge tube through the column three times, 700 μL each time, centrifuge at 10,000 g for 1 min each time;

[0117] ⑨ After all plasmid DNA is adsorbed, use New Wash (containing 1 mM EDTA, 0.1 M NaCl, 10 mM Tris-HCl (pH 7.5), 50% anhydrous ethanol (v / v), and water as the balance) to remove non-specifically bound impurities. Add 600 μL each time and centrifuge at 10,000 g for 1 min. Centrifuge again after washing to remove residual ethanol.

[0118] ⑩ Remove the inner tube of the adsorption column and place it in a new 1.5 mL centrifuge tube. Carefully add 80 μL of TE aqueous solution (containing a final concentration of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA) to the tube, ensuring that the liquid covers the adsorption membrane at the bottom of the column. Allow the tube to stand for at least 30 minutes to allow the plasmid to fully dissolve. Centrifuge at 10,000 g for 1 minute to collect the purified plasmid DNA. Measure the concentration and set aside.

[0119] To further confirm whether the connected recombinant plasmid is correct, 10 μg of plasmid was sent to a sequencing company for DNA sequencing to ensure that the connected DNA fragment did not produce mutations.

[0120] 1.2 Construction of sgRNA recombinant plasmid

[0121] When constructing sgRNA recombinant plasmids, except that the vector was replaced with lentiCRISPR v2, the restriction enzyme was replaced with Bsm BI, and the primer sequences were replaced with the primers shown in Table 2, the other conditions were the same as those for constructing shRNA recombinant plasmids.

[0122] Table 2: sgRNA primer sequences and their target sequences

[0123] 2. Lentivirus packaging and viral infection of U251 cell line

[0124] 2.1 Lentivirus packaging

[0125] Taking HEK-293T cells cultured in 6-well plates for virus packaging as an example, the lentiviral packaging steps are as follows:

[0126] ① Divide HEK-293T cells into 6-well plates for culture. The cell density required for virus packaging is around 90%. Before formally packaging the lentivirus, the medium should be changed 2-3 hours in advance to keep the cells in better condition and facilitate virus packaging.

[0127] ② The plasmid required for viral packaging (the recombinant plasmid constructed in "1. Construction of recombinant plasmid" in Example 1) and the lentiviral packaging auxiliary plasmid (PMDL:VSVG:REV) were transfected into HEK-293T cells using a PEI transfection reagent with a molecular weight of 40,000. The transfection system is: 180 μL HBS buffer (containing a final concentration of 100 mM NaCl, a final concentration of 10 mM HEPES (pH 7.4), and the balance is water), 1.5 μg core plasmid (shRNA recombinant plasmid or sgRNA recombinant plasmid), 1.5 μg Mix (PMDL:VSVG:REV = 5:3:2 (mass ratio)) and 20 μL 10 μM PEI (HBS dissolved) solution, mixed thoroughly and let stand for 20 minutes;

[0128] ③ Slowly and evenly add the transfection mixture to the culture dish, mix well, and culture in an incubator. After 4-8 hours, replace the cells with 2 mL of fresh culture medium.

[0129] ④ After culturing the cells for 36 hours, if the cells are still in good condition, add 1 mL of fresh culture medium and continue culturing for another 24 hours.

[0130] ⑤ After about 60 hours, a large amount of "pseudovirus" particles will be secreted in the culture medium. Collect the culture medium into a 2 mL cryovial and centrifuge at 3000 rpm for 5 minutes to allow the HEK-293T cells suspended in the culture medium to sink to the bottom of the tube. Transfer the supernatant, aliquot the virus, and store at -80°C for later use.

[0131] 2.2 Lentiviral infection of U251 cell line

[0132] Mix the virus obtained in step 5 of "2.1 Lentivirus Packaging" with the cell culture medium containing the U251 cell line for infection. The cell density during infection should be adjusted based on the cell growth rate. Generally, it is best to allow the cells to reach confluence 24 hours after infection. Replace the cells with fresh culture medium before infection. To improve the efficiency of viral infection, add the negative charge scavenging agent polybrene (final concentration 10 μg / mL) to remove negative charges on the surface of cells and viral particles, thereby reducing electrostatic repulsion. The culture medium can be replaced or the cells can be passaged 24 hours after infection.

[0133] 3. siRNA transfection of U251 cell line

[0134] siRNA can be transfected into cells using a lipofectamine transfection reagent to knock down target gene expression. Mix 1 μg of the synthesized siRNA listed in Table 3, 3 μL of Lipofectamine 3000, and 200 μL of serum-free DMEM. Let the mixture stand for 20 minutes. Then, add the mixture to U251 cells cultured in a 6-well plate at 50% density.

[0135] Table 3: siRNA sequences

[0136] 4. Western blot detection of D2HGDH protein expression

[0137] Western blot (WB) is a protein quantitative detection method that integrates multiple technologies such as polyacrylamide gel electrophoresis, protein transfer technology, antigen-antibody adsorption technology, enzyme-linked antibody technology, and exposure and development technology. Its main steps are as follows:

[0138] ① Prepare protein samples. Protein samples requiring Western blotting (WB) testing primarily include cultured cells and animal tissues. For cultured cells, aspirate the culture medium and add an appropriate amount of cell lysis buffer containing protease inhibitors. Scrape the cells from the culture dish and transfer them to a new 1.5mL centrifuge tube. Ultrasonicate and centrifuge at 12,000g for 10 minutes at 4°C. For animal tissue samples, homogenize the sample after adding the cell lysis buffer, ultrasonicate, and centrifuge at 12,000g for 10 minutes at 4°C. Transfer the supernatant to a new 1.5mL centrifuge tube. Take 2μL and use the G250 Coomassie Brilliant Blue staining method to determine the total protein concentration. Normalize the samples based on the detected protein concentration to ensure a consistent total protein concentration of 3-5μg / μL for each sample. Add 5× SDS sample preparation buffer and heat at 100°C for 10 minutes to prepare the WB protein sample.

[0139] ② Denaturing gel polyacrylamide gel electrophoresis. Prepare polyacrylamide gel of different concentrations (8-15%) according to the molecular weight of the target protein. After preparation, set it up with an electrophoresis rack, place it in a vertical electrophoresis tank, and add electrophoresis buffer. According to the experimental design, add the protein samples to the lanes of the gel in sequence. Separate the protein samples at a voltage of 100V, and determine the running time of the gel according to the size of the target protein under the instructions of the pre-made protein maker (usually 60-120 minutes);

[0140] ③ Protein transfer. Cut a PVDF membrane of appropriate size based on the size of the target protein. Activate the membrane with ethanol before transfer. Arrange the negative electrode, filter paper, gel, PVDF membrane, filter paper, and anode in this order. Place the transfer clip in the vertical electrophoresis tank, add transfer buffer, and transfer at 100V for 30-90 minutes. To prevent the buffer from overheating during transfer, add pre-chilled ice packs to the tank. If the transfer time exceeds 60 minutes, replace the ice packs.

[0141] ④ Antibody incubation. Before incubation, block the untransferred areas of the PVDF membrane with 5% skim milk or 5% BSA. After washing three times with TBS-T, incubate with the primary antibody (anti-D2HGDH, anti-IDH1 R132H, anti-Flag, or anti-GAPDH) and secondary antibody (Goat Anti-Rabbit IgG Antibody or Goat Anti-Mouse IgG Antibody).

[0142] ⑤ Exposure and development. After washing the secondary antibody with TBS-T, add an exposure solution made from a mixture of ECLA and ECLB to the PVDF membrane. Quickly place the membrane soaked in the exposure solution into an exposure clip covered with plastic wrap. Collect the signal using film in a darkroom, then develop and fix the film. After washing, dry the film.

[0143] Main reagent formula:

[0144] 10% separating gel: 66.8 mL 30% Acr-Bis, 15.8 mL 65% sucrose, 20 mL 10× lower buffer (containing a final concentration of 3.5 M Tris-HCl (pH 8.8) and 1% SDS), and dilute to 200 mL with water;

[0145] 26.6 mL of 4% stacking gel, 30% Acr-Bis, 50 mL of 4× stacking buffer (containing a final concentration of 0.5 M Tris-HCl (pH 6.8) and 0.4% SDS), and dilute to 200 mL with water.

[0146] Electrophoresis buffer: 25 mM Tris base, 0.2 M glycine, 1.0% SDS;

[0147] Transfer buffer: 25 mM Trisbase, 0.2 M glycine, 10% anhydrous ethanol;

[0148] TBS-T: 0.1% Tween-20, 0.242% Tris base, 0.8% NaCl pH 7.6;

[0149] ECLA: 0.396mM P-coumaric, 25μM Luminol, 100mM Tris-HCl (pH 8.5);

[0150] ECLB: 5.4mM H2O2, 100mM Tris-HCl (pH 8.5).

[0151] 5. Screening of effective sequences for D2HGDH knockdown or knockout

[0152] Western blot analysis was used to analyze the expression level of D2HGDH in U251 cells infected with viruses expressing shRNA or sgRNA or transfected with siRNA 72 h later. The shRNA, sgRNA or siRNA sequences with the best knockdown or knockout effect of D2HGDH were screened based on the Western blot results.

[0153] Results: The final results of Example 1 are shown in Figure 1. In Figure 1A, shRFP is a negative control, targeting the red fluorescent protein (RFP) gene from the Branchiostoma floridae species. Figure 1A shows that shRNA-1, shRNA-2, and shRNA-3 had a significant knockdown effect, while shRNA-4, shRNA-5, and shRNA-6 had no significant knockdown effect.

[0154] The siRFP negative control in Figure 1B contains an RNA sequence targeting the red fluorescent protein (RFP) gene from the Branchiostoma floridae species. The results in Figure 1B show that siRNA-1, siRNA-2, and siRNA-3 have a significant knockdown effect, while siRNA-4, siRNA-5, and siRNA-6 have no significant knockdown effect.

[0155] In Figure 1C, sgCTRL is a negative control, a Lenti V2 plasmid vector that does not contain an sgRNA sequence. The results in Figure 1C show that sgRNA-1, sgRNA-2, and sgRNA-3 have good knockout effects, while sgRNA-4, sgRNA-5, and sgRNA-6 have no significant knockout effect.

[0156] Example 2: Effect of knockdown of D2HGDH expression on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0157] The present invention uses the following glioma cell lines: U251 overexpressing wild-type IDH1 (abbreviated as U251 IDH1 WT), U118 overexpressing wild-type IDH1 (abbreviated as U118 IDH1 WT), U251 overexpressing IDH1 R132H mutant glioma cell line (abbreviated as U251 IDH1 R132H), U118 overexpressing IDH1 R132H mutant glioma cell line (abbreviated as U118 IDH1 R132H), JURKAT overexpressing wild-type IDH2 leukemia cell line (abbreviated as JURKAT IDH2 WT), and JURKAT overexpressing IDH2 R140Q mutant leukemia cell line (abbreviated as JURKAT IDH2 R140Q), as well as primary IDH mutation chondrosarcoma cells HT1080 (carrying IDH1 R132C mutation) and SW1353 (carrying IDH2 R172S mutation) and other cells were used as experimental subjects for research.

[0158] 2.1 Construction of stable cell lines overexpressing wild-type IDH1 or IDH2 (IDH1 WT or IDH2 WT) and overexpressing mutant IDH1 or IDH2 (IDH1 R132H or IDH2 R140Q)

[0159] Primers for specific amplification of IDH1 or IDH2 genes were designed (primer sequences are: forward primer for amplifying IDH1: Seq ID NO.52: 5′-AGAGAATTCGGATCCATGTCCAAAAAAATCAGT-3′, reverse primer: Seq ID NO.53: 5′-CTTCCATGGCTCGAGAAGTTTGGCCTGAGCTAG-3′. Forward primer for amplifying IDH2: Seq ID NO.54: 5′-AGAGAATTCGGATCCATGGCCGGCTACCTGCGG-3′, reverse primer: Seq ID NO.55: 5′-CTTCCATGGCTCGAGCTGCCTGCCCAGGGCTCT-3′), and the gene fragments of IDH1 or IDH2 were amplified using cDNA obtained from U251 cells as experimental materials as templates. Then, the same method as in Part 1 of Example 1 was used to construct the plasmid plv-EF1-puro-Flag-IDH1 WT overexpressing IDH1 WT or the plasmid plv-EF1-puro-Flag-IDH2 WT overexpressing IDH2 WT.

[0160] Based on this, primer-designed point mutation cloning technology was used to obtain overexpression plasmids carrying the IDH1 R132H mutation, plv-EF1-puro-Flag-IDH1 R132H, or the IDH2 R140Q mutation, plv-EF1-puro-Flag-IDH2 R140Q. The forward primer for IDH1 R132H was constructed using Seq ID NO.56: 5′-ATAGGTCATCATGCTTATGGGGATC-3′, and the reverse primer was constructed using Seq ID NO.57: 5′-AGCATGATGACCTATGATGATAGGT-3′. The forward primer for IDH2 R140Q was constructed using Seq ID NO.58: 5′-CTATCCGGAACATCCTGGGGGG-3′, and the reverse primer was constructed using Seq ID NO.59: 5′-TGTTCCGGATAGTTCCATTGGGAC-3′.

[0161] Six cell lines (U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, and JURKAT IDH2 R140Q) were constructed by lentiviral infection using the same method as in Part 2 of Example 1. Western blot analysis was used to examine the expression of wild-type and mutant IDH1 or IDH2 (Flag), as well as the expression of D2HGDH in these six cell lines.

[0162] To further confirm that the constructed IDH1 mutant or IDH2 mutant cell lines possess the basic characteristics of IDH1 mutant or IDH2 mutant cells, we analyzed the D-2HG levels in the culture medium of the above six cells using a D-2HG detection kit. The specific steps were as follows:

[0163] ① Digest the above 6 cells with trypsin and count the cells. Take 1.0×10 5 cells, and plated them into 12-well plates, with 3 biological parallels set for each cell;

[0164] ②After 24 hours of culture, replace 1 mL of cell culture medium;

[0165] ③ After 72 hours of continuous culture, take 100 μL of cell culture medium into a 200 μL centrifuge tube and incubate at 100°C for 20 minutes using the incubation function of the PCR instrument. After the incubation, centrifuge at 1000 rpm for 10 minutes and transfer the supernatant for later use;

[0166] ④ Take 10uL of supernatant and perform the test according to the instructions of the D-2HG detection kit;

[0167] ⑤ The protein concentration of the cultured cells was detected according to the method in Section 4 of Example 1 for normalization of D-2HG concentration. At the same time, Western blot samples were prepared for analysis of the expression of IDH1 WT protein, IDH1 R132H protein, IDH2 WT protein, IDH2 R140Q protein, and D2HGDH protein.

[0168] Results: The experimental results are shown in Figure 2.

[0169] Conclusion: The results show that IDH1 WT protein, IDH1 R132H protein, IDH2 WT protein, and IDH2 R140Q protein are all expressed in U251, U118, and JURKAT cells, and the expression levels of wild-type and mutant types are comparable. At the same time, the protein expression level of D2HGDH is significantly increased in cells with IDH1 mutation or IDH2 mutation. IDH1 mutation or IDH2 mutation cells may be more dependent on the function of D2HGDH. This experimental result is one of the important theoretical foundations of the present invention. In addition, cells overexpressing IDH1 R132H or IDH2 R140Q can produce high concentrations of D-2HG. This shows that the constructed IDH1 mutant or IDH2 mutant cell lines have the basic characteristics of IDH1 mutant or IDH2 mutant cells and can be used for research related to IDH mutations.

[0170] 2.2 Cell proliferation curve analysis of the effect of knockdown of D2HGDH expression on the proliferation of IDH1 or IDH2 wild-type and mutant cells

[0171] The present invention is illustrated by taking three pairs of shRNAs that can effectively inhibit D2HGDH as an example. As described in Example 1, after obtaining the optimal shRNA sequences (shRNA-1, shRNA-2, and shRNA-3), U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HT1080, and SW1353 cell lines that knock down D2HGDH were constructed by lentiviral infection. In the present invention, a cell counter is used to count cells and draw a cell proliferation curve. The steps are as follows:

[0172] ① Digest the cells to be tested for proliferation with trypsin and count the cells. Take 10,000 cells and plate them into a 12-well plate, setting up 3 biological replicates for each cell.

[0173] ② Count cells every 24 hours for a total of 120 hours;

[0174] Results: The experimental results of this embodiment are shown in FIG3 .

[0175] Conclusion: These results demonstrate that knocking down D2HGDH expression in IDH1 or IDH2 mutant cell lines significantly inhibits cell proliferation. However, knocking down D2HGDH expression in IDH1 wild-type or IDH2 wild-type cells has limited effect on their proliferation. This suggests that knocking down D2HGDH can specifically inhibit the proliferation of cells harboring IDH1 or IDH2 mutations.

[0176] Example 3: Effect of D2HGDH enzyme activity on proliferation of IDH1 or IDH2 wild-type and mutant cells

[0177] Given that the main function of D2HGDH is to catalyze D-2HG, the present invention further describes the effect of D2HGDH enzyme activity on the cell proliferation of IDH1 wild-type cells, IDH2 wild-type cells, IDH1 mutant cells, and IDH2 mutant cells.

[0178] 3.1 Construction of wild-type and enzyme-deficient D2HGDH overexpression plasmids

[0179] Primers specifically amplifying the D2HGDH gene were designed (forward primer: Seq ID NO. 60: 5′-AGAGAATTCGGATCCATGCTGCCCCGTCGGCCT-3′, reverse primer: Seq ID NO. 61: 5′-CTTCCATGGCTCGAGGGCCTGGCTGGGCAGC-3′). The D2HGDH gene fragment was amplified using cDNA obtained from U251 cells as a template. The wild-type D2HGDH overexpression plasmid, plv-EF1-puro-Flag-D2HGDH WT, was then constructed using the same method as in Part 1 of Example 1.

[0180] To investigate the function of D2HGDH enzyme activity in cells with IDH1 or IDH2 mutations, we needed to construct a plasmid lacking D2HGDH enzyme activity. Clinically, D-dihydroxyglutaric aciduria type II is a genetic disorder caused by mutations in the D2HGDH gene that abolish its catalytic activity, resulting in abnormally elevated levels of D-2HG. Based on previous reports, we selected the D2HGDH G477R mutant as a D2HGDH enzyme-deficient mutant. Using primer-designed point mutation cloning technology, we constructed the plv-EF1-puro-Flag-D2HGDH G477R mutant plasmid. The forward primer for constructing D2HGDH G477R is Seq ID No. 62: 5′-GCGCGGAGCACCGAGTGGGC-3′, and the reverse primer is Seq ID No. 63: 5′-CTGAAGCCCACTCGGTGCTC-3′.

[0181] 3.2 Cell proliferation curve analysis of the effect of D2HGDH enzyme activity on the proliferation of IDH1 or IDH2 wild-type and mutant cells

[0182] To study the effect of D2HGDH enzyme activity on the proliferation of wild-type and mutant IDH1 or IDH2 cells, based on Example 2, we constructed a knockdown D2HGDH by lentiviral infection (using shRNA-1 lentivirus to knock down D2HGDH) and then complemented the wild-type D2HGDH (rWT) U251 IDH1 WT, U118 IDH1 WT, U118 IDH1 WT, JURKAT IDH2 WT, HT1080 and SW1353 cell lines, and D2HGDH enzyme activity-deficient D2HGDH mutant (rG477R) U251 IDH1 R132H, U118 IDH1 R132H, JURKAT IDH2 R140Q, HT1080 and SW1353 cell lines. Cells were counted using a cell counter to draw a cell proliferation curve. The steps are as follows:

[0183] ① Digest the cells to be tested for proliferation with trypsin and count the cells. Take 10,000 cells and plate them into a 12-well plate, setting up 3 biological replicates for each cell.

[0184] ② Count the cells every 24 hours for a total of 120 hours.

[0185] Results: The experimental results of this embodiment are shown in FIG4 .

[0186] Conclusion: These results demonstrate that the decreased cell proliferation caused by knockdown of D2HGDH expression in IDH1 or IDH2 mutant cell lines can be restored to levels comparable to the control shRFP by complementing wild-type D2HGDH. However, complementation of D2HGDH lacking enzymatic activity failed to restore proliferation to levels comparable to the control shRFP. This suggests that the decreased proliferation of IDH1 or IDH2 mutant cells caused by D2HGDH knockdown is primarily due to the loss of D2HGDH enzyme activity. Therefore, inhibition of D2HGDH enzyme activity, such as through small molecule inhibitors, can also inhibit the proliferation of IDH1 or IDH2 mutant cells.

[0187] Example 4: Effect of knockdown of D2HGDH expression on the tumorigenicity of IDH1 mutant cells in vivo

[0188] The HT1080 cell line is a primary chondrosarcoma cell line harboring the IDH1 R132C mutation and is an important cell line resource for studying IDH1- and IDH2-mutant tumors. Therefore, the present invention used HT1080 as the research subject and utilized the D2HGDH knockdown, wild-type D2HGDH complementation, and enzyme-deficient mutant HT1080 cell lines constructed in Example 3 to analyze the effects of D2HGDH knockdown and D2HGDH enzyme activity on the in vivo tumorigenicity of IDH1-mutant tumor cells.

[0189] 4.1 Nude mouse subcutaneous tumor formation assay to analyze the effects of knocking down D2HGDH expression and D2HGDH enzyme activity on the tumorigenicity of IDH1 mutant cells in vivo

[0190] Ten 6-week-old female BALB / C nude mice were randomly divided into two groups, with 5 mice in each group. A subcutaneous tumor-forming mouse model was established according to the following steps:

[0191] ① The constructed D2HGDH knockdown and complemented wild-type D2HGDH and enzyme activity-deficient mutant HT1080 cell lines were expanded and cultured into two 100 mm cell culture dishes.

[0192] ② Take HT1080 cells in the logarithmic growth phase, digest them with trypsin, count the cells using a cell counter, and dilute the cells to 1.0×10 7 Place the cell suspension at a concentration of 100 μg / mL on ice until ready for use.

[0193] ③2.0×10 6 Each cell type (200 μL) was injected into one side of the thigh of nude mice using a 1 mL medical syringe (different cells were injected on the left and right sides), and 5 nude mice were injected with each cell type.

[0194] ④ Four to six weeks after injection, when the largest subcutaneous tumor reaches approximately 1 cm, euthanize the mouse. Take photos to record the size of the tumor. After removing the subcutaneous tumor, take photos to record the size, weigh it, and measure its volume.

[0195] Results: The experimental results of this embodiment are shown in FIG5 .

[0196] Conclusion: Knockdown of D2HGDH expression significantly inhibited the subcutaneous tumorigenicity of HT1080 cells. Replenishment of wild-type D2HGDH restored this ability, whereas repletion of a mutant lacking enzymatic activity failed to restore this ability. This suggests that D2HGDH and its enzymatic activity are crucial for the in vivo tumorigenicity of IDH1-mutant tumor cells. Knockdown of D2HGDH or inhibition of D2HGDH enzymatic activity with a small molecule inhibitor significantly inhibited the tumorigenicity of IDH1-mutant tumor cells.

[0197] Example 5: Effects of D2HGDH Inhibitors on Proliferation of IDH1 or IDH2 Wild-Type and Mutant Cells

[0198] Examples 2-4 demonstrate that the enzymatic activity of D2HGDH plays a crucial role in the proliferation of tumors harboring IDH1 or IDH2 mutations. Targeting D2HGDH may be a therapeutic target for tumors harboring IDH1 or IDH2 mutations. In light of this, the present inventors designed a high-throughput screening strategy for D2HGDH inhibitors and identified several effective inhibitors from a pool of over 8,000 small molecules.

[0199] 5.1 Construction of D2HGDH inhibitor screening system

[0200] The primary function of D2HGDH is to catalyze the conversion of D-2HG to α-KG and FADH2. The rate of this reaction can be measured by measuring the rate of FADH2 formation: FADH2 transfers hydrogen ions to the hydrogen acceptor INT (iodonitrotetrazolium violet). INT is reduced to produce Furazan, which has a characteristic absorption peak at OD530. By measuring changes in this absorption peak, D2HGDH enzyme activity can be quantitatively analyzed. To more effectively screen for drugs, we optimized the enzyme activity reaction system by adjusting the substrate concentration, pH buffer system, hydrogen acceptor INT content, and reaction time, ensuring a signal-to-noise ratio greater than 5 and a linear reaction time as long as possible.

[0201] Results: The results of this example are shown in FIG6 .

[0202] Conclusion: The optimal system for screening D2HGDH inhibitors is: 1.0 μg D2HGDH protein, 200 μM D-2HG, 50 μM FAD+, 1.5 mM INT, 0.02% castor oil (Cermophor) and 100 mM Hepes pH 8.0.

[0203] 5.2 High-throughput screening of D2HGDH inhibitors

[0204] After building a high-throughput drug screening system, the Plate&Worker automated screening system was used to screen D2HGDH small molecule inhibitors. The enzyme activity reaction system was divided into two parts. +First add the buffer system Hepes to the 384-well plate, then add the small molecule compound to the 384-well plate (add DMSO to the first two columns and the last two columns as a negative control) to make the final concentration of the small molecule 20μM. Place it in a 37℃ incubator for 10 minutes to allow the small molecule compound to bind to the D2HGDH protein as much as possible. Finally, add the other components of the reaction system to the 384-well plate, mix them and immediately measure the OD530 as the initial value of the reaction OD5300. Place the 384-well plate in a 37℃ incubator and measure the OD530 again after 60 minutes of reaction. Count and obtain △OD530=OD530 60 -OD5300. The inhibition efficiency of small molecule compounds on D2HGDH enzyme activity was calculated based on the detected △OD530, inhibition efficiency = 1-△OD530 小分子 ÷△OD530 DMSO Small molecules with inhibition efficiencies exceeding 50% or 80% were revalidated, and half-inhibition curves were drawn.

[0205] Results: The results of this example are shown in FIG7 (due to space limitations, only preferred results are shown here).

[0206] Conclusion: From a library of more than 8,000 small molecule compounds, we screened out ginkgolic acid (C15:1) and NSC87877, which can effectively inhibit the activity of D2HGDH protein.

[0207] Effects of D2HGDH Inhibitors on the Proliferation of IDH1 or IDH2 Wild-Type and Mutant Cells

[0208] We treated U251 IDH1 WT, U251 IDH1 R132H, U118 IDH1 WT, U118 IDH1 R132H, JURKAT IDH2 WT, JURKAT IDH2 R140Q, HuCCT1 IDH1 WT, HuCCT1 IDH1 R132H, HT1080, and SW1353 cell lines with gradient concentrations of Ginkgolic acid (C15:1) and NSC87877. The specific steps are as follows:

[0209] ① 1.0×10 4 Cells were plated into 12-well plates;

[0210] ② After the cells adhered for 24 hours, the culture medium was replaced with a gradient of Ginkgolic acid (C15:1) and NSC87877. The concentrations of the compounds used were: 0 (DMSO), 5 μM, 10 μM, 20 μM, and 50 μM.

[0211] ③ Cell counts were performed 24 hours and 120 hours after treatment with small molecule compounds. The cell numbers at these two time points were recorded as N1 and N2. The cell proliferation rate = (N2-N1)÷N1.

[0212] Results: The results of this example are shown in Figures 8 and 9.

[0213] Conclusion: Ginkgolic acid (C15:1) and NSC87877 can specifically inhibit the proliferation of IDH1 mutation or IDH2 mutation tumor cells, while the inhibitory effect on IDH1 wild-type or IDH2 wild-type cells is relatively small.

[0214] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. Use of a nucleic acid molecule that specifically inhibits the transcription or translation of the D2HGDH gene, or an inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene, in the preparation of a drug for treating tumors, wherein the nucleic acid molecule is shRNA, siRNA, and / or sgRNA.

2. The use according to claim 1, wherein the target sequence of the nucleic acid molecule comprises Seq ID NO.1, Seq ID NO.4, Seq ID NO.7, Seq ID NO.13, Seq ID NO.16, or Seq ID NO.19; and / or the shRNA comprises shRNA-1, shRNA-2, or shRNA-3; the shRNA-1 comprises a sense strand and an antisense strand, the sense strand of the shRNA-1 contains Seq ID NO.2, and the antisense strand of the shRNA-1 contains Seq ID NO.3; the shRNA-2 comprises a sense strand and an antisense strand, the sense strand of the shRNA-2 contains Seq ID NO.5, and the antisense strand of the shRNA-2 contains Seq ID NO.6; the shRNA-3 comprises a sense strand and an antisense strand, the sense strand of the shRNA-3 contains Seq ID NO.8, and the antisense strand of the shRNA-3 contains Seq ID NO.9; and / or the siRNA comprises siRNA-1, siRNA-2, or siRNA-3; the sequence of siRNA-1 is Seq ID NO.10; the sequence of siRNA-2 is Seq ID NO.11; the sequence of siRNA-3 is Seq ID NO.12; and / or the sgRNA comprises sgRNA-1, sgRNA-2, or sgRNA-3; the sgRNA-1 comprises a sense strand and an antisense strand, the sense strand of the sgRNA-1 contains Seq ID NO.14, and the antisense strand of the sgRNA-1 contains Seq ID NO.15; the sgRNA-2 comprises a sense strand and an antisense strand, the sense strand of the sgRNA-2 contains Seq ID NO.17, and the antisense strand of the sgRNA-2 contains Seq ID NO.18; the sgRNA-3 comprises a sense strand and an antisense strand, the sense strand of the sgRNA-3 contains Seq ID NO.20, and the antisense strand of the sgRNA-3 contains Seq ID NO.

21.

3. The use according to claim 1, wherein the inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene comprises at least one of ginkgolic acid C15:1 and NSC87877.

4. The use according to any one of claims 1-3, wherein the tumor is selected from tumors carrying IDH1 mutation or IDH2 mutation.

5. The use according to claim 4, wherein the tumor comprises glioma, acute myeloid leukemia, angioimmunoblastic T-cell lymphoma, chondrosarcoma, cholangiocarcinoma, or sinus cancer.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule is the nucleic acid molecule as described in the use according to any one of claims 1-2 and 4-5.

7. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule according to claim 6 and a viral vector.

8. The recombinant vector according to claim 7, wherein the viral vector comprises a retroviral vector, a lentiviral vector or an adenoviral vector.

9. A nucleic acid molecule delivery system, characterized in that, The nucleic acid molecule delivery system comprises the nucleic acid molecule according to claim 6.

10. The nucleic acid molecule delivery system according to claim 9, wherein the nucleic acid molecule delivery system comprises at least one of N-acetylated galactosamine conjugate modification, lipid nanoparticles, polymer nanoparticles (such as polyethyleneimine and chitosan), and exosomes.

11. A host cell, characterized in that, Comprises the nucleic acid molecule according to claim 6 or the recombinant vector according to any one of claims 7-8.

12. A composition, characterized in that, Comprises at least one of the nucleic acid molecule according to claim 6, the recombinant vector according to any one of claims 7-8, the nucleic acid molecule delivery system according to any one of claims 9-10, the host cell according to claim 11, and an antibody against the protein encoded by D2HGDH or an inhibitor that inhibits the activity of the protein encoded by the D2HGDH gene.

13. The composition according to claim 12, wherein the inhibitor comprises at least one of ginkgolic acid C15:1 and NSC87877.

14. The composition according to any one of claims 12-13, wherein the composition further comprises a pharmaceutically acceptable adjuvant.

15. The composition according to claim 14, wherein the pharmaceutically acceptable adjuvant comprises dimethyldioctadecylammonium and / or monophosphoryl lipid A and / or physiological saline or other adjuvants.

16. The composition according to claim 15, wherein the other adjuvant comprises an aluminum adjuvant or a Freund's adjuvant.

17. A method for delivering nucleic acid molecules to cells for non-therapeutic purposes, characterized in that, Comprises contacting a cell with at least one of the nucleic acid molecule according to claim 6, the recombinant vector according to any one of claims 7-8, the nucleic acid molecule delivery system according to any one of claims 9-10, the host cell according to claim 11, or the composition according to any one of claims 12-16.

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