CN111671904B - Medicine containing endonuclease inhibiting function and anti-tumor application thereof - Google Patents

Abstract

The invention discloses an inhibitory drug of ENDOD1 endonuclease and an SSA enhancer, and application of the inhibitory drug of ENDOD1 endonuclease and the SSA enhancer in the preparation of drugs for treating or preventing human tumors and hepatitis B virus infection diseases. The endonuclease is an endonuclease encoded by an ENDOD1 gene, the inhibitory drug inhibits the gene expression, activity and/or function of the ENDOD1 endonuclease, and the SSA enhancer upregulates the activity or function of single-stranded DNA fusion repair (SSA). The ENDOD1 endonuclease inhibitory drug or SSA enhancer can effectively inhibit or kill tumor cells with DNA homologous recombination, DNA damage response, DNA damage repair, gene mutation of cancer suppressor genes, gene expression or function defect, and has no toxicity to the genes or normal non-tumor cells.

Description

A drug containing the function of inhibiting endonuclease and its anti-tumor application

technical field

The invention belongs to the field of medicine, and in particular relates to the use of the function of inhibiting ENDOD1 endonuclease in the preparation of medicines for treating or preventing human tumors.

Background technique

Malignant tumors are highly resistant to chemical drugs and are prone to drug resistance. It is very difficult to achieve the goal of temporarily controlling tumor growth and prolonging the survival time of patients. Therefore, the clinical needs of developing a new generation of tumor targeted therapy are particularly urgent. Drugs that can effectively kill tumor cells but cause relatively little damage to normal non-tumor cells. One of the drug development strategies to achieve this goal is synthetic lethality (synthetic lethality), that is, to discover a specific defect in tumor cells, and to determine that destroying another cell function in this defective state will lead to the death of tumor cells (see: O' Neil, N.J., M.L. Bailey, and P. Hieter, Synthetic lethality and cancer. Nat Rev Genet, 2017.18(10): p. 613-623.).

The genome of normal cells faces the threat of DNA damage factors at any time, leading to mutations of oncogenes and tumor suppressor genes and uncontrolled cell proliferation, which promotes tumorigenesis. In this process, DNA damage response and DNA repair mechanism play a key role, inhibit tumor-related genomic mutations, eliminate cells with abnormal genome structure or replication defects, and ensure genome stability and non-cancerous organ tissue (see: Jeggo, P.A., L.H. Pearl, and A.M. Carr, DNA repair, genome stability and cancer: a historical perspective. Nat Rev Cancer, 2016.16(1): p.35-42.). Common tumor suppressor genes include PTEN, CDKN2A, TP53, APC, VHL, p16INK4A, p14ARF, LKB1, FBXW7, NF1, NF2 and WT-1, etc., whose mutations lead to glioma, neurofibroma, lung cancer, gastric cancer, liver cancer , melanoma, colorectal cancer, nasopharyngeal cancer, kidney cancer and lymphoma (see: Wang, L.H., et al., Loss of Tumor Suppressor Gene Function in Human Cancer: An Overview. Cell Physiol Biochem, 2018.51(6): p .2647-2693.). Especially the TP53 gene, whose mutation is widely present in 50-80% of tumors, and can be disrupted by viral agents such as human papillomavirus (HPV), hepatitis B virus (HBV) (see: Aubrey, B.J., A.Strasser , and G.L. Kelly, Tumor-Suppressor Functions of the TP53 Pathway. Cold Spring Harb Perspect Med, 2016.6(5).).

In human cells, the DNA damage response is mainly regulated by ATM and ATR kinases, which activate CHK1 and CHK2 through signal transduction pathways such as γH2AX, 53BP1, MDC1 and CDC25, control cell cycle progression, and promote the activation of DNA damage repair mechanisms. Known DNA damage repair mechanisms include non-identical end joining (NHEJ), homologous recombination repair (HR), single-strand DNA fusion (SSA), nucleotide excision repair (NER), base excision repair (BER), single Strand DNA break repair (SSR), etc. (See: Chatterjee, N. and G.C. Walker, Mechanisms of DNA damage, repair, and mutagenesis. Environ Mol Mutagen, 2017.58(5): p.235-263.). Inactivation of these mechanisms leads to the development of skin cancer, breast cancer, ovarian cancer, liver cancer, cholangiocarcinoma, colorectal cancer, prostate cancer and leukemia.

In the process of cell proliferation, DNA replication produces a large amount of DNA damage, which easily leads to gene mutation. These damages are mainly eliminated by repair mechanisms relying on homologous sequences, such as HR, SSA. HR is mainly regulated by factors such as BRCA1, BRCA2, MRE11, NBS1, BLM, 53BP1, RAD51, RPA, WDR70 and Fanconi (FANC), while SSA depends on related repair factors such as RAD52, MRE11, RAD54B and RPA (see: Haber, J.E., DNA Repair: The Search for Homology. Bioessays, 2018.40(5): p.e1700229.).

The SSA mechanism is extremely conserved during evolution, mainly occurring in homologous sequences near DNA breakpoints. The process relies on repair factors such as RAD52 to combine with DNA breakpoints to form nucleoprotein complex synapses, invade and fuse homologous sequences near DNA breakpoints. source sequence. RAD52 is a key molecule of the SSA pathway, which can directly bind to the broken end of the processed DNA, especially the single-stranded DNA product, protect the processed DNA from nuclease degradation, and promote the fusion of homologous sequences on the single-stranded DNA (see : Wu, Y., et al., The DNA binding preference of RAD52 and RAD59 proteins: implications for RAD52 and RAD59 protein function in homologous recombination. J Biol Chem, 2006.281(52): p.40001-9.). RAD52 also promotes interstrand exchange during late homologous recombination repair. RAD52 can also bind to the single-stranded DNA formed by the replication fork block, maintain the stability of the replication fork, prevent the newly replicated DNA strand from being degraded, and play an important role in the stability of DNA during the replication process (see: Malacaria, E., et al., Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation. Nat Commun, 2019.10(1):p.1412.). Due to the possibility of loss of homologous sequences, SSA is considered a low-fidelity or even toxic repair method. Thus, disturbances in the regulation of the SSA pathway may lead to defects in DNA response and damage repair, resulting in genomic instability.

Contents of the invention

The purpose of the present invention is to provide an ENDOD1 endonuclease inhibitory drug or SSA enhancer and its use in the preparation and manufacture of drugs for the prevention or treatment of tumors, especially for the treatment of DNA homologous recombination defects and DNA damage response defects , DNA repair defects, TP53 and other tumor suppressor gene mutations, gene expression or functional defects in tumors.

The ENDOD1 endonuclease of the present invention is as a brand-new broad-spectrum antitumor drug target, and this endonuclease is the protein encoded by the ENDOD1 gene, wherein the mRNA sequence of the ENDOD1 is SEQ ID No.1 (corresponding NCBI reference number NM_015036.3), the encoded amino acid sequence of the protein is the sequence corresponding to NCBI accession number NP_055851.1.

The ENDOD1 endonuclease inhibitory drug herein may also be referred to as an ENDOD1 endonuclease inhibitor.

In one embodiment, the present invention provides a method for treating or preventing tumors, comprising administering an effective amount of ENDOD1 endonuclease inhibitory drugs or SSA enhancers to tumor patients, and the inhibitory drugs inhibit ENDOD1 endonuclease The function of the enzyme, the endonuclease is the endonuclease encoded by the ENDOD1 endonuclease gene. The SSA enhancer can improve the function of single strand DNA fusion repair (single strand annealing, SSA).

In the above treatment method of the present invention, the tumor contains DNA homologous recombination defects, DNA damage response defects, DNA repair defects, gene mutations, gene expression or functional defects of tumor suppressor genes such as TP53. Specifically includes lung cancer, breast cancer, colorectal cancer, stomach cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma or/and Cholangiocarcinoma.

In the above treatment method of the present invention, the ENDOD1 endonuclease inhibitory drug or SSA enhancer can be any substance that has an inhibitory effect on ENDOD1 endonuclease, and the SSA enhancer can be any substance that improves single-stranded DNA fusion repair. (SSA) substances, including but not limited to antibodies, organic or natural compounds, small nucleic acids, small RNAs, proteins, polypeptides, antisense acids, and inorganic substances.

The above-mentioned treatment method of the present invention further includes using in combination with or forming a composition with one or more other anti-tumor treatment methods. The other one or more anti-tumor treatment methods include surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, virus therapy, RNA therapy, targeted therapy, adjuvant therapy and/or immunotherapy.

In some embodiments, the present invention provides a pharmaceutical composition for treating or preventing tumors, comprising an ENDOD1 endonuclease inhibitory drug or an SSA enhancer, and the ENDOD1 inhibitory drug can inhibit the gene of ENDOD1 endonuclease Expression, activity or function, the endonuclease is the endonuclease encoded by the ENDOD1 endonuclease gene. The SSA enhancer can up-regulate the function of single-strand DNA fusion repair (SSA). The tumor has DNA homologous recombination defect, DNA damage response defect, DNA repair defect, gene mutation, gene expression or function defect of tumor suppressor genes such as TP53. Specifically lung cancer, breast cancer, colorectal cancer, stomach cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma or/and Cholangiocarcinoma.

In the above-mentioned pharmaceutical composition of the present invention, the ENDOD1 endonuclease inhibitory drug can be any substance that has an inhibitory effect on ENDOD1 endonuclease, and the SSA enhancer can be any up-regulated single-strand DNA fusion repair (SSA) Functional substances specifically include but are not limited to drugs selected from antibodies, synthetic organic compounds, natural organic compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, and inorganic substances.

In some embodiments, the ENDOD1 endonuclease inhibitory drug or SSA enhancer of the present invention is used in the manufacture of a drug for treating and/or preventing tumors, and the ENDOD1 inhibitory drug can inhibit the gene expression of ENDOD1 endonuclease , activity or function, the endonuclease is the endonuclease encoded by the ENDOD1 endonuclease gene. The SSA enhancer can up-regulate the function of single-strand DNA fusion repair (SSA).

In the above application of the present invention, the tumor has DNA homologous recombination defect, DNA damage response defect, DNA repair defect, gene mutation, gene expression or function defect of tumor suppressor genes such as TP53. The tumors include but not limited to lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma , sarcoma and/or cholangiocarcinoma.

In the above-mentioned purposes of the present invention, the ENDOD1 endonuclease inhibitory drug can be any substance that has an inhibitory effect on the gene expression, activity or function of the ENDOD1 endonuclease, and the SSA enhancer can be an up-regulated single-stranded DNA fusion Any substance that restores (SSA) function, including but not limited to antibodies, organic or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, and inorganic substances.

The above-mentioned use of the present invention further includes using the ENDOD1 endonuclease inhibitory drug or SSA enhancer in combination with another or more anti-tumor drugs or radiotherapy or forming a composite composition.

In the above application of the present invention, the other or more anti-tumor drugs are selected from chemotherapy drugs, polypeptide drugs, antibody drugs, TKI (kinase inhibitors), small nucleic acid and protein drugs. Preferably, the chemotherapeutic drugs are selected from platinum, mitomycin C, camptothecin, PARP inhibitors, DNAPK inhibitors, radioisotopes, vinca alkaloids, anti-tumor alkylating drugs, monoclonal antibodies and anti- metabolize drugs.

In the above-mentioned use of the present invention, the combined use refers to the simultaneous or sequential administration of ENDOD1 endonuclease inhibitory drugs or SSA enhancers and another or more anti-tumor agents.

In the above application of the present invention, the DNA response-deficient tumors include gene mutations or functional defects of ATM, ATR, CHK1, CHK2, and DNAPK, or overexpression of CDC25A, CDC25B, CDC25C, Cyclin E, Cyclin B1, and Cyclin D1 characteristic tumors.

In some specific embodiments, a method for treating or preventing tumors of the present invention comprises administering an effective amount of an ENDOD1 endonuclease inhibitory drug or an SSA enhancer to the subject, and the inhibitory drug can inhibit ENDOD1 endonuclease The gene expression, activity or function of Dicer, the SSA enhancer can up-regulate the single-strand DNA fusion repair (SSA) function, and the tumor has gene mutation, gene expression or function defect of DNA damage repair. Preferably, the DNA damage repair defect includes homologous recombination repair defect, base excision repair defect, nucleotide excision defect, single-strand DNA break repair defect, and a gene mutation or gene related to the repair mechanism Expression and function deficits. The ENDOD1 endonuclease inhibitory drug is any substance that can inhibit the function of ENDOD1 endonuclease, and the SSA enhancer can be any substance that up-regulates single-strand DNA fusion repair (SSA) function.

In some specific embodiments, a pharmaceutical composition for treating or preventing tumors of the present invention includes an ENDOD1 endonuclease inhibitory drug or an SSA enhancer, and the ENDOD1 inhibitory drug can inhibit the gene of the ENDOD1 endonuclease Expression, activity or function, the SSA enhancer can up-regulate the single-strand DNA fusion repair (SSA) function, and the tumor has gene mutation, gene expression or function defect of tumor suppressor gene. Preferably, the tumor suppressor gene expression or functional defects include gene mutations or gene expression and functional defects of genes or proteins such as TP53, PTEN, CDKN2A, APC, p16INK4A, p14ARF, LKB1, FBXW.

In the above-mentioned pharmaceutical composition, the ENDOD1 endonuclease inhibitory drug can be any substance that has an inhibitory effect on the activity or function of ENDOD1 endonuclease, specifically including but not limited to antibodies, organic compounds or natural compounds, small nucleic acid , small ribonucleic acid, protein, polypeptide, antisense acid and inorganic substances and other substances.

In the above-mentioned pharmaceutical composition, the SSA enhancer can be any substance that up-regulates the function of single-strand DNA fusion repair (SSA), specifically including but not limited to antibodies, organic compounds or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides , antisense acid and inorganic substances and other substances.

In another embodiment, the present invention provides a method for treating and preventing hepatitis B virus infection, comprising administering an effective dose of ENDOD1 endonuclease inhibitory drug or SSA enhancer to the subject, the endonuclease The enzyme is an endonuclease encoded by the ENDOD1 endonuclease gene.

The above-mentioned treatment method or pharmaceutical composition also includes using in combination with or forming a composition with another or more antiviral treatment methods. The one or more antiviral treatment methods include antiviral metabolic drug therapy, gene therapy, DNA therapy, RNA therapy, targeted therapy, adjuvant therapy and/or immunotherapy. The endonuclease is an endonuclease encoded by the ENDOD1 endonuclease gene, and the ENDOD1 endonuclease inhibitory drug can be any substance that inhibits the activity or function of the ENDOD1 endonuclease, so The SSA enhancer can be any substance that up-regulates the function of single-strand DNA fusion repair (SSA), specifically including but not limited to antibodies, organic compounds or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids and inorganic substances and other substances.

In another specific embodiment, the present invention provides a pharmaceutical composition for the treatment and prevention of hepatitis B virus infection, containing an effective dose of ENDOD1 endonuclease inhibitory drugs or SSA enhancers, and pharmaceutical excipients, The endonuclease is an endonuclease encoded by the ENDOD1 endonuclease gene. Preferably, the ENDOD1 endonuclease inhibitory drug can be any substance that inhibits the activity or function of ENDOD1 endonuclease, and the SSA enhancer can be any substance that up-regulates single-strand DNA fusion repair (SSA) function , including but not limited to antibodies, organic or natural compounds, small nucleic acids, small RNAs, proteins, polypeptides, antisense acids, and inorganic substances.

In the above-mentioned pharmaceutical composition of the present invention, the ENDOD1 endonuclease inhibitory drug or SSA enhancer includes small molecule compounds, antibodies, polypeptides or antisense compounds.

In yet another embodiment, the use of ENDOD1 endonuclease inhibitory drugs or SSA enhancers in the manufacture of medicines for the treatment and prevention of hepatitis B virus infection, the endonuclease is encoded by the ENDOD1 endonuclease gene endonuclease. It may further include using ENDOD1 endonuclease inhibitory drugs or SSA enhancers simultaneously or sequentially in combination with other antiviral treatments.

In the above method, the ENDOD1 endonuclease inhibitory drug acts to inhibit the expression level of ENDOD1 endonuclease and/or its regulatory gene, inhibits the endonuclease activity of ENDOD1 endonuclease, and blocks its DNA Repair activity, destroy its subcellular localization, destroy its post-translational modification of protein degradation, ubiquitination, phosphorylation, or interfere with the biological functions of ENDOD1 endonuclease and its regulatory protein cleavage and signal peptide maturation.

In the above method, the SSA enhancer can up-regulate the single-strand DNA fusion repair (SSA) function, enhance the recruitment of RAD52, MRE11, and RAD54B at the DNA break site, and enhance the binding of RAD52 to the end of the DNA break point and single-stranded DNA , enhance the combination of RAD52 and RPA70, RPA32, or enhance RAD52 to promote the fusion of homologous sequences on single-stranded DNA.

The present invention also provides a method for screening anti-tumor drugs or anti-hepatitis B infection drugs, comprising contacting the screening target substance with cells expressing ENDOD1 endonuclease, and selecting for inhibition of the activity, function or expression of ENDOD1 endonuclease active substance.

The present invention also provides a method for screening anti-tumor drugs or anti-hepatitis B infection drugs, including contacting the screening target substance with target cells, and selecting the one that has an up-regulation effect on the activity, function or expression of single-stranded DNA fusion (SSA) repair factors substance.

Homologous genes of human ENDOD1 endonuclease (NCBI Gene ID, 23052) widely exist in higher eukaryotes, but not in lower eukaryotes such as yeast, Drosophila and nematodes.

The human ENDOD1 endonuclease gene encodes a protein product of 500 amino acid residues (AA), which belongs to the His-Cys endonuclease family. Its ENDOD1 endonuclease protein has five isoforms, which are produced due to post-translational cleavage, signal peptide cleavage and ubiquitination modification of ENDOD1 endonuclease.

The human ENDOD1 endonuclease localizes to the nuclear membrane and nucleus in normally growing cells. After DNA damage, ENDOD1 endonuclease can enter DNA breakpoints to form aggregation points, tightly bind to chromatin, and recruit around 2.5Kb of DNA breakpoints.

Human ENDOD1 endonuclease participates in DNA repair, promotes non-homologous end joining (NHEJ) and homologous recombination (HR) repair functions, and more importantly, inhibits the function of single-strand DNA fusion (SSA). The function of ENDOD1 endonuclease to inhibit SSA requires the participation of protein (RAD52), and ENDOD1 endonuclease can also promote the accumulation of NHEJ factors (γH2AX and 53BP1, etc.) and HR factors (BLM, MRE11, etc.) at the damage site. The function of ENDOD1 endonuclease to regulate DNA damage repair depends on its endonuclease activity.

ENDOD1 endonuclease has endonuclease activity and can cut DNA molecules with structural damage, such as ultraviolet damage, DNA double-strand breaks (5' and 3' overhang), oxidative damage (hydrogen peroxide treatment), etc.

ENDOD1 endonuclease gene expression or functional inhibition is combined with multiple DNA damage response defects, including homologous recombination-related proteins, specifically including but not limited to CHK1, ATM, CHK2, DNAPK, etc.

Inhibition of ENDOD1 endonuclease gene expression or function is lethal in combination with defects in multiple DNA damage repair functions, including BRCA1, BRCA2, MRE11, ARID1A, ARID1B, CTIP, EXO1, FANC, and WDR70.

Simultaneous inhibition of ENDOD1 endonuclease and DNA damage repair functions (such as BRCA1) can lead to more severe DNA repair defects.

Inhibition of ENDOD1 endonuclease function combined with TP53, PTEN and other tumor suppressor genes is lethal.

The ENDOD1 endonuclease is a good tumor-specific therapeutic target for the following reasons:

1) ENDOD1 endonuclease gene expression or functional inhibition has no effect on the proliferation of normal non-tumor cells (such as RPE-1, MRC-5 and L02).

2) ENDOD1 endonuclease gene expression or functional inhibition can effectively kill tumor cells from various tissues, accounting for 76% (19/25) of the tested tumor cell lines.

3) ENDOD1 endonuclease gene expression or functional inhibition can effectively kill tumor cells with DNA repair gene mutations or functional defects.

4) ENDOD1 endonuclease gene expression or functional inhibition can effectively kill tumor cells with TP53 tumor suppressor gene mutation or functional defect.

5) ENDOD1 endonuclease gene expression or functional inhibition can synergistically enhance the antitumor activity of conventional radiotherapy and chemotherapy, while drug treatment under the same conditions has no significant cytotoxic effect on normal cells.

6) In addition to inhibiting the expression level of the ENDOD1 endonuclease gene, specifically eliminating the nuclease active region of the ENDOD1 endonuclease can also effectively kill tumor cells.

One of the examples illustrates that patients with DNA homologous recombination defects, DNA damage response defects, DNA repair defects, TP53 and other inhibitory drugs can be specifically treated or prevented by administering sufficient doses of ENDOD1 endonuclease inhibitory drugs to the treated subjects. Tumors with defective gene mutations, gene expression, or function of oncogenes, but have no killing effect on normal non-tumor cells. The same principle is also applicable to drugs that can increase the activity of SSA (SSA enhancers), because it can be predicted that ENDOD1 inhibitory drugs can effectively enhance the activity of SSA, and killing tumors depends on the increase of SSA activity, so it can be inferred that other SSA enhancers also should have the same therapeutic effect.

The present invention finds that to maintain the survival of tumor cells, the expression or activity of ENDOD1 endonuclease and the functions of tumor suppressor genes TP53 and PTEN cannot be lost at the same time, and the increase of single-strand fusion repair (SSA) activity cannot simultaneously lose tumor suppressor genes TP53 and PTEN and other functions. Silencing ENDOD1 endonuclease expression gene, or enhancing single-strand fusion repair (SSA), can significantly lead to the rapid death of TP53, PTEN and other deficient tumor cells. Consistent with the cytological results, mouse models of TP53-deficient tumors were completely suppressed by in vivo silencing of the ENDOD1 endonuclease gene. TP53, PTEN and other tumor suppressor gene deficient tumors include any reduction, slowdown, damage and prevention of TP53 gene expression or protein level reduction, as well as defects in the normal function of tumor suppressor genes, including deletion, point mutation, frameshift mutation, gene expression reduction (gene site methylation) and other variations. These mutations are those detectable by any formal sequencing assay. Without being bound by any definitive theory, inhibition of the ENDOD1 endonuclease protein or enhancement of single-strand fusion repair (SSA) function can enhance tumor cell death in cancers deficient in TP53, PTEN, and other tumor suppressor genes.

The present invention also finds that to maintain the survival of tumor cells, the ENDOD1 endonuclease activity and the function of homologous recombination repair genes (such as BRCA1, BRCA2, WDR70 and FANC, etc.) cannot be lost simultaneously, and the single-strand fusion repair (SSA) activity cannot be increased. Homologous recombination repair is also lost. Silencing the ENDOD1 endonuclease protein expression gene can significantly lead to the rapid death of homologous recombination-deficient tumor cells. Consistent with the cytological results, mouse models of homologous recombination-deficient tumors were completely suppressed by in vivo silencing of the ENDOD1 endonuclease gene. Homologous recombination-deficient tumors include any defects that reduce, slow down, damage and prevent homologous recombination repair gene expression or protein levels, and perform the normal repair function of homologous recombination, including deletions, point mutations, frameshift mutations, gene expression reductions (gene site methylation) and other variations. These mutations are those detectable by any formal sequencing assay. Without being bound by any definitive theory, inhibition of the ENDOD1 endonuclease protein or enhancement of single-strand fusion repair (SSA) function may enhance homologous recombination-deficient tumor cell death.

Human tumor cells harboring an integrated hepatitis B virus (HBV) genome or expressing HBx sensitive to inhibition of ENDOD1 endonuclease function disrupted the function of a homologous recombination repair factor (CRL4WDR70) (see: Ren, L., et al., The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B. Hepatology, 2019.69). Silencing the ENDOD1 endonuclease protein expression gene can significantly lead to the rapid death of HBV positive or HBx expressing cells; while inhibiting the function of ENDOD1 endonuclease has no equivalent toxic effect on liver cells that are negative for hepatitis B virus and do not express the HBx gene. Consistent with the cytological results, tumor models of HBV-positive cells were completely suppressed by in vivo silencing of the ENDOD1 endonuclease gene. Silencing ENDOD1 endonuclease gene expression can effectively reduce the titer and viral load of HBV antigen in mouse serum.

The therapeutic range of ENDOD1 endonuclease inhibitory drugs or SSA enhancers includes human lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer (viral or non-viral liver cancer), ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer Carcinoma, melanoma, endometrial cancer, glioma, sarcoma and cholangiocarcinoma and other diseases, as well as abnormal physiological indicators caused by tumor growth, respiratory system, circulatory system, nervous system, digestive system, hematopoietic system, motor system symptoms.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers can treat tumors with homologous recombination deficiency in the above tumor types.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers can treat tumors with mutations or functional defects in tumor suppressor genes such as TP53 and PTEN among the above tumor types.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers can treat tumors that are not sensitive to radiation and chemotherapy in the above tumor types.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers can treat tumors with significant expression of the ENDOD1 endonuclease gene and single-strand fusion repair (SSA) factors in the above tumor types.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers are suitable for tumors, including but not limited to breast cancer with homologous recombination repair defects (BRCA1, BRCA2, PTIP, ARID1A, ARID1B, Fanconi gene, WDR70, etc.) , ovarian cancer, prostate cancer, gastric cancer and osteosarcoma and other tumors.

ENDOD1 endonuclease inhibitors or SSA enhancers are applicable to tumors, including but not limited to colorectal cancer and other tumors with base excision repair defects (MLH1, MSH2 mutations or functional defects).

The range of tumors for which ENDOD1 endonuclease inhibitory drugs or SSA enhancers are applicable includes, but is not limited to, skin cancers with nucleotide excision repair defects (XPA, XPB, XPC, XPD, XPE, and XPF mutations or functional defects) and Tumors such as melanoma.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers are applicable to tumors, including but not limited to HBV-positive or HBx-expressing virus infection, liver cancer and cholangiocarcinoma, and hepatobiliary diseases caused by HBV virus infection.

ENDOD1 endonuclease inhibitory drugs or SSA enhancers are suitable for tumor range, and other types of tumors such as lung cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, glioma, etc. These tumors have DNA damage response and repair defects, including but not limited to tumors with mutations in the following DNA damage response and DNA repair genes, such as BRCA1, BRCA2, PTIP, CHK1, CHK2, ATM, ATR, MLH1, MSH2, WDR70, FANCA, FANCC, FANCD2, FANCF, EMSY, XPA, XPB, XPC, XPD, XPE, XPF, DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, etc.; or tumor suppressor gene mutation Such as TP53, PTEN, CDKN2A, APC, p16INK4A, p14ARF, LKB1, FBXW, VHL and WT-1, etc.

Another object of the present invention can be used to reduce or eradicate HBV-infected cells or hepatitis cells expressing HBx gene, reduce the serum virus antigen titer and viral load of HBV carrier patients, and reduce infectivity. The method comprises administering to the patient an effective amount of an ENDOD1 endonuclease inhibitory agent or an SSA enhancer.

Here, the above-mentioned ENDOD1 endonuclease inhibitory drugs include any agent that can reduce, reduce, hinder, damage and prevent ENDOD1 endonuclease activity in cells relative to the control solvent; or any agent that can reduce or reduce ENDOD1 Agents of endonuclease gene expression levels and protein levels. In particular, the ENDOD1 endonuclease protein includes different protein activity regulatory regions, so related drugs should also include agents that can inhibit the activity of this region. The protein active region includes signal peptide processing, protein cleavage, subunits of the nucleus, Golgi and nuclear membrane Cellular localization, and ENDOD1 endonuclease degradation and ubiquitination, etc. Specifically, these drugs also include agents that can reduce, slow down, damage and prevent the ENDOD1 endonuclease activity protein region.

The target of ENDOD1 endonuclease inhibitory drugs is to inhibit the function of ENDOD1 endonuclease, including reducing or completely eliminating ENDOD1 endonuclease gene expression and/or protein level, or inhibiting its endonuclease activity, interfering with Its DNA damage repair function blocks its protein cleavage, processing and maturation, destroys its subcellular localization, destroys its ubiquitination regulation, or interferes with biological functions such as protein cleavage and maturation of ENDOD1 endonuclease. These drugs also include agents that reduce, slow down, impair or prevent the active protein region of the ENDOD1 endonuclease.

Here, the above-mentioned SSA enhancer includes any agent that can up-regulate and improve the activity of single-strand fusion repair (SSA) in cells relative to the control solvent; or any agent that can up-regulate and reduce or reduce single-strand fusion repair (SSA) gene expression level and protein level agents. In particular, SSA enhancers include different regulatory regions of protein activity, so related drugs should also include agents that can enhance the activity of this region, including protein maturation, nucleus, subcellular localization of chromatin and DNA breakpoints, and SSA regulation. Phosphorylation, acetylation, degradation and ubiquitination of factors. Specifically, these drugs also include agents that can affect the active protein regions of single-strand DNA fusion repair (SSA) such as RAD52, MRE11 and RAD54B. ENDOD1 endonuclease inhibitory drugs or SSA enhancers can be small molecules, antibodies and antibody fragments, gene editing, polypeptides, and antisense compositions in any form.

Specifically, the ENDOD1 endonuclease inhibitory drugs can be a class of molecules targeting ENDOD1 endonuclease mRNA and DNA, which function to reduce and prevent the expression of ENDOD1 endonuclease protein. Specifically, ENDOD1 endonuclease-related drugs can interfere with the nucleic acid sequence of DNA or RNA of ENDOD1 endonuclease, such as siRNA, shRNA, dsRNA, miRNA, amiRNA, antisense oligonucleotides, or use CRISPR/CAS9 or Derivative methods enable gene editing or gene knockout to reduce the expression level of the ENDOD1 endonuclease or its regulatory genes. Such drugs may be in modified or unmodified form. Such modified drugs may contain modified nucleotides, sugar groups, modified backbone carriers and any previous modifications. For example, modifications include, but are not limited to, R2'O-Me modification, nucleotide 2'-fluoro (2'-F) modification, substitution of nucleotide analogues, and backbone phosphorylation modification.

The present invention also provides an siRNA or shRNA that inhibits the expression of the ENDOD1 gene, which is a single-stranded or double-stranded RNA molecule of 12 to 100 nucleotides, one of which contains 12 to 24 continuous or discontinuous ENDOD1 gene A sequence of nucleotide matches, preferably, the siRNA that inhibits the expression of the ENDOD1 gene is a single-stranded or double-stranded RNA with a length of 18 to 28 nucleotides, more preferably, the siRNA that inhibits the expression of the ENDOD1 gene , whose nucleotide sequence is selected from the group consisting of SEQID No.2-No.353 sequences.

The present invention also identifies nucleotide targeting sequences that disrupt ENDOD1 endonuclease genome structure and gene expression by CRISPR/CAS9, including but not limited to:

5'-CCAGCGCGAGCCAGCGCGCGG-3'

5'-CAGCCTCTTCGCCCTGGCTGG-3'

5'-TGTCACATTCGCCAAAGCCGG-3'

5'-CCCGGGTGCTGTAGAGGGTGG-3'

The above-mentioned ENDOD1 endonuclease inhibitory drug or SSA enhancer, including the composition contained therein, is used to treat tumors and related diseases. ENDOD1 endonuclease inhibitory drugs or SSA enhancers can be used in combination with other anti-tumor treatments with synergistic effects (such as anti-cancer drugs, surgery, transplantation and radiation therapy). Further, the synergistic use with other drugs includes all agents and administration methods that can increase the anticancer effect when used in combination. The above-mentioned synergistic agents include any cancer treatment methods, including but not limited to agents, therapeutic combinations, and medication techniques (such as surgery, radiotherapy, etc.). Specific anticancer therapies include, but are not limited to, surgery, radiotherapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, and immunotherapy in combination and alone.

The combined drug can be that the two drugs are taken at intervals or at the same time, or the ENDOD1 endonuclease inhibitory drug or SSA enhancer and other targeted anticancer drugs form a pharmaceutical composition.

The combination or composition of the above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancers, further including platinums (including but not limited to cisplatin, carboplatin, oxaliplatin, setterbo, picoplatin, neda Platinum, Triplatin, Lipoplatin), camptothecin, mitomycin and other DNA toxic anticancer drugs in combination. The combined medication can be taken together, or taken at intervals one after the other.

The combination or composition of the above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancers, further including PARP inhibitors, DNAPK inhibitors, HDAC inhibitors, ATMi inhibitors, vinca alkaloids, anti-tumor alkaloids, single Anti- and antimetabolite drugs. The combined medicine can be taken together, or taken at intervals one after the other.

The combination or composition of the above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancers, further including combination with radiation therapy, including but not limited to ⁶⁷ Cu, ⁶⁷ Ga, ⁹⁰ Y, ¹³¹ I, ¹⁷⁷ LU, ¹⁸⁶ Re , ¹⁸⁸ Re, α_Particle emitter, ²¹¹ At, ²¹³ Bi, ²²⁵ Ac, Auger-electron emitter, ¹²⁵ I, ²¹² Pb, and ¹¹¹ In. The combined drug can be taken simultaneously with radiation therapy, or taken one after the other at intervals.

Further, examples of anti-tumor alkylating agents include, but are not limited to: nitrogen mustard, cyclophosphamide, methyldioxyethylamine or mustin (HN2), uramustine or uracil, mustard gas, lysate Sarcomatin, chloramphenicol, ifosfamide, bendamustine, nitrosoureas, carmustine, lomustine, streptozotocin, alkylsulfonates, butyl dimethanesulfonate esters, thiotepa, promethazine, hexamethylmelamine, triazine, dacarbazine, mitozolomide, and temozolomide. The combination medicine can be taken together, or taken one after the other at intervals.

Examples of further anti-cancer monoclonal antibodies include, but are not limited to, necitumumab, dinutuximab, nivolumab, blinatumab, pembrolizumab, ramucirumab, obinutuzumab, adotrastuzumab emtansine, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumaxomab, bevacizumab, cetuximab, tositumomab , alemtuzumab, gemtuzumab, trastuzumab and rituximab, vinca alkaloids and derivatives analogues. The combined medication can be taken together, or taken at intervals one after the other.

Further combination or composition of the above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancers, including but not limited to anti-metabolite agents. Examples of agents include, but are not limited to, fluorouracil, cladribine, capecitabine, 6-mercaptopurine, pemetrexed, fludarabine, gemcitabine, hydroxyuria, methotrexate, nelarbine, clofarabine, arabine Glycosides, decitabine, deazaaminopterin, 5-fluorouracil deoxynucleoside, 6-thioguanine.

Further combination or composition of above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancers, including but not limited to cellular immunotherapy, antibody therapy or cytokine therapy. ENDOD1 endonuclease inhibitory drugs work synergistically with immunotherapy. Examples of cellular immunotherapy include, but are not limited to, dendritic cell therapy and Sipuleucel-T. Examples of antibody therapy include, but are not limited to, necitumumab, dinutuximab, nivolumab, blinatumab, pembrolizumab, ramucirumab, obinutuzumab, adotrastuzumab emtansine, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumaxomab, bevacizumab, cetuximab, tositumomabrit-Il31, tiib. Examples of cytokine therapy include, but are not limited to, interferons (e.g., IFNa, IFNp, IFNy, IFNX) and interleukins. In some embodiments, immunotherapy includes one or more immune checkpoint inhibitors. Examples of immune checkpoint proteins include, but are not limited to, CTLA-4 and its ligands CD80 and CD86, PD-1 and its ligands PD-L1 and PD-L2, and 4-1BB. The combination medicine can be taken together, or taken one after the other at intervals.

Further, other examples of anticancer therapies include, but are not limited to, acetate dicarboxylate (e.g., ZYTIGA), ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ado-trastuzumab antancina (e.g., KADCYLA), afatinib diformyl ester (eg, GILOTRIF), aldesleukin (eg, PROLEUKIN), alemtuzumab (eg, CAMPATH), anastrozole (eg, ARIMIDEX), arsenic trioxide (eg, TRISENOX), asparaginase (eg, ERWINAZE), axitinib (eg, INLYTA), azacitidine (eg, MYLOSAR, VIDAZA), BEACOPP, belinostat (eg, BELEODAQ), bendamustine hydrochloride (e.g., TREANDA) BEP, bevacizumab (e.g., AVASTIN), bicalutamide (e.g., CASODEX), bleomycin (e.g., BLENOXANE), blinatumomab (e.g., BLINCYTO), bortezomib (e.g., VELCADE), Bosutinib (eg, BOSULIF), brutuximab (eg, ADCETRIS), busulfan (eg, BUSULFEX, MYLERAN), cabazitaxel (eg, JEVTANA), cabozantinib malate (eg, COMETRIQ), CAF, capecitabine (e.g., XELODA), CAPOX, carboplatin (e.g., PARAPLAT, PARAPLATIN), carboplatin paclitaxel, carfilzomib (e.g., KYPROLIS), carbamycin (e.g., BECENUM, BICNU , CARMUBRIS), cardamycin implants (eg, GLIADEL WAFER, GLIADEL), ceritinib (eg, ZYKADIA), cetuximab (eg, ERBITUX), chloramphenicol (eg, AMBOCHLORIN, AMBOCLORIN, LEUKERAN, LINFOLIZIN ), chloramphenicol, prednisone, CHOP, cisplatin (e.g., PLATINOL, PLATINOL-AQ), clofluridine (e.g., CLOFAREX, CLOLAR), CMF, COPP, COPP-AB V, crizotinib (e.g., XALKORI), CVP, cyclophosphamide (eg, CLAFEN, CYTOXAN, NEOSAR), cytarabine (CYTOSAR-U, TARABINE PFS), dabrafenib (eg, TAFINLAR), dacarbazine (eg, DTIC-DOME), actin Bacteroids (such as COSMEGEN), dasatinib (e.g., SPRYCEL), daunorubicin hydrochloride (e.g., CERUBIDINE), decitabine (e.g., DACOGEN), degarelix, denileukin diftitox (e.g., ONTAK), denosumab (e.g., PROLIA, XGEVA), dinutuximab (e.g., UNITUXIN), docetaxel (e.g., , TAXOTERE), doxorubicin hydrochloride (e.g., ADRIAMYCIN PFS, ADRIAMYCIN RDF), doxorubicin hydrochloride liposome (e.g., DOXIL, DOX-SL, EV ACET, LIPODOX), enzalutamide (e.g., XT ANDI), epirubicin hydrochloride (e.g., ELLENCE), EPOCH, erlotinib hydrochloride (e.g., TARCEVA), etoposide (e.g., TOPOSAR, VEPESID), etoposide phosphate (e.g., ETOPOPHOS), everolimus (e.g., AFINITOR DISPERZ, AFINITOR), exemestane (e.g., AROMASIN), FEC, fludarabine phosphate (e.g., FLUDARA ), fluorouracil (eg, ADRUCIL, EFUDEX, FLUOROPLEX), irinotecan, irinotecan plus bevacizumab, irinotecan plus cetuximab, oxaliplatin triplet chemotherapy, oxaliplatin , FU-LV, fulvestrant (eg, FASLODEX), gefitinib (eg, IRESSA), gefitinib hydrochloride (eg, GEMZAR), gefitinib in combination with cisplatin, gefitinib In combination with oxaliplatin, goserelinacetate (e.g., ZOLADEX), Hyper-CVAD, ibrutinib (e.g., ZEVALIN), ibrutinib (e.g., IMBRUVICA), ICE, idelalisib (e.g., ZYDELIG), ifosfamide (e.g., CYFOS, IFEX, IFOSFAMIDUM), imatinib mesylate (eg, GLEEVEC), imiquimod (eg, ALDARA), ipilimumab (eg, YER VO Y), irinotecan hydr ochloride (e.g., CAMPTOSAR), ixabepilone (e.g., IXEMPRA), lanreotide acetate (e.g., SOMATULINE DEPOT), lapatinib ditosylate (e.g., TYKERB), lenalidomide (e.g., REVLIMID), lenvatinib (e.g., LENVIMA), letrozole (e.g., FEMARA ), leucovorin calcium (e.g., WELLCOVORIN), leuprolide acetate (e.g., LUPRON DEPOT, LUPRON DEPOT-3 MONTH, LUPRON DEPOT-4MONTH, LUPRON DEPOT-PED, LUPRON, VIADUR), liposomal cytarabine (e.g., DEPOCYT), lomustine (e.g., , CEENU), mechlorethamine hydrochloride (e.g., MUSTARGEN), megestrolacetate (e.g., MEGACE), mercaptopurine (e.g., PURINETHOL, PURIXAN), methotrexate (e.g., ABITREXATE, FOLEX PFS, FOLEX, METHOTREXATE LPF, MEXATE, MEXATE-AQ), silk Mitomycin C (e.g., MITOZYTREX, MUTAMYCIN), mitoxantrone hydrochloride, MOPP, nelarabine (e.g., ARRANON), nilotinib (e.g., TASIGNA), nivolumab (e.g., OPDIVO), obinutuzumab (e.g., GAZYVA), OEPA, ofatumumab (e.g., , ARZERRA), OFF, olaparib (eg, LYNPARZA), omacetaxine mepesuccinate (eg, SYNRIBO), OPPA, oxaliplatin (eg, ELOXATIN), paclitaxel (eg, TAXOL), paclitaxel (SYNRIBO), paclitaxel nanoparticles Agents (eg, ABRAXANE), PAD, palbociclib (eg, IBRANCE), pamidronate disodium (eg, AREDIA), panitumumab (eg, VECTIBIX), panobinostat (eg, FARYDAK), pazop anib hydrochloride (eg, VOTRIENT), pegaspargase (eg, ONCASPAR), alfa-2b peginterferon (eg, PEG-INTRON, SYLATRON), pembrolizumab (eg, KEYTRUDA), pemetrexed disodium (e.g., ALIMTA), pertuzumab (e.g., PERJETA), plerixafor (e.g., MOZOBIL), pomalidomide (e.g., POMALYST), ponatinib hydrochloride (e.g., ICLUSIG), pralatrexate (e.g., FOLOTYN), prednisone, procarbazine hydrochloride (e.g., For example, MATULANE), radium 223dichloride (for example, XOFIGO), raloxifene hydrochloride (for example, EVISTA, KEOXIFENE), ramucirumab (for example, CYRAMZA), R-CHOP, recombinant human papillomavirus bivalent vaccine (for example, CERVARIX), recombinant human Papillomavirus (eg, HPV) monovalent vaccine (eg, GARD AS IL 9), nonavalent vaccine (eg, HPV) quadrivalent vaccine (g.g., GARDASIL), alfa-2b recombinant leukocyte interferon (eg, INTRON A), Rui Griffinib (e.g., STIVARGA), rituximab (e.g., RITUXAN), romidepsin (e.g., ISTODAX), ruxolitinib phosphate (e.g., JAKAFI), siltuximab (e.g., SYLVANT), sipuleucel-t (e.g., , PROVENGE), sorafenib tosylate (e.g., NEXAVAR), STANFORD V, sunitinib malate (e.g., SUTENT), TAC, tamoxifen citrate (e.g., NOVALDEX), temozolomide (e.g., eg METHAZOLASTONE, TEMODAR), temsirolimus (eg, TORISEL), thalidomide (eg, SYNOVIR, THALOMID), thiotepa, topotecan hydrochloride (eg, HYCAMTIN), toremifene (eg, FARESTON), tositumomab and radioactive iodine (I131) plus tositumomab (eg, BEXXAR), TPF, trametinib (eg, MEKINIST), trastuzumab (eg, HERCEP TIN), VAMP, vandetanib (eg, CAPRELSA), VEIP, vemurafenib (eg, ZELBORAF), vinblastinesulfate (eg, VELBAN, VELSAR), vincristine sulfate (eg, VINCAS AR PFS), vincristine sulfate liposome (eg, MARQIBO), vinorelbine tartrate (g.g., NAVELBINE), vismodegib (eg, ERIVEDGE), vorinostat (eg, ZOLINZA), XELIRI, XELOX, ziv-aflibercept (eg, ZALTRAP), zoledronic acid (eg, ZOMETA), or any combination thereof.

Furthermore, anticancer therapies can be selected from epigenetic and transcriptional regulators. Examples include but are not limited to DNA alpha transferase inhibitors, histone acetylation inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (eg, taxanes and vinca alkaloids), hormones Receptor modulators (eg, estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors, cell signaling pathway inhibitors, protein stability modulators (eg, protease inhibitors), Hsp90 inhibitors, sugars Corticosteroids, trans retinoids, and other pro-differentiation factors.

Specifically, ENDOD1 endonuclease inhibitory drugs or SSA enhancers can independently participate in any anticancer therapy. Such anticancer therapies include, but are not limited to, surgery, radiation therapy, transplantation (stem cell transplantation, myelosuppression), immunotherapy, and chemotherapy.

Specific tumors that can be applied to the above-mentioned ENDOD1 endonuclease inhibitory drugs or SSA enhancer regimens include but are not limited to lung cancer (e.g., bronchial cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma carcinoma); kidney cancer (eg, nephroblastoma, also known as Wilms tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal carcinoma; anal carcinoma; benign monoclonal gammopathy; gallbladder cancer; bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glial Glioma, glioma (eg, astrocytoma, oligodendroglioma), medulloblastoma); bronchial carcinoma; carcinoid tumor; cervical cancer (eg, adenocarcinoma, squamous carcinoma); gallbladder carcinoma; chordoma; Craniopharyngioma; colorectal cancer (eg, colon, rectal, colorectal adenocarcinoma); connective tissue carcinoma; epithelial carcinoma; ependymoma; endothelial sarcoma (eg, Kaposi's sarcoma, multiple idiopathic hemorrhage endometrial cancer (eg, uterine cancer, uterine sarcoma); esophageal cancer (eg, esophageal adenocarcinoma, Barrett's esophageal adenocarcinoma); Ewing's sarcoma; eye cancer (eg, intraocular melanoma, retinoblastoma) ; eosinophilia; gallbladder cancer; gastric cancer (eg, gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell carcinoma; head and neck cancer (eg, head and neck squamous cell Squamous cell carcinoma), throat cancer (such as laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, throat cancer); heavy chain disease (such as alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharyngeal cancer; Inflammatory myofibroblastic tumor; immune cell amyloidosis; liver cancer (eg, hepatocellular carcinoma (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (eg, systemic mastocytosis); Muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myelodysplastic disease (MPD) (eg, polycythemia vera (PV), essential thrombocythemia (ET), nonprimary myeloid Myelofibrosis (AMM), myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelogenous leukemia (CML), chronic neutrophil leukemia (CNL), hypereosinophilic syndrome (HES), Neuroblastoma; neurofibroma (eg, neurofibromatosis (Nr) type 1 or 2, schwannomatosis); neuroendocrine carcinoma (eg, gastrointestinal neuroendocrine tumor (GEP-NET), benign tumors); osteosarcoma (eg, bone cancer); ovarian cancer (eg, ovarian cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer; intraductal papillary myxoma (IPMN); tumor; penile cancer (eg, penoscrotal Paget's disease); pineal tumor; primitive neuroectodermal tumor; Cell tumors; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; (BCC)); small bowel cancer (eg, appendix carcinoma); soft tissue sarcomas (eg, malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma cancer of the small intestine; cancer of the sweat glands; synovoma; cancer of the testis (eg, seminoma, embryonal carcinoma of the testis); cancer; vaginal cancer; vulvar cancer (eg, Paget's disease of the vulva).

Specifically, in the treatment of cancer with ENDOD1 endonuclease inhibitory drugs or SSA enhancers, "treatment" refers to reversing, alleviating, or delaying the occurrence of cancer or the progression of cancer. Specifically, the treatment can be performed after one or more signs or symptoms of the disease have developed or can be observed; it can also be performed in asymptomatic and signal diseases. For example, susceptible individuals can be treated pre-symptomatically (eg, based on history of symptoms and/or exposure to the pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay and/or prevent relapse.

Specifically, "administration" of ENDOD1 endonuclease inhibitory drugs or SSA enhancers in the treatment of cancer refers to implanting, oral administration, absorption, injection of DO inhibitory drugs or SSA enhancers, or their components, as described herein , inhaled or introduced into the body or outside the body of the subject in any other way.

In the scheme of the present invention, the term "administration" refers to the ENDOD1 endonuclease inhibitory drug or SSA enhancer described herein, or its composition implantation, oral administration, absorption, injection, inhalation or any other way into the treatment inside or outside the subject.

The term "single-strand DNA fusion repair", that is, single strand annealing (SSA), refers to a repair method that mainly relies on RAD52-mediated and requires single-stranded DNA and homologous sequences. Its regulatory pathway also includes factors such as RAD52, MRE11, RAD54B and RPA.

The term "inhibit" refers to the ability of a compound to reduce, slow down, stop and/or prevent the activity of a specific biological process in a cell relative to a carrier. "Control" or "prevention" means that the activity is inhibited, hindered, controlled or prevented by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, compared to the control activity. 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. Or 100%. Specifically, "inhibiting, hindering, controlling or preventing" means that the target expression of the inhibitor is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% compared with the control .45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. Or 100%. Specifically, "inhibit, hinder, control or prevent" means that the target activity (such as biological enzyme activity) of the inhibitor is reduced by at least 5%, 10%, 15%, 20%, 25%, 30% compared with the control , 35%. 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%. 90%. 95%. Or 100%.

"Effective dose" refers to a dose capable of inducing a desired biological response. Such reactions can be understood by conventional techniques in the art. Effective doses of the components of the present invention may vary depending on the following factors: the expected biological endpoint, the pharmacokinetics of the compound, the treatment environment, the mode of administration, the age and health factors of the subject to be treated. Effective dosages include, but are not limited to, dosages necessary to slow, reduce, inhibit, ameliorate, or reverse one or more cancer-related symptoms. Specifically, "effective dose" refers to the dose at which a drug (such as an ENDOD1 inhibitory drug or an SSA enhancer) can cause tumor cells to reduce the expression and/or activity of ENDOD1. The effective dosage range of "reducing the expression or/and activity of ENDOD1" includes any multiple between 2 times and 100 times, and any value between 100% and 1%. Specifically, the effective dose in cancer treatment refers to the dose that achieves the lack of expression of ENDOD1 protein or/and its protein activity in tumor cells.

If two or more inhibitors are administered to the subject, the effective amount may be a combined selected amount. When a first inhibitor is used with a second and optionally a third inhibitor, the selective amount of the first inhibitor may vary. When two or more inhibitors are used simultaneously, the effective amount of each inhibitor may be the same as when used alone or. The effective amount of each agent may be less than the effective amount when used alone, provided that the desired effect can be achieved at lower doses. Specifically, when the subject can better tolerate one or more inhibitors, and higher doses can provide greater therapeutic benefit, the effective amount of each drug may be greater than the effective amount when used alone. Specifically, an effective amount of a compound may vary from about 0.001 mg/kg to about 1000 mg/kg, based on one or more administrations over one or several days (depending on the mode of administration). In certain embodiments, the effective dose varies from about 0.001 mg/kg to about 1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg. From about 0.1 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, from about 10.0 mg/kg to about 150 mg/kg. An appropriate therapeutically effective amount can be determined empirically by one of ordinary skill in the art.

A "subject" or "patient" or "subject" includes, but is not limited to, humans and animals capable of developing cancer, or any disease capable of directly or indirectly involving cancer. Research subjects include mammals. Such as humans, dogs, cows, horses, pigs, sheep, goats, cats, rats, rabbits, mice and genetically modified non-human animals. Specifically, "subject" or "patient" or "subject for treatment" includes companion animals such as dogs, cats, rabbits and rats. Specifically, "subject" or "patient" or "subject for treatment" includes livestock such as cattle, pigs, sheep, goats and rabbits. Specifically, "subject" or "patient" or "subject for treatment" includes purebred or exhibition animals such as horses, pigs, cows and rabbits. In particular, a "subject" or "patient" or "subject for treatment" is a human being, e.g., a human being who is or may be at risk of cancer.

The compounds described in this patent can be administered to a subject in any order. The first therapeutic agent, such as an ENDOD1 inhibitor, can be administered at (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour. 2 hours, 4 hours, 6 hours. 12 hours, 24 hours, 48 hours, 72 hours , 96 hours. 1 week. 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks ago), with or after (eg 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) A second therapeutic agent is administered to the subject. Accordingly, the ENDOD1 inhibitor can be administered separately, sequentially or simultaneously with a second therapeutic agent (such as a chemotherapeutic agent as described herein).

The compounds described herein may be administered by any route, including enteral (eg, oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal. interaction specification. Rectal, intravaginal, intraperitoneal, topical (eg, powder, ointment, salves, and/or drops), mucosal, nasal, oral, sublingual; intratracheal instillation, bronchial instillation, and/or inhalation; and/or oral spray spray, nasal spray and/or aerosol. In particular, routes contemplated include oral, intravenous (eg, systemic intravenous), regional administration via the blood and/or lymphatic supply, and/or direct administration to the site of a lesion or suspected lesion. Specifically, the most suitable route of administration depends on a variety of factors, including the nature of the formulation (e.g., stability in the gastrointestinal environment) and/or the condition of the subject (e.g. whether the subject can tolerate administered orally). The specific amount of compound required (to achieve an effective amount) will vary from subject to subject, depending, for example, on the subject's species, age and health, the severity of side effects or disturbances in an individual's biological system, the nature of the particular compound, the medicine, etc. The desired dosage may be administered three times daily, twice daily, once daily, every other day, every third day, every week, every two weeks, every three weeks or every four weeks. Specifically, the required dose can be administered using multiple administrations (such as two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more times) medicine). Specifically, the effective amount of the compound administered one or more times per day to a 70 kg adult may comprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg. From about 0.001 mg to about 1000 mg, from about 0.01 mg to about 1000 mg, from about 0.1 mg to about 1000 mg, from about 1 mg to about 1000 mg, from about 1 mg to about 100 mg, from about 10 mg to about 1000 mg, or from about 100 mg to about 1000 mg, of the compound per unit dosage form.

Specifically, the compounds provided herein may be sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably Delivered at dose levels from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg of the subject's body weight, more preferably From about 1 mg/kg to about 25 mg/kg, one or more times a day to obtain the desired therapeutic effect. It is to be noted that the dosage ranges as described herein provide guidance for the administration of the provided pharmaceutical ingredients to an adult. The specific amount administered to a child or adolescent can be determined by a doctor or a person skilled in the art, and may be lower than or equal to the amount administered to an adult.

In the present invention, the term "ENDOD1 endonuclease" refers to the gene (NCBI Gene ID, 23052), mRNA (NM_015036.3) and protein (NP_055851.1) of human ENDOD1 endonuclease. .

In the scheme of the present invention, the ENDOD1 endonuclease inhibitory drug can be administered through parenteral routes such as oral or injection, or can also be administered through biological media such as lentivirus and adenovirus.

On the other hand, another object of the present invention is to determine the cell biology screening methods and strategies of ENDOD1 endonuclease inhibitory drugs, including but not limited to the cell biology methods described in the present invention, including ion ray or chemotherapeutic drug treatment , using immunofluorescence or fluorescent fusion protein to measure the aggregation ability of functional proteins in cells at DNA breakpoints, such as ENDOD1 endonuclease, MRE11, gH2AX, 53BP1 and single-stranded DNA generation signal reduction, RAD52, ATM-pSerine1981, BLM, etc. Aggregation signal enhancement.

In yet another aspect, another object of the present invention is to determine the screening methods and strategies for ENDOD1 inhibitory drugs, including the in vitro endonuclease activity assay method of the present invention, the screening method for abnormal function of SSA at the cellular level, and ENDOD1 gene expression inhibitors Screening method. Specific assay signals include but are not limited to fluorescence, biotin, absorbance, chemiluminescence, electrical signals, radiographic imaging, etc. Derivative assay methods include enzyme activity competitive inhibition, flow cytometry, scintillation proximity assay (SPA), fluorescence Polarization detection (FPA), surface plasmon resonance technology, etc.

Description of drawings

Figure 1. Human ENDOD1 endonuclease gene and protein sequence characteristics

(A-B) Coding sequences of mRNA (NM_015036.3) and protein (NP_055851.1) of human ENDOD1 endonuclease gene (23052); (C) protein domain including signal peptide, endonuclease and active site ( Residues C190, C224 and C269), transmembrane region I-III; (D) structure of ENDOD1 endonuclease gene, including 2 exons and 1 intron sequence; (E) ENDOD1 drawn by Genetree software Evolutionary tree of endonuclease genes whose homologs exist in higher eukaryotes but not in lower eukaryotes such as yeast, Drosophila and nematodes;

Figure 2. Characteristics and post-translational modifications of human ENDOD1 endonuclease

(A) CRISPR/CAS9 gene editing of the ENDOD1 endonuclease gene, the gRNA target is shown in A1, and the underlined nucleotides are PAM sequences. A2 shows the structure of the CAS9-gRNA gene editing plasmid used; (B) 3 small interfering RNA sequences (B1) and silencing effects (B2) used for ENDOD1 endonuclease gene silencing, si001 is a commonly used siRNA in this study. The * number shown is a non-specific band; (C) Western blot assay of the protein product of ENDOD1 endonuclease in RPE-1 cells, the main band size is about 55Kd (full length, a). The marked * is a non-specific band; (D) RPE-1 cell line with ENDOD1 endonuclease gene knockout (2F4) or mutation (2F6) obtained by CAS9-gRNA gene editing. 2F4 is completely knocked out, and Western blotting cannot detect any ENDOD1 endonuclease protein bands. 2F6 is a protein processing mutation, and only expresses band a, while bands b and c are missing; (E) Western blot detection of siRNA (si001) gene silenced RPE-1 cells. ENDOD1 endonuclease protein bands a, b, c, d, and e were all weakened; (F) Ubiquitination experiment proved that the protein subtype e of ENDOD1 endonuclease was produced by ubiquitination modification, and the modification methods included Polyubiquitination modification of lysine 48 (K48) and lysine 63 (K63); (G) ENDOD1 endonuclease signal peptide position (residue 22) predicted by bioinformatics software (PrediSci), LEG -RL is a possible protease cleavage site; (H) NetSurfP-2.0 software predicts the low sequence region (disorder) of ENDOD1 endonuclease, and finds that residues 300-325 are potential protease cleavage sites, and residues LEG- The cleavage activity of the RL sequence together produces the b, c, d subtypes of the ENDOD1 endonuclease;

Figure 3. Binding of human ENDOD1 endonuclease to DNA damage sites

(A) Immunofluorescence shows that the ENDOD1 endonuclease is mainly localized in the cytoplasm and nuclear membrane in normal growing cells. After DNA damage (irradiation with ion rays), ENDOD1 endonuclease can enter the DNA breakpoint in 40 minutes to form ENDOD1 endonuclease aggregation point, and persist until 2 hours after irradiation. Nuclear DNA was counterstained with DAPI; (B) Immunofluorescence showed that ENDOD1 endonuclease could also enter DNA breakpoints to form aggregation points after treatment with camptothecin (CPT) and hydroxyurea (HU); (C) Immunofluorescence showed , ENDOD1 endonuclease can co-localize with γH2AX at the DNA damage accumulation point after ion ray irradiation, as shown by the arrow; (D) Chromatin immunoprecipitation experiment proves that ENDOD1 endonuclease is located at a distance of 0.5, Binding at 1 and 2.5Kb; (E) Chromatin immunoprecipitation assay of single-strand DNA binding factor (RPA32) proved that RPA32 has no obvious defect after ENDOD1 endonuclease silencing, but co-expression in homologous recombination repair factor (WDR70) Under silencing conditions, the binding of RPA at DNA breakpoints can be eliminated; (F) Chromatin separation experiments prove that the a, b, c, and d forms of ENDOD1 endonuclease can tightly bind to chromatin;

Figure 4. Human ENDOD1 endonuclease participates in DNA repair

(A) After silencing the ENDOD1 endonuclease gene in RPE-1 cells, I-Sce1 induced DNA breaks on pcDNA3-NHEJ, pcDNA3-HR, and pcDNA3-SSA plasmids to measure DNA breakpoint repair efficiency. ENDOD1 endonuclease gene silencing can slightly inhibit the repair function of non-homologous end joining (NHEJ) and homologous recombination (HR), and more importantly, promote the function of single-strand DNA fusion (SSA); (B) As shown in A, DNA break repair efficiency was tested in RPE-1 wild-type, 2F4 and 2F6 cells; (C) Doxycycline-induced wild-type ENDOD1 endonuclease plasmid was transfected in ENDOD1 endonuclease gene knockout 2F4 cells (Wt), can correct the above repair abnormalities; while the introduction of endonuclease inactive ENDOD1 endonuclease plasmid (ND, nuclease dead; ΔEND: endonuclease deleted) has no correction function. It shows that ENDOD1 endonuclease depends on its endonuclease activity to regulate DNA repair; (D) RPE-1 cells silence ENDOD1 endonuclease and/or SSA functional gene (RAD52), and test DNA break repair efficiency. RAD52 silencing can reverse the hyperfunction of SSA caused by ENDOD1 endonuclease deficiency; (E-F) Immunofluorescence shows that knockdown (E) or silencing (F) of the ENDOD1 endonuclease gene in RPE-1 cells can enhance the binding of RAD52 to DNA breakpoints clustering, indicating that the ENDOD1 endonuclease inhibits SSA repair efficiency by reducing the recruitment of RAD52 to sites of DNA damage. Both 2F4 and 2F6 were defective in RAD52 hyperfunction compared with wild-type RPE-1. E1, F1: Immunofluorescence staining pictures, the white circles indicate the position of the nucleus. E2, F2: counts of RAD52 aggregated at the injury site. Cellular DNA was counterstained with DAPI. Unless otherwise specified, the statistical methods described in the article use the two-tailed T test to verify the P value, and P≤0.05 is considered to have a significant difference. NS: no statistical difference;

Figure 5. The regulation mechanism of ENDOD1 endonuclease on other DNA repair factors

(A) Silencing ENDOD1 endonuclease gene expression inhibits IR-induced aggregation of γH2AX and 53BP1; (B) RPE-1 cells (2F4 and 2F6) with knockout or mutant ENDOD1 endonuclease cannot form IR-induced FK2 Focal point; (C) Silencing ENDOD1 endonuclease gene expression enhances IR-induced ATM autophosphorylation (pS1981) focal point; (D-E) Silencing ENDOD1 endonuclease gene expression inhibits IR-induced BLM (D) and MRE11 (E) gathering point;

Figure 6. Cutting effect of ENDOD1 endonuclease on damaged DNA

(A) ENDOD1 endonuclease-STREP tagged protein purified from BL21 bacteria. Includes the full length (FL) of the ENDOD1 endonuclease, a fragment of residues 22-233 and 1-344. SN: cell lysate; Pu: fraction of purified ENDOD1 endonuclease; (B) 0.5 μg of purified ENDOD1 endonuclease endonuclease domain fragment (22-344 residues) added to 0.37 μg of double-stranded DNA Fragments or DNA/RNA hybrid strands in digestion buffer (100mM NaCl, 50mM Tris-HCl, 10mM MgCl2, 100μg/ml BSA, pH 7.9). DNA double strands include three forms of blunt end (30bp), 3'(28bp) and 5'-overhang(28bp); (C) 0.5 μg of purified ENDOD1 endonuclease protein was added to 0.2 μg of variously treated pEGFP-C3 Plasmid DNA, in vitro digestion reaction (37°C, 16 hours) in restriction buffer. ENDOD1 endonuclease can cleave plasmids with structural damage, such as UV damage, DNA double-strand breaks (5' and 3' overhang), oxidative damage (hydrogen peroxide treatment). HTRA2 purified protein was used as a control without nuclease activity;

Figure 7. ENDOD1 endonuclease gene knockout or silencing does not affect the proliferation of normal non-tumor cells

(A) Normal somatic cells (RPE-1, MRC-5, GES-1 and L02) cells silenced the ENDOD1 endonuclease gene (siEN), and measured its proliferation curve; (B) ENDOD1 endonuclease wild-type and Proliferation curves of gene-edited RPE-1 cells (2F4 and 2F6);

Figure 8. ENDOD1 endonuclease gene silencing inhibits the proliferation of tumor cells

(A-B) The tumor cells shown in (A-B) were transfected with ENDOD1 endonuclease-specific siRNA (si001), and the proliferation rate was measured. Tumor cells show two characteristics of sensitive (A) and insensitive (B). MDA-MB-231, NCI-H1299 and HL60 cells were calculated to calculate the cell proliferation ratio at the end point of the experiment; (C) Part of the test cells were stained with crystal violet at the end point of the experiment, and the dark color showed the remaining living cells; (D) Western blot showed that siENDOD1 was in Silencing effect in insensitive cells (HO8910-PM, SW480, GES-1, L02); (E) Silencing of ENDOD1 in SKOV3 cells, while doxycycline induced ENDOD1 wild-type or nuclease inactivation overexpressing anti-siENDOD1 (ND), detection of cell proliferation multiples; (F) simultaneous transfection of siRNA of ENDOD1 endonuclease and RAD52 in SKOV3 cells, determination of cell proliferation multiples;

Figure 9. Joint lethal effect of ENDOD1 endonuclease and DNA damage response and homologous recombination repair genes

(A) RPE-1 and 2F4 cells were treated with DNA damage response factor inhibitors or gene silencing, and their proliferation ability was determined. Viable cells were stained by crystal violet; (B) ENDOD1 endonuclease was co-silenced with homologous recombination repair genes such as BRCA1, BRCA2, BLM, MRE11, CTIP, EXO1, ARID1A/RID1B, and WDR70, and its proliferation ability and joint lethality were determined Effects; (C) Quantification of joint lethal effects after co-silencing of the ENDOD1 endonuclease and the indicated DNA repair genes. The vertical axis represents the multiplication factor of the cells at the end of the experiment (12 days); (D) the joint lethal effect of 3 different ENDOD1 endonuclease siRNAs and BRCA1 gene co-silencing on RPE-1 cells; (E) knockout cells (2F4 ) to silence BRCA1, FANCD2 or FANCC genes, and determine their combined lethal effects;

Figure 10. The joint lethal effect of ENDOD1 endonuclease and tumor suppressor gene

(A) TP53 gene was silenced in RPE-1 cells or 2F4 cells that silenced ENDOD1 endonuclease, and the surviving cells were stained with crystal violet after 12 days; (B) The tumor cell line with high level of TP53 (HO8910-PM) was simultaneously Silencing of ENDOD1 endonuclease and TP53, determination of proliferation multiples and crystal violet staining at the end of the experiment; (C) Simultaneous transfection of wild-type TP53 expression in TP53 pure and mutant cells (NCI-H1975) with silenced ENDOD1 gene Plasmid (oeTP53), carry out gene complementation, determine the multiplication factor and crystal violet staining at the end point of the experiment; (D) silence the tumor suppressor gene PTEN in RPE-1 and 2F4 cells, determine the multiplication factor and crystal violet staining at the end point of the experiment;

Figure 11. Correlation analysis between tumor cell gene mutation and ENDOD1 endonuclease gene silencing cytotoxicity

(A) Protein levels of ENDOD1 endonuclease in normal and tumor cell lines detected by Western blot; (B) Protein levels of ENDOD1 endonuclease in normal human tissues shown in the Human Protein Atlas database; (C) Expression levels of ENDOD1 endonuclease in Human Protein Atlas database in human tumor tissues from various sources; (D) Effect of ENDOD1 endonuclease gene silencing on tumors with TP53 and other gene mutations (such as BRCA1, BRCA2, etc.) The cytostatic effect was significantly higher than that of non-mutated tumor cells. Differences between groups were verified by two-tailed T-test, and P≤0.05 was considered significant difference;

Figure 12. The sensitization effect of ENDOD1 endonuclease gene silencing on chemotherapy

Normal cells (RPE-1) and tumor cells (A549) were treated with targeted inhibitors such as CPT, cisplatin, and DNAPK after silencing ENDOD1 endonuclease, and their proliferation curves were measured;

Figure 13. The therapeutic effect of ENDOD1 endonuclease gene silencing on tumor-bearing mice

(A) Nude mouse subcutaneous tumor models of SKOV3, HCC1937 and OVCAR-8TR cells were established, and control or ENDOD1 endonuclease-specific siRNA (si001) was injected in vivo twice a week to measure tumor volume. The three cell lines all carry TP53 or BRCA1 gene mutation; (B) the inhibition rate (T/C%) of silencing the ENDOD1 gene on the three tumor models;

Figure 14. Combined lethal effect of ENDOD1 endonuclease function inhibition and active hepatitis B virus on cells

(A) Combined lethal effect of ENDOD1 endonuclease and WDR70 co-silencing on HBV-negative hepatocytes (L02); (B) silencing of the ENDOD1 endonuclease gene in HBV-positive (T43) or negative cells (L02), assay its proliferative capacity. PARP inhibitor (olaparib, 0.5uM) was used as a positive control for T43 cytotoxicity; (C) T43 subcutaneous tumor model was established in nude mice, and the control or ENDOD1 endonuclease siRNA was injected twice a week to measure the tumor volume (C1). The control and ENDOD1-silenced tumors were dissected and isolated at the end of the experiment, showing a group of tumor anatomy samples at the end of the experiment (C2); (D) Nude mice were inoculated with HepG2.2.15 in the tail vein, and the HBV antigens in the serum at the indicated time points were measured by ELISA ( HBsAg) titer; (E) nude mice were inoculated with HepG2.2.15 through the tail vein, and the titer of HBV virus DNA in the serum of the indicated time point was measured by using the method of utilizing fluorescent quantitative PCR to determine the HBV copy number in the blood;

Figure 15. Titration of ENDOD1 endonuclease activity in vitro

Flowchart of screening small molecule inhibitors of ENDOD1 endonuclease activity by competitive inhibition method: add ENDOD1 protein, linear double-stranded DNA substrate and small molecule inhibitor library to a 384-well PCR skirt plate, perform enzyme digestion reaction and add the containing Fluorescent quantitative PCR master mix of primers, and perform fluorescent quantitative PCR to detect the remaining amount of DNA substrate and enzyme cutting efficiency, and screen effective small molecule inhibitors.

detailed description

The following non-limiting examples serve to further clarify and understand the essence of the present invention. It should be understood that variations in the proportions of the components shown and methods of manipulation will be obvious to those skilled in the art and thus fall within the scope of the present invention.

In the process of studying the DNA damage response mechanism, the present invention unexpectedly finds that the endonuclease encoded by the ENDOD1 endonuclease gene has the function of inhibiting the SSA repair mediated by RAD52, and has less impact on other DNA repair mechanisms. According to literature reports, ENDOD1 endonuclease is a tumor suppressor gene, and silencing the function of this gene can promote tumor proliferation (see: Qiu, J., et al., Identification of endonuclease domain-containing 1 as a noveltumor suppressor in prostate cancer. BMC Cancer , 2017.17(1): p.360.). However, our study unexpectedly found that inhibition of ENDOD1 endonuclease function can be strongly associated with DNA damage response defects (such as ATM, CHK1), DNA repair defects (such as BRCA1, BRCA2, MRE11, FANC and hepatitis B virus positivity, etc.) Synthetic lethal effect, can also be synthetically lethal with cells with tumor suppressor gene mutations (such as TP53, PTEN), and can effectively kill tumor cells with these defects; in vivo with the gene mutations or functional defects In tumor models, inhibition of ENDOD1 endonuclease function curbs the progression of these tumors. In contrast, in non-tumor cells with normal DNA damage response, DNA repair mechanisms, and tumor suppressor genes such as TP53, inhibition of ENDOD1 endonuclease function, or even complete knockout of the ENDOD1 endonuclease gene, resulted in no synthetic lethality at all. effect without affecting the proliferation of these cells. Moreover, inhibiting the expression or function of ENDOD1 endonuclease can make tumor cells with or without the above-mentioned defects more sensitive to cytotoxic treatment (radiotherapy or chemotherapy), and has a synergistic sensitization effect, while the same drug treatment has no toxic effect on normal cells . Therefore, these studies prove that ENDOD1 endonuclease can be combined with defects in DNA damage response, DNA repair mechanism and tumor suppressor genes to kill tumor cells, and it is a new broad-spectrum anti-tumor drug target.

The types and sources of the cell lines in the following examples are shown in Table 1, and the sources are donated by ATCC, the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences or the Chengdu Institute of Biology of the Chinese Academy of Sciences). ENDOD1 endonuclease gene-edited RPE-1 cells (2F4 and 2F6) were constructed by Cong Liu’s laboratory at West China Second Hospital, Sichuan University. Nude mice (BALB/c-Nude strain, SPF grade, female, from Experimental Animal Center of Sichuan University) are used in experiments in the present invention.

The bacterial strains used in the examples are common bacterial strains such as DH5a and BL21. The pET28a, pLVX-IRES-ZsGreen1, pcDNA3, pLVX-Tet3G, pLVX-TRE3G and other plasmids used for molecular cloning and cell expression of ENDOD1 endonuclease, as well as various wild-type and mutants were all experimented by Liu Cong, West China Second Hospital, Sichuan University. room manufacturing. The plasmid of the I-Sce1 DNA break induction system was provided by Professor Chen Jun of Zhejiang University. The CRISPR/CAS9 gene editing system used was constructed by Beijing Weishang Lide Biotechnology Co., Ltd. Small interfering RNA (siRNA) was synthesized by Shanghai Ruibo Biotechnology Co., Ltd. Molecular cloning primers were synthesized by Chengdu Qingke Zixi Biotechnology Co., Ltd.

Lipofectamine 3000 (Invitrogen, L3000015), rabbit anti-human ENDOD1 endonuclease antibody (Abcam, Ab121293), RAD52 (LSBio, LS-C176555/122111), ATM-pSerine1981 (Cell Signaling, 526S), FK2 used in the examples (Enzo, BML-PW8810), 53BP1 (bethyl, A300-272A), γH2AX (Millipore, 05-636), MRE11 (Cell Signaling, 4895), BLM (Bethyl, A300-110A-2), BRCA1 (Bethyl, A300 -001A), RPA32-pSerine33 (Novus, NB100-544), cisplatin (131102, Supertrack Bio-pharmaceutical), hydroxycamptothecin (Selleck, S2423), hydroxyurea (Selleck, S1896), ATM inhibitor (Ku55933, Selleck , S1092), MRE11 inhibitor (Mirin, Selleck, S8096), CHK2 inhibitor (BML-277, Selleck, S8632), DNAPK inhibitor (NU7026, Selleck, S2893), CGK733 (Selleck, S7136), ATR inhibitor ( Caffeine, Sigma, C1778-5VL), 4-OHT ((Z)-4-hydroxy tamoxifen) and various other reagents are used in experiments in the present invention. Doses of common reagents: IR, 8Gy; CPT, 0.0125uM; HU, 0.2mM; cisplatin, 2ug/mL; 4-OHT, 300nM. The dose of transfected plasmid is 0.25ug/100,000 cells; the dose of transfected siRNA is 0.2ug/100,000 cells.

The information on the genome mutation sites of the cell lines mentioned in the examples is provided by literature or ATCC.

Hepatitis B virus surface antigen diagnostic kit (S10910113, Shanghai Kehua Bioengineering Co., Ltd.), hepatitis B virus (HBV) nucleic acid amplification (PCR) fluorescence quantitative detection kit (S20030059, Haikehua Bioengineering Co., Ltd. company) and other various reagents are used for experiments in the present invention, and are not used for other purposes.

Table 1. Cell lines and culture conditions used

The gene name, ID, genome and chromosome location involved in the present invention are shown in Table 2.

Table 2. Gene list

The terms or abbreviations used in the following examples are as follows:

IRIF (ionizing radiation-induced foci): radiation-induced focus, that is, DNA damage site;

AA (amino acid): amino acid (residue);

Kb (kilobase): kilobase pairs;

bp(base pair): base pair

ul: microliter;

nM: Nanomole;

uM: micromole;

Kd: kilodalton;

Input: internal control;

ChIP: chromatin immunoprecipitation;

Ub: ubiquitin;

siScramble: random siRNA control;

NHEJ: non-homologous end joining;

HR: homologous recombination;

SSA: single-stranded DNA fusion;

Ctrl: control;

Example 1. Human ENDOD1 endonuclease gene and protein characteristics.

Experimental method: Search the ENDOD1 endonuclease gene and protein sequence through the NCBI database, and use bioinformatics software to draw the phylogenetic tree of the ENDOD1 endonuclease gene in different species and the protein functional domain of the ENDOD1 endonuclease. Use CRISPR/CAS9 technology to edit the ENDOD1 endonuclease gene to obtain ENDOD1 endonuclease gene knockout or mutant cell lines; use siRNA technology to silence the ENDOD1 endonuclease gene to knock down the expression of the ENDOD1 endonuclease gene. Extract the protein from RPE-1 cells, separate the protein by SDS-PAGE electrophoresis, transfer to polyvinylidene fluoride membrane, and use specific rabbit anti-human ENDOD1 endonuclease and goat anti-rabbit HRP secondary antibody to detect ENDOD1 endonuclease protein levels.

CRISPR/CAS9 Gene Editing

Design the gRNA target sequence located in Exon 1 of the ENDOD1 endonuclease gene: 5'-CAGCCTCTTCGCCCTGGCTGG-3', clone it into the pCAG1-CAS9-U6-sgRNA vector, and transfect it into RPE- 1 cell, 48 hours later, add 3 μg/mL puromycin to select transfected positive cells. Positive cell clones were isolated and ENDOD1 endonuclease gene knockout or mutant cell lines were screened by Western blotting.

siRNA interference technology

Design and synthesis of RNA interference (RNAi) targets for the ENDOD1 endonuclease gene:

siENDOD1-001: 5'-GCAAGCGGATTGGCTACAA-3';

siENDOD1-002: 5'-GGATGAAGAACGAATGGTA-3';

siENDOD1-003: 5'-GTGGCCACATTTACTCTCA-3';

The specific implementation of ENDOD1 endonuclease gene intracellular silencing is as follows: Ultrapure water (Invitrogen, 10977-015) dissolves ENDOD1 endonuclease siRNA powder and control siRNA powder (Guangzhou Ruibo), and prepares the stock solution (5nM) in Store at -40°C. The specific preparation of the transfection mixture in each well of the 24-well plate is as follows: 5ul siRNA (5nM) and 3ul Lipofectin 3000 (Invitrogen, L300015) were sequentially added to 30ul serum-free medium opti-MEM, mixed evenly and left to stand for 15min, then evenly added dropwise into a 24-well plate. Add 470ul complete medium to the 24-well plate cells in advance. 24 hours after transfection, the medium was changed to 1ml of complete medium. After 48 hours, the samples were collected, the cells were dissolved in SDS sample buffer, and the silencing efficiency was detected by western blotting.

western blot

Collect an equal amount of cells (about 500,000), wash twice with PBS, and centrifuge at 500x g each time to collect the cell pellet. Add 100 microliters of SDS loading buffer (50mM Tris-HCl [pH6.8], 2% SDS, 0.1% bromophenol blue, 100% glycerol, 5% β-mercaptoethanol) to lyse the cells and treat them in a metal bath at 100°C 5 minutes. Proteins were separated by polyacrylamide gel electrophoresis (80 volts/15 minutes followed by 150 volts/55 minutes) and blotted onto polyvinylidene fluoride membranes (150 mA/3 hours). Place the membrane in blocking buffer (10mM Tris-HCl [pH7.5], 150mM NaCl, 0.1% Tween, 5% BSA) for blocking for 1 hour, add rabbit anti-human ENDOD1 endonuclease antibody (1:1000), After incubation at room temperature for 4 hours, wash with washing buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween) three times, ten minutes each time. Continue to incubate the membrane in blocking buffer containing goat anti-rabbit HRP secondary antibody (1:3000) for 1 hour, wash 3 times, ten minutes each time. The color was developed using ECL luminescent liquid, and the image was collected by BIO-RAD gel imaging system (chemidoc XRS).

Experimental results:

1. The mRNA sequence of the human ENDOD1 endonuclease gene is shown in Figure 1A, and the coding region (ORF) has a total of 1503 nucleotides. The protein sequence of ENDOD1 endonuclease is shown in Figure 1B, with a total of 501 amino acids. ENDOD1 endonuclease protein domain as shown in Figure 1C, including signal peptide (1-22 amino acid residues (AA)), endonuclease domain (49-257AA), 3 transmembrane sequences (I:345 -364AA, II:411-435AA and III:455-482AA). Cysteine C190, C224 and C269 are the conserved active sites of the His-Cys endonuclease family. The ENSEMBL database shows that the ENDOD1 endonuclease genomic locus contains two exons and one intron (Fig. 1D). Genetree software analyzed the evolutionary relationship of the ENDOD1 endonuclease gene in different species, and found that its homologous genes exist in higher eukaryotes such as corals, fish, vertebrates and humans, but not in yeast, fruit flies and nematodes, etc. lower eukaryotes (Fig. 1E).

2. The characteristics and post-translational modification of human ENDOD1 endonuclease.

The protein product of ENDOD1 endonuclease in RPE-1 cells was determined by western blotting ( FIG. 2C ), and the electrophoresis size of its main band was about 55Kd. Protein electrophoresis showed that the ENDOD1 endonuclease protein had four modified or cut isoforms, which were 47Kd (residues 23-500, b), 42Kd (residues 23-340, c), 35Kd (residues 23- 290, d) and the >90Kd covalently modified form (e). and >90Kd covalently modified form (e). After CISPR/CAS9 gene editing (Fig. 2A) or siRNA gene silencing (Fig. 2B), all electrophoretic forms of ENDOD1 endonuclease disappeared or decreased (Fig. 2D-E). The ubiquitin (ubiquitin) or its K48R, K63R mutants and the ENDOD1 endonuclease expression plasmid (Flag-ΔN22-ENDOD1 endonuclease) with the deletion of the signal peptide were transferred into RPE-1 cells, and it was found that the ENDOD1 endonuclease Type e is due to covalent modification by ubiquitination (Fig. 2F). Analyzing the protein structure characteristics of ENDOD1 endonuclease by using bioinformatics software such as PrediSci and NetSurfP-2.0, it was found that residues 300-325 are low-order regions of the protein and potential protease cleavage sites (Figure 2G-H), which can be compared with The cleavage activity of the LEG-RL signal peptide sequence near residue 22 co-generates b, c, d isoforms of the ENDOD1 endonuclease protein.

Example 2. Human ENDOD1 endonuclease is involved in DNA repair.

Experimental methods: Immunofluorescence, chromatin immunoprecipitation (ChIP) and nucleocytoplasmic separation were used to characterize the subcellular localization and distribution of the ENDOD1 endonuclease protein. ChIP experiments were carried out in DiVA cells, and the expression of AsiSI-ER endonuclease was induced by 4-OHT to generate DNA breaks at chromosome Chr1:89,458,595-89,458,603. After 4 hours of 4-OHT induction, the corresponding proteins were precipitated using specific antibodies (anti-ENDOD1 endonuclease or anti-Flag). Protein enrichment levels at DNA break sites were determined by PCR amplification. The cell component separation experiment (cytoplasm/nuclear membrane/chromatin) refers to the method described in the literature (see: Yu, Z., Z. Huang, and M.J.B.P. Lung, Subcellular Fractionation of Cultured Human Cell Lines. Bio Protocol, 2013.3 (9)) , add 0.1% Triton-X100 to dissolve the nuclear membrane and Golgi apparatus step.

Immunofluorescence

Cells were fixed and cultured on glass griddles by conventional cell culture, cells were fixed with 4% PFA for 10 minutes, washed with PBS and then punched with 0.3% Triton/PBS. The primary antibody was diluted proportionally in blocking buffer (2% BSA, 0.3% Triton/PBS), incubated at 37°C for 3 minutes, and then washed twice with PBS. The secondary antibody was diluted proportionally in blocking buffer (2% BSA, 0.3% Triton/PBS), and the cells were incubated at 37°C for 30 minutes and washed twice with PBS. The slides were mounted with DAPI-containing antifade dye (Vector Laboratories) and observed under an upright fluorescent microscope (Olympus, BX51) to collect pictures. The number of foci in the nuclei (DAPI staining) was counted, and the counting results were visualized by Graphpad Prism (v8.4.0.671) software. Differences between groups were performed using a two-tailed T test, and P≤0.05 was considered to be significantly different.

Chromatin immunoprecipitation (ChIP)

About 5 million cells were collected and cross-linked in 1% formaldehyde for 15 min, then 2.5M glycine was added to terminate the cross-linking, washed twice with PBS and resuspended in 300ul ChIP lysate (50mM HEPES[pH 7.4]; 140mM NaCl; 1% TritonX100 ; 0.1% NaDeoxycholate; protease inhibitors), lyse at 4°C for 30 minutes, centrifuge at 10000x g, wash the pellet once in 1ml ChIP lysate, resuspend in 300ul ChIP lysate, and break the DNA at 500bp with a Biorupter ultrasonic breaker About, centrifuge at 21000x g to discard the precipitate, add protein G magnetic beads (invitrogen) after antibody incubation for 1 hour, or directly add anti-Flag M2 magnetic beads (Sigma), and incubate overnight at 4°C. ChIP lysate, ChIP high salt lysate (50mM HEPES [pH 7.4]; 500mM NaCl; 1% Triton X100; 0.1% NaDeoxycholate), ChIP washing solution (10mM Tris [pH 8.0]; 250mM LiCl; 0.5% NP- 40; 0.5% NaDeoxycholate; 1mM EDTA), TE buffer (10mM Tris-HCl [pH 8.0]; 1mM EDTA) to wash the magnetic beads twice, then add ChIP elution buffer (50mM Tris [pH 8.0]; 1% SDS; 10 mM EDTA) at 65° C. for 1 hour, the eluted liquid was purified by a DNA purification kit (Omega) and recovered in 30 ul of water, and 5 ul was taken for quantitative analysis.

nucleoplasm separation

Collect about 2 million cells, wash twice with PBS and resuspend in 200 μL Lysis Solution A (10mM HEPES[pH 7.9], 10mM KCl, 1.5mM MgCl2, 0.34M sucrose, 10% glycerol, 1mM DTT, 0.1% Triton, protease inhibitor) After incubation on ice for 5 minutes, centrifuge at 1500x g for 5 minutes, and collect the supernatant and pellet respectively. The supernatant is the cell membrane and cytoplasmic proteins, directly add 50 μL 5X SDS loading buffer to extract the proteins. The pellet was washed once in 1 mL of Triton-free lysate A, centrifuged at 1500x g for 5 minutes, then resuspended in 200 μL of lysate A, incubated on ice for 5-10 minutes, and then centrifuged at 1500x g for 5 minutes. The supernatant part is the nuclear membrane part, and 50 μL of 5X SDS sample buffer is directly added to extract the protein. The pellet was washed once in 1 mL of Triton-free Lysis Solution A, centrifuged at 1500x g for 5 minutes, then resuspended in 200 μL of Lysis Solution B (3mM EDTA, 0.2mM EGTA, 1mM dithiothreitol, protease inhibitor), incubated on ice for 10 minutes, 2000x Centrifuge at g for 5 minutes. The supernatant is soluble nuclear protein, directly add 50μL 5X SDS loading buffer to extract protein. The pellet was washed once in 1 mL of lysate B, centrifuged at 13,000 x g for 1 minute, the supernatant was discarded, and the pellet was dissolved in 100 μL of SDS loading buffer. All samples were treated in a metal bath at 100°C for 5 minutes and detected by western blot. Total: total cell protein, the loading amount is 2.5% of the total; Membrane: nuclear membrane and Golgi apparatus, etc., the loading amount is 10% of the total; Chromatin: chromatin protein, the loading amount is 10% of the total .

Experimental results:

In order to analyze the biological function of ENDOD1 endonuclease, the present invention confirmed through a series of biological experiments that ENDOD1 endonuclease aggregates and activates at DNA damage sites, participates in DNA repair, and ensures the stability of genetic material.

1. Human ENDOD1 endonuclease binds directly to DNA damage sites

When DNA is damaged, the proteins involved in DNA repair will be activated and aggregated to the damaged site. This aggregation can be used to show the localization changes of various signals and repair factors after DNA damage by immunofluorescent staining. Immunofluorescence showed that ENDOD1 endonuclease was mainly localized in the cytoplasm and nuclear membrane in normal growing cells. After DNA damage (irradiation with ion rays), ENDOD1 endonuclease can enter DNA breakpoints within 40 minutes to form ENDOD1 endonuclease aggregation sites, which persist until 2 hours after irradiation (Figure 3A). ENDOD1 endonuclease could also form aggregates after treatment with DNA replication poisons (CPT and HU), suggesting that ENDOD1 endonuclease is also involved in the clearance of DNA replication errors (Fig. 3B). Moreover, the aggregation point of ENDOD1 endonuclease at the DNA break can co-localize with the classical marker protein (γH2AX) at the site of DNA damage (Fig. 3C), indicating that after DNA damage, ENDOD1 endonuclease actively acts as a repair factor Recruitment to DNA breakpoints.

2. ENDOD1 endonuclease directly binds to the end of the DNA breakpoint

In the ChIP experiment, ENDOD1 endonuclease was bound at 0.5Kb, 1Kb and 2.5Kb from the DNA breakpoint (Figure 3D), further demonstrating the direct accumulation of ENDOD1 endonuclease to the end of the DNA damage site. ChIP experiments also demonstrated that one of the functions of ENDOD1 endonuclease at DNA breakpoints is to participate in DNA end resection: although silencing ENDOD1 endonuclease alone did not weaken the single-stranded DNA binding protein (RPA32) at the breakpoint However, in the absence of other terminal back-cutting regulators (such as WDR70), ENDOD1 endonuclease silencing can exacerbate the enrichment of RPA32 to DNA breakpoints, resulting in complete inhibition of RPA32 recruitment (Fig. 3E ). These experiments demonstrate that simultaneous loss of homologous recombination repair and ENDOD1 endonuclease function results in severe DNA repair defects.

Through the separation experiments of cytoplasm-nuclear membrane-chromatin components, it was proved that the a, b, c, and d forms of ENDOD1 endonuclease protein can be tightly combined with chromatin, and the distribution of ENDOD1 endonuclease on chromatin accounts for the total 25% of protein (Fig. 3F).

Taken together, these results suggest that the ENDOD1 endonuclease protein actively responds to and recruits DNA breakpoints after cellular DNA damage, participating in the regulation of DNA repair responses.

Example 3. Human ENDOD1 endonuclease inhibits SSA repair function.

Experimental method: I-Sce1 endonuclease was used to induce DNA breakage (see: Liu, J., et al., Development of novel visual-plus quantitative analysis systems for studying DNA double-strand break repairs in zebrafish. J Genet Genomics, 2012.39(9):p.489-502.), Fluorescent quantitative PCR was used to determine the relative efficiency of HR, NHEJ and SSA repair. Use doxycycline-induced wild-type (pLVX-TRE3G-ENDOD1 endonuclease, ENDOD1 endonuclease) or mutant ENDOD1 endonuclease plasmid (pLVX-TRE3G-ENDOD1 endonuclease loss live, ie ND; pLVX-TRE3G-ENDOD1 endonuclease deletion, ie ΔEND) for functional complementation experiments (complementation) on ENDOD1 endonuclease deficient cells (2F4). After DNA damage (IR), the aggregation ability of various related DNA repair factors at DNA damage sites was determined by immunofluorescence experiments.

Determination of DNA break repair efficiency

Use I-Sce1 endonuclease (NEB) to digest pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids in vitro (16°C, 16 hours), add 2 times the volume of absolute ethanol and 1/20 volume of 3M acetic acid Sodium precipitates DNA. Adjust the recovered DNA to a concentration of 1 μg/μL. The cells were first treated with siRNA or other methods, and 24 hours later, the linearized pcDNA3-NHEJ, pcDNA3-HR, and pcDNA3-SSA plasmids were transfected (Lipo3000) into differently treated cells, and the cells were collected after 24 hours of culture (about 100 10,000), using a high-purity DNA extraction kit (Roche) to recover the total DNA of cells, perform real-time fluorescent quantitative PCR (BIO-RAD), and analyze the efficiency of different repair pathways.

Construction and expression of ENDOD1 endonuclease and its mutant plasmid

The ENDOD1 endonuclease gene and its corresponding mutants ND and ΔEND were obtained by DNA synthesis (Shanghai Sangon Bioengineering Co., Ltd.), and subcloned into the pLVX-TRE3G vector through the EcoR1 site after PCR amplification. The corresponding plasmid and pLVX-Tet3G were co-transfected (Lipo3000) into the cells, and doxycycline (200 ng/mL) was used to induce the expression of the corresponding gene after 24 hours of culture.

Experimental results:

DNA breaks are mainly repaired through three pathways: non-homologous end joining (NHEJ), homologous recombination (HR) and single-strand DNA fusion (SSA), and are regulated by a variety of repair factors. The degree of single-stranded DNA is an important regulatory event in the selection of these repair pathways. On the basis of the findings in Example 2, more experiments have proved that the ENDOD1 endonuclease is directly recruited to the DNA breakpoint and regulates the back cutting of the DNA end, which is an important molecule regulating the selection of the repair pathway.

1. ENDOD1 endonuclease inhibits the SSA repair pathway

pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids were digested with I-Sce1 in vitro to form linearized plasmids with site-specific DNA fragmentation, and transfected into RPE-1 cells that silenced the ENDOD1 endonuclease gene , the repair efficiency of the three pathways of DNA breakpoints can be determined. It was found that ENDOD1 endonuclease gene silencing could slightly inhibit non-homologous end joining (NHEJ) and homologous recombination (HR) repair functions, and more importantly, promote single-strand DNA fusion (SSA) functions (Fig. 4A). Similarly, the repair efficiency of I-Sce1-induced DNA breaks was tested in RPE-1 wild-type, 2F4 and 2F6 cells, and it was also found that the repair efficiency of SSA in ENDOD1 endonuclease-deficient 2F4 and 2F6 cell lines was higher than that of wild-type RPE- 1 cells were significantly elevated (Fig. 4B). These results suggest that the ENDOD1 endonuclease factor can inhibit the repair pathway of SSA at the site of DNA damage.

2. ENDOD1 endonuclease effectively inhibits the SSA repair pathway through its endonuclease function

Transfection of the wild-type ENDOD1 endonuclease plasmid (Wt) that can be induced by doxycycline in 2F4 cells knocked out of the ENDOD1 endonuclease gene can reduce the level of SSA to the level of normal cells; and the introduction of nucleic acid The nickase-inactive ENDOD1 endonuclease mutant plasmids (ND and ΔEND) had no corresponding correction function (Fig. 4C), indicating that ENDOD1 endonuclease inhibited the SSA repair pathway dependent on its endonuclease activity.

3. ENDOD1 endonuclease inhibits SSA function by regulating RAD52

RAD52 is an important protein that regulates SSA repair. In order to analyze the specific function of ENDOD1 endonuclease involved in the SSA pathway, ENDOD1 endonuclease and/or SSA functional gene (RAD52) was silenced in RPE-1 cells, and the two were tested in I- Sce1 induced the repair function after DNA breaks, and found that RAD52 silencing could completely reverse the hyperfunction of SSA caused by ENDOD1 endonuclease deficiency (Figure 4D), indicating that the function of ENDOD1 endonuclease to inhibit SSA was dependent on RAD52.

Immunofluorescence experiments showed that ENDOD1 endonuclease gene knockout (Fig. 4E) or silenced (Fig. 4F) RPE-1 cells showed a higher level of IR-induced RAD52 aggregation, further proving that ENDOD1 endonuclease inhibited The repair function of RAD52 at DNA breakpoints. Compared with wild-type RPE-1, both 2F4 and 2F6 are defective in RAD52 hyperfunction, indicating that in addition to the main band (55Kd) of the ENDOD1 endonuclease protein, its post-translational processing and b, c, d subtypes are also involved Regulation of RAD52 and SSA.

4. The regulatory effect of ENDOD1 endonuclease on other DNA repair factors

Silence the expression of ENDOD1 endonuclease gene in RPE-1 cells for 48 hours and perform IR treatment to measure the aggregation level of various DNA repair factors at the damage site. The aggregation levels of γH2AX/53BP1 (Fig. 5A), FK2 (Fig. 5B), BLM (Fig. 5D) and MRE11 (Fig. 5E) were found to be lower in RPE-1 cells silenced or edited for the ENDOD1 endonuclease than in wild-type cells , while the aggregation ability of ATM autophosphorylation was increased in the defective cells (Fig. 5C). These results indicate that ENDOD1 endonuclease cooperates with MRE11, FK2, BLM, γH2AX and 53BP1 to regulate DNA repair, and antagonizes the autophosphorylation function of ATM.

It can be seen that the main function of ENDOD1 endonuclease is to participate in DNA break repair, cooperate with MRE11, γH2AX and 53BP1, etc. to inhibit the SSA pathway regulated by RAD52, and this process depends on its endonuclease activity.

Example 4, ENDOD1 endonuclease has endonuclease activity.

experimental method:

Expression and purification of ENDOD1 endonuclease-STREP protein

The full length of the ENDOD1 endonuclease gene and its mutants were amplified by PCR and subcloned into the pGEX4T1-2strep vector, and transformed into BL21 Escherichia coli strain. The bacteria carrying the ENDOD1 endonuclease plasmid were cultured at 37°C to the logarithmic growth phase (OD=0.6), 1mM IPTG was added to induce protein expression, and the bacteria were collected by centrifugation after continuing to culture at 18°C for 16 hours. The bacteria were resuspended in NETN lysate (100mM NaCl, 20mM Tris-Cl [pH 8.0], 0.5mM EDTA0.5% Nonidet P-40), and the cells were disrupted by ultrasonication and centrifuged at 21000xg to collect the supernatant. Add Strep-Tactin XT magnetic beads (iba) pre-washed with NETN lysate to the supernatant, incubate at 4°C for 2 hours, discard the supernatant, wash the beads 5 times with NETN lysate, and add SDS loading buffer Extract purified protein. Purified proteins were detected by western blot.

DNA in vitro cleavage reaction

DNA double-stranded fragments (28-30bp) or DNA/RNA hybrid strands (30bp) were synthesized by Shanghai Sangon Bioengineering Co., Ltd. The pEGFP plasmid was cut with restriction endonucleases ApaI, BamHI and SmaI to obtain a plasmid with DNA double-strand breaks (5', 3'overhang and blunt ends), and the plasmid was passed through ultraviolet light (1200J/m ² ) and hydrogen peroxide (0.03% , 1h) treatment to obtain the damaged plasmid. The treated DNA was recovered using a DNA purification kit and subjected to an enzyme digestion reaction. The total reaction volume was 20 microliters: 50mM Tris-HCl [pH 7.9], 100mM NaCl, 10mM MgCl2, 100μg/ml BSA, 0.5μg ENDOD1 protein, 0.2 μg (plasmid) or 0.4 μg (synthetic double-stranded DNA fragments) substrate, digested in vitro at 37°C for 16 hours (plasmids) or 4 hours (synthetic double-stranded DNA fragments), then performed agarose gel or sequencing gel electrophoresis, using Gel -Stain dye to stain DNA and acquire images.

Experimental results: ENDOD1 endonuclease has a classic endonuclease domain. In order to verify its endonuclease activity, we expressed and purified ENDOD1 endonuclease-STREP protein in bacteria, including ENDOD1 endonuclease Full-length (FL) and polypeptide fragments (1-344, 22-344) (Figure 6A), and their enzyme cleavage reactions to different types of DNA were detected. 0.5 μg endonuclease domain fragment (22-344 residues) of ENDOD1 with 0.37 μg DNA double-stranded fragment (blunt-ended, 3' and 5'-overhang) or DNA/RNA hybrid strand in cleavage buffer An in vitro digestion reaction (37°C, 2 hours) was performed. The ENDOD1 fragment can cleave these nucleic acid substrates to form smaller fragments (Fig. 6B). Add 0.5 μg of purified ENDOD1 endonuclease (1-344 residues and full-length (FL)) to 0.2 μg of various pEGFP-C3 plasmid DNA, and perform in vitro digestion reaction in buffer (37°C, 16 hours). The results show that ENDOD1 Endonucleases can cleave DNA molecules with structural damage, such as UV damage, DNA double-strand breaks (5' and 3' overhang), oxidative damage (hydrogen peroxide treatment), but have no cutting effect on intact plasmids (Figure 6C ).

Example 5, ENDOD1 endonuclease gene function inhibition specifically attacks tumor cells, but does not affect the proliferation of normal non-tumor cells.

Experimental method: Knock out or silence the ENDOD1 endonuclease gene in normal or tumor cells, and continuously passage or silence the cells, and measure their proliferation curves. The experimental period for most cells is 21 days. The number of surviving cells was counted and recorded graphically at the indicated time points and at the end of the experiment. The value-added rate was calculated according to the cell count results: value-added rate=(N _t −N ₀ )/N _t *100%, where N _t is the absolute value of the cell count at the end of the experiment, and N ₀ is the initial cell inoculation number. All measured cell line control group and experimental group experimental end-point proliferation rate by two-tailed t-test test. P<0.05 considered significant statistical difference.

Crystal Violet Stain:

Dissolve 10 g of crystal violet (Solarbio, C8470) powder in methanol to prepare a 10% stock solution. At the end of the experiment, the cells were fixed with methanol for 10 minutes and dried in the air. The crystal violet stock solution was diluted 1:10 with PBS to prepare the working solution. After fixation, the cell samples were stained with working solution for 10 min, washed with water, and dried in the air. Observe and scan stored images.

Experimental results:

DNA damage response is an important mechanism for eukaryotic cells to maintain genome stability. When the damage cannot be repaired normally, it will lead to cell death. Example 3 has confirmed that the silencing of the ENDOD1 endonuclease gene leads to abnormal regulation of the SSA pathway by RAD52, affects DNA repair, and may affect cell survival. Therefore, we used cell proliferation assays to determine the effect of ENDOD1 endonuclease on the proliferation of different types of cells, and found that ENDOD1 endonuclease gene silencing had a specific inhibitory effect on tumor cells, but did not affect the survival of normal cells, indicating that ENDOD1 Inhibition of endonuclease function has potential tumor therapeutic effects. The specific results are as follows:

1. Knockout or silencing of the ENDOD1 endonuclease gene does not affect the proliferation of normal non-tumor cells

Normal somatic cells (RPE-1, MRC-5, GES-1 and L02) were measured for their 21-day proliferation curves after silencing or knocking out ENDOD1 endonuclease gene expression (Fig. 7A-B). It can be seen that there is no statistical difference (NS) in the proliferation rate of ENDOD1 endonuclease wild-type and gene function-inhibited non-tumor cells.

2. ENDOD1 endonuclease gene silencing inhibits the proliferation of tumor cells

As shown in Figure 8A, after the ENDOD1 endonuclease was silenced, the proliferation of various tumor cells was completely or partially inhibited, and even cell death was statistically different; Effect (Fig. 8A-C). Moreover, it could be demonstrated that in cells insensitive to ENDOD1 endonuclease (HO8910-PM, SW480, GES-1 and L02), ENDOD1 gene expression could be effectively silenced (Fig. 8D), indicating that these cells were not due to gene transfection The difficulty in showing resistance to siENDOD1 is rather due to other genetic mechanisms showing differences in survival.

3. ENDOD1 endonuclease function reduces toxicity to tumor cells depending on endonuclease activity

Silencing ENDOD1 endonuclease (si001) in SKOV3 cells, while transfecting doxycycline-induced and si001-resistant ENDOD1 endonuclease wild-type or endonuclease mutant (ND), found that ENDOD1 endonuclease The cytotoxicity of si001 could be rescued by the enzyme wild-type plasmid, but not by the ENDOD1 endonuclease plasmid with loss of endonuclease function (Fig. 8E), indicating that the toxicity of ENDOD1 endonuclease to tumor cells requires functional ENDOD1 Endonuclease activity.

4. The toxicity of ENDOD1 endonuclease function to tumor cells depends on the increase of SSA activity

In SKOV3 cells, it was found that simultaneous silencing of RAD52 gene expression could reduce the toxicity of ENDOD1 silencing on SKOV3 (Fig. 8F). It shows that the toxicity of ENDOD1 endonuclease to tumor cells depends on the function of RAD52, and also shows that the hyperactivity of SSA function can mediate the toxicity of ENDOD1 endonuclease to tumor cells.

Therefore, Example 5 illustrates that by administering sufficient doses of ENDOD1 endonuclease inhibitory drugs to the treated subjects, tumors can be specifically treated or prevented, but there is no killing effect on normal non-tumor cells. The same principle also applies to drugs that can increase the activity of SSA (SSA enhancers), because it can be predicted that ENDOD1 inhibitory drugs can effectively enhance the activity of SSA, killing tumors depends on the increase of SSA (such as RAD52) activity, and increase the level of SSA repair Cytotoxic effects similar to inhibition of ENDOD1 endonuclease function or gene expression can also be obtained. Therefore, it can be inferred that other SSA enhancers should also have the same therapeutic effect.

Example 6. Joint lethal effect of ENDOD1 endonuclease with DNA damage response and homologous recombination repair genes.

Experimental method: In RPE-1 and 2F4 cells, ENDOD1 endonuclease was co-silenced with other functional genes, or used in conjunction with enzyme inhibitors, and the cell proliferation ability was measured by cell counting or crystal violet staining.

Experimental results:

Most tumor cells have defects in DNA damage repair. In order to study the mechanism by which ENDOD1 endonuclease gene deletion specifically inhibits tumor cell proliferation, the present invention uses gene silencing or enzyme inhibitors to interfere with DNA damage response or the function of repair factors to simulate tumors. Intracellular DNA response defects, found that ENDOD1 endonuclease gene silencing combined lethality with defects in most DNA damage response factors including ATM, CHK1 and homologous recombination repair factors, the specific results are as follows:

1. ENDOD1 endonuclease gene silencing combined with DNA damage response factor lethality.

Add inhibitors of DNA damage response factors to RPE-1 and 2F4 cells, including ATM kinase inhibitor (Ku55933, 20uM); PIKK inhibitor (CGK733, 1ug/ml); ATR inhibitor (Caffeine, 10ug/ml); DNAPK kinase Inhibitor (NU7026, 1uM); CHK2 kinase inhibitor (BML-277, 1uM); MRE11 nuclease inhibitor (Mirin 20uM); or silence CHK1 expression with siRNA. Live cell staining was performed 12 days later and it was found that ENDOD1 endonuclease gene silencing combined with ATM inhibitor, MRE inhibitor and CHK1 silencing was lethal to RPE-1 cells (Fig. 9A). It is suggested that inhibition of ENDOD1 endonuclease function can produce selective therapeutic effect on tumors with mutation or dysfunction of ATM, MRE11 and CHK1 genes.

Therefore, it can be used to treat or prevent tumors with DNA response defects, including those with ATM, ATR, CHK1, CHK2, DNAPK Gene mutations, gene expression or function defects, or tumors with CDC25A, CDC25B, CDC25C, Cyclin E, Cyclin B1, Cyclin D1 overexpression characteristics.

2. ENDOD1 endonuclease gene silencing combined with homologous recombination repair factors to kill.

In RPE-1 cells, the expression of ENDOD1 endonuclease gene was silenced, and DNA repair genes such as BRCA1, BRCA2, BLM, MRE11, CTIP, EXO1, MSH1, ARID1A/RID1B and WDR70 were silenced at the same time, and their proliferation ability was determined (Fig. 9B-C) . Co-silencing of the ENDOD1 endonuclease with BRCA1, BRCA2, CTIP, EXO1, ARID1A/RID1B, and WDR70 genes showed joint lethality, but no significant combined lethal effect with BLM. In addition, the three specific ENDOD1 endonuclease siRNAs can all produce a joint lethal effect with siBRCA1 (Figure 9D), and the ENDOD1 endonuclease knockout 2F4 cells are also lethal in combination with BRCA1 and FANCD2/FANCC gene silencing (Figure 9E ). The joint lethal effect of ENDOD1 endonuclease and homologous recombination repair factors was further confirmed. These evidences suggest that inhibition of ENDOD1 endonuclease gene expression or protein function can have a selective therapeutic effect on tumors with homologous recombination gene mutations or dysfunctions such as BRCA1, BRCA2, FANC, CTIP, EXO1, ARID1A/RID1B, and WDR70.

Therefore, it can be used to treat or prevent tumors with DNA damage repair defects, including homologous recombination repair defects, base excision repair defects, by administering sufficient doses of ENDOD1 endonuclease inhibitory drugs or SSA enhancers , nucleotide excision defects, single-strand DNA break repair defects, and tumors with genetic mutations or functional defects associated with these repair mechanisms. These tumors include but are not limited to the following characteristics: DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, BRCA1, BRCA2, PTIP, WRN, Fanconi, EMSY, ARID1A, ARID1B and WDR70 and other gene mutations, Defects in gene expression or function; or overexpression of oncogenes that inhibit DNA repair function, such as CDK12; or liver or cholangiocarcinoma with hepatitis B virus infection and viral gene expression.

Example 7. Combined lethal effect of ENDOD1 endonuclease and tumor suppressor gene.

Experimental method: co-silence ENDOD1 endonuclease and TP53 functional gene in RPE-1 or tumor cells, and measure cell proliferation ability. The TP53 expression plasmid was transferred into the tumor cells, and the expression of the ENDOD1 endonuclease gene was silenced simultaneously, and the cell count or crystal violet staining was used to determine the rescue effect of the TP53 gene recovering the function on the cytotoxicity of the ENDOD1 endonuclease.

Experimental results:

1. The joint lethal effect of ENDOD1 endonuclease and tumor suppressor gene TP53

A large number of studies have shown that TP53, PTEN, etc. are important tumor suppressor genes, which are mutated or inactivated in 60-80% of human tumors. To further understand the mechanism by which ENDOD1 endonuclease gene silencing specifically inhibits tumor cell proliferation, ENDOD1 endonuclease and TP53 genes were silenced in RPE-1 wild-type, or ENDOD1 endonuclease gene knockout (2F4) cells Cell survival was determined after silencing TP53 in the medium. It was found that ENDOD1 endonuclease dysfunction resulted in cells that were highly sensitive to loss of TP53 gene function (Fig. 10A). Similarly, simultaneous silencing of ENDOD1 endonuclease and TP53 in a cell line with high levels of TP53 (HO8910-PM) revealed loss of cell viability and proliferation (Fig. 10B). These data suggest that intact ENDOD1 endonuclease gene function is an essential factor for maintaining viability in TP53 mutant or loss-of-function tumor cells.

2. Restoring TP53 function in TP53 mutant cells can inhibit the cytotoxic effect of silencing ENDOD1 endonuclease

Further, the wild-type TP53 was expressed by plasmid transfection, and it was found that the cell line (NCI-H1975) with a homozygous mutation of the TP53 gene and sensitive to ENDOD1 endonuclease gene silencing became insensitive, indicating that the function of TP53 was restored The cytotoxic effect of inhibition of ENDOD1 endonuclease function on tumor cells that lost TP53 function could be effectively abrogated ( FIG. 10C ). These experiments further illustrate that the loss of TP53 gene function directly leads to the sensitivity of tumor cells to the inhibition of ENDOD1 endonuclease function, and also suggests that the inhibition of ENDOD1 endonuclease function can have a selective therapeutic effect on tumors with TP53 gene mutation or dysfunction.

4. Combined lethal effect of other tumor suppressor genes and ENDOD1 endonuclease gene silencing

The PTEN tumor suppressor gene was silenced in RPE-1 and 2F4 cells, and it was found that 2F4 had relatively high sensitivity to PTEN silencing (Fig. 10D). It shows that inhibition of tumor suppressor genes other than TP53 can also be lethal in combination with inhibition of ENDOD1 endonuclease function.

Therefore, it can be used to treat or prevent tumors with TP53, PTEN and other tumor suppressor gene mutations, gene expression or function defects by taking sufficient doses of ENDOD1 endonuclease inhibitory drugs or SSA enhancers to the treated subjects.

Example 8. Correlation analysis between tumor cell gene mutation and ENDOD1 endonuclease gene silencing cytotoxicity.

experimental method:

Protein levels of ENDOD1 endonuclease in human normal and tumor cell lines were detected by immunoblotting. The protein abundance of the ENDOD1 endonuclease in normal and tumor tissues was collected from the Human Protein Atlas database.

Calculation of Proliferation Inhibition Rate

The formula for calculating the inhibition rate of tumor cell proliferation shown in Table 3: inhibition rate=(1-Nt/Nc)%; wherein Nt represents the cell count of the drug group at the end of the experiment, and Nc represents the cell count of the control group at the end of the experiment.

Experimental results:

1. Western blotting detected ENDOD1 endonuclease protein in different tissue sources of normal (RPE-1,) and tumor cell lines (SKOV-3, NCI-H1975, NCI-H1299, HL60, MRC-5, RPE-1 , MDA-MB-231, U2OS, MDA-MB-468, Raji, OVCAR-8TR) were ubiquitously expressed ( FIG. 11A ).

2. The Human Protein Atlas database shows that ENDOD1 endonuclease has a medium to high level of protein expression in most normal tissues (Figure 11B); ENDOD1 endonuclease also has a significant protein expression level in more than half of tumor tissues (Figure 11C) , including glioma, thyroid cancer, rectal cancer, head and neck cancer, gastric cancer, pancreatic cancer, lung cancer, kidney cancer, prostate cancer, breast cancer, endometrial cancer, ovarian cancer, melanoma and skin cancer. The expression ratio of ENDOD1 endonuclease is low in individual tumors (such as testicular cancer and lymphoma).

3. Table 3 shows the cytotoxicity of ENDOD1 endonuclease to 25 kinds of tumor cells tested and the gene mutation carried by the cells. Statistical analysis showed that tumor cells with TP53 or other gene mutations (BRCA1, BRCA2, or with HBV, HPV viruses, etc.) were much more sensitive to ENDOD1 endonuclease gene silencing than those without these mutations Tumor cells (Fig. 11D). SPSS software was further used to analyze the Pearson correlation coefficient between TP53 mutation and other types of tumor gene mutations and ENDOD1 gene silencing inhibition rate, and found that there was a high correlation between the two (PearsonCorrelation: 0.708, p<0.0001), indicating that patients with these mutations Tumor cells are far more sensitive to ENDOD1 gene silencing than non-mutated cells.

Table 3. The relevant mutation characteristics of various human normal and tumor cell lines and the inhibition rate of cell proliferation after ENDOD1 endonuclease gene silencing. (wt: wild type; mu: mutant; null: full deletion mutation; homo: pure and mutant)

Example 9. The sensitization effect of ENDOD1 endonuclease function inhibition on tumor chemotherapy.

experimental method:

10,000 cells (RPE-1 or A549) were inoculated in a 24-well plate, and the ENDOD1 endonuclease gene silencing experiment was performed 24 hours later, and continuous drug treatment was performed (see Figure 12 for drug concentration). At the end of the experiment (14 days), the cell proliferation multiple was calculated.

Experimental results:

Normal cells (RPE-1) silenced by ENDOD1 siRNA were not sensitive to chemotherapeutic drugs, ENDOD1 gene silencing or combined treatment (Fig. 12, left), but tumor cells (A549) were more sensitive to chemotherapeutic drugs, and silencing ENDOD1 Sensitivity to camptothecin (CPT), cisplatin (cisplatin), and DNAPK inhibitor (Nu7062) were greatly improved after endonuclease (Fig. 12, right).

Therefore, administering sufficient doses of ENDOD1 endonuclease inhibitory drugs to the treated subjects can sensitize low-dose chemoradiotherapy for the treatment of tumors that have no effective clinical response to radiation or chemotherapeutic drugs alone. Potential combination therapy drugs include platinum, mitomycin C, camptothecin, PARP inhibitors, DNAPK inhibitors, radioisotopes, vinca alkaloids, anti-tumor alkylating drugs, monoclonal antibodies, antimetabolites . These treatment methods can be used simultaneously or sequentially, reflecting a synergistic effect, so that the therapeutic effect is better than the method of using drugs that inhibit the function of ENDOD1 or using antitumor drugs alone.

Example 10. The therapeutic effect of ENDOD1 endonuclease gene silencing on a mouse tumor-bearing model.

experimental method:

Tumor-bearing model in nude mice

Preclinical animal tumor-bearing experiments were carried out in the Experimental Animal Center of West China Second Hospital of Sichuan University (SPF level). Week-old athymic male nude mice (purchased from Nanjing Model Animal Research Center) were randomly divided into 3 groups after one week of isolated observation. Each mouse was inoculated with tumor cells in the armpit and groin, and the inoculation amount was 4x10 ⁶ .

in vivo gene silencing

When inoculated with a tumor volume of about 100 mm ³ , the ENDOD1 endonuclease gene was silenced in vivo. The specific implementation is as follows: Ultrapure water (invitrogen, 10977-015) dissolves ENDOD1 endonuclease in vivo siRNA powder and control siRNA powder (Guangzhou Ruibo), prepares a working solution (1ug/ul) and stores at -40°C. 10% glucose was prepared with ultrapure water, filtered, and stored at -40°C for later use. In vivo gene silencing was administered by tail vein injection, and the administration frequency was 2 times/week. The injection should be injected immediately after preparation. The injection solution for each mouse was specifically prepared as follows: 50ul of siRNA was mixed with 50ul of 10% glucose to make 1 solution; 25ul of ultrapure water was mixed with 50ul of 10% glucose and 25ul of in vivo transfection reagent (Engreen, 18668-11-1). Make 2 liquids, let stand for 2 minutes, mix 1 liquid and 2 liquids, let stand for 15 minutes.

Tumor Measurements and Statistical Analysis

Tumor size was measured from the start day of administration, and the measurement frequency was 3-5 days. The length and width of the tumor body surface were measured using a vernier caliper. After the experiment, the data was sorted out, and the drug effect was calculated and evaluated according to the following standard according to the "Technical Guidance for Non-clinical Research of Cytotoxic Antineoplastic Drugs": Tumor volume calculation formula: V=1/2×a×b2. Where a and b represent the length and width of the tumor body surface, respectively. Tumor efficacy evaluation formula: relative tumor proliferation rate T/C%=TRTV/CRTV*100%. T/C%<40% can be judged that the treatment method is effective. The lower the T/C% value, the more significant the drug effect. Among them, TRTV means the RTV of the treatment group; CRTV means the RTV of the negative control group. RTV stands for relative tumor volume (RTV), and its calculation formula is: RTV=Vt/V0. Where V0 is the initial tumor volume, and Vt is the tumor volume at the observation time point. In this experiment, Vt is the set experimental endpoint corresponding to the tumor volume of the mouse.

In this embodiment, the difference between the experimental endpoint control group and the experimental group was compared using T-test two-tailed test, and P>0.05 shows that there is a significant difference between the two groups. The experimental period was set to 21 days. According to ethical requirements, when the end point of the 21-day experiment is not reached, the mouse tumor volume is greater than or equal to 2000mm ³ and the mouse must also be killed, and the sample is terminated to continue the experiment.

Experimental results:

The growth of mouse tumors in the ENDOD1 endonuclease silencing experimental group was significantly inhibited compared with the control group. Animal models have effectively proved that ENDOD1 endonuclease gene silencing can significantly inhibit a variety of TP53-deficient (SKOV3, HCC1937, OVCAR-8TR) and BRCA1-deficient (HCC1937, BRCA1 pure and mutant) (Figure 13A), and the inhibition rate (T/C%) were SKOV3, 15.2%; HCC1937, 23.7%; OVCAR-8TR, 25.7% (T/C%≤40% indicates that the drug has a significant effect on the tumor) ( FIG. 13B ).

Therefore, it can be used to treat tumors with corresponding gene, genetic, and protein function defects by administering sufficient doses of ENDOD1 endonuclease inhibitory drugs to the treated subjects.

Example 11. The therapeutic effect of ENDOD1 endonuclease function inhibition on active hepatitis B virus.

Previous findings suggest that the hepatitis B virus-encoded oncogene (HBx) inhibits homologous recombination repair by disrupting the CRL4WDR70 ubiquitinase (see: Ren, L., et al., The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B. Hepatology ,2019.69). In this example, by measuring the sensitivity of HBV-positive cells to ENDOD1 endonuclease gene silencing, it is proved that inhibition of ENDOD1 endonuclease function can control HBV infection indicators.

experimental method:

Cell Proliferation Assay:

50,000 cells were seeded in 24-well plates, and gene silencing experiments were performed 24 hours later. The experimental cycle was set to 21 days, and subculture was performed every 3 days. ENDOD1 endonuclease siRNA transfection experiments were performed 24 hours after each passage. Accurately count and record with a cell counter during subculture, and draw a proliferation curve at the end of the experiment.

HBV positive cell animal model experiments:

For the evaluation of the treatment effect of patients with hepatitis B virus, the detection index of virus copy number and surface antigen (HBsAG) in serum can be adopted.

Three 6-week-old athymic female nude mice were used, and each mouse was inoculated with HepG2.2.15 cells in the armpit and groin to establish the HBV mouse infection model. The number of inoculated cells was 8x10 ⁶ . After cell inoculation, take blood from the tail vein at different time points, and use the hepatitis B virus (HBV) nucleic acid amplification (PCR) fluorescent quantitative detection kit to monitor the changes in the copy number of HBV in the blood, and wait until the copy number rises to the highest value in the serum 4000UI/ml or so began to silence the ENDOD1 endonuclease gene in vivo.

The specific implementation is as follows: ENDOD1 endonuclease in vivo siRNA powder and control siRNA powder (Guangzhou Ruibo) were dissolved in ultrapure water (invitrogen, 10977-015), prepared into a working solution (1ug/ul) and stored at -40°C. 10% glucose was prepared with ultrapure water, filtered, and stored at -40°C for later use. In vivo gene silencing was administered by tail vein injection, and the administration frequency was 2 times/week. The injection should be injected immediately after preparation.

The specific preparation of each mouse injection is as follows: 50ul of siRNA and 50ul of 10% glucose are mixed to make 1 liquid, 25ul of ultrapure water is mixed with 50ul of 10% glucose and 25ul of in vivo transfection reagent (Engreen, 18668-11-1) Make 2 liquids, let stand for 2 minutes, mix 1 liquid and 2 liquids, let stand for 15 minutes.

On days 1, 2, 3, 4, 5, 7, 9, 15, and 20, blood was collected from the tail vein to monitor the copy number of HBV genome in the serum, and ELISA method was used to detect the change of HBsAg titer. After stopping the drug on the 16th day, continue to monitor the copy number and HBsAg titer in the serum for five days. The titer curve was made at the time point of the results. Unless otherwise specified, in this embodiment, the calculation of differences between all groups in cell experiments and zoology experiments adopts t-test two-tailed test. P>0.05 indicates that there is a significant difference between the two groups.

Experimental results:

1. Simultaneous silencing of the combined lethal effect of ENDOD1 endonuclease and WDR70

Cell proliferation curves were determined by silencing ENDOD1 endonuclease and/or WDR70 in normal hepatocytes (L02). It was found that ENDOD1 gene silencing had no obvious toxicity to L02 cells, but it could inhibit cell proliferation in combination with siWDR70 (Fig. 14A). Inhibition of ENDOD1 endonuclease and WDR70 function has a combined lethal effect.

2. Cytotoxic effect of silencing ENDOD1 endonuclease on HBV positive cells

Silencing the expression of ENDOD1 endonuclease gene in hepatocytes (T43) integrating the HBV genome, and measuring the ability of cell clone formation (Fig. L02) colony formation was not affected at all. Nude mice were inoculated subcutaneously with T43 to establish a tumor model, and it was found that ENDOD1 gene silencing could effectively inhibit the growth of T43 tumors (Figure 14C), indicating that inhibiting the function of ENDOD1 endonuclease could eliminate HBV-positive cells with CRL4WDR70 functional defects.

3. Silencing ENDOD1 endonuclease reduces HBV infection indicators

The tail vein of nude mice was inoculated with HepG2.2.15, and the titer of HBV antigen (HBsAg) in the serum at the indicated time points was measured by ELISA method, and the titer of HBV virus DNA in the serum was measured by fluorescent quantitative PCR method. The experiment found that the silencing of ENDOD1 endonuclease in vivo can make the HBV-DNA content and HBsAg antigen OD value in the serum of model mice from ~1.5 (at this time, the HBsAg antigen content in the serum reaches the highest value, indicating that HepG2.2.15 cell liver cancer mice In the model, the virus enters a stable high-level active replication period) and drops to about 0.05, which is close to or lower than the index of clinical negative (>0.05, at which time the OD value is the lowest threshold for detection). Within 5 days after the cessation of ENDOD1 endonuclease silencing in vivo, the HBV copy number and HBsAg expression in mouse serum were always in a stable negative range. The results of these cytology and animal experiments showed that: silencing of ENDOD1 endonuclease in vivo can permanently eliminate HBV virus-infected host cells, suggesting that down-regulating the function of ENDOD1 endonuclease can eliminate HBV or HBx in HBV-infected patients The hepatic cells expressing it can realize a real clinical cure for hepatitis B virus.

Therefore, the hepatitis B virus infection can be eradicated or controlled by taking sufficient doses of ENDOD1 endonuclease inhibitory drugs to the treated subjects to reduce the hepatitis B virus serum antigen index and virus titer. This treatment method can also be used in combination with various antiviral treatment methods or form a composition, such as antiviral metabolic drugs, gene therapy, DNA therapy, RNA therapy, targeted therapy, adjuvant therapy and immunotherapy, etc., these treatment methods can Simultaneous or sequential use, the therapeutic effect is better than that of using drugs that inhibit the function of ENDOD1 or using antiviral drugs alone.

Example 12. Screening process for ENDOD1 endonuclease inhibitors.

experimental method

Screening of small molecule inhibitors of ENDOD1 endonuclease activity by competitive inhibition method. Add 0.1 μg bacterially expressed ENDOD1 protein, 100ng linear double-stranded DNA substrate (sequence:

ATTCTCAGCCTGAAAGCCAGGTTCTAGAGGATGATTCTGGTTCTCACTTCAGTATGCTATCTCGACACCTTCCTAATCTCCAGACGCACAAAGAAAATCCTGTGTTGGATGTTGTGTCCAATCCTGAACAAACAGCTGGAGAAGAACGAGGAGACGGTAATAGTGGGTTCAATGAACATTTGAAAGAA) and a library of small molecule inhibitors or DMSO controls were reacted in Cut Smart 1 buffer for 6 hours at 37°C. Using a mechanical multi-channel pipette (eppendorf), add an equal volume of 2x fluorescent quantitative PCR master mix (Promega, containing specific primers using DNA substrates as templates, the sequence is F: 5'-ATTCTCAGCCTGAAAGCCAGG-3'; R: 5'-TTCTTTCAAATGTTCATTGAA-3'), perform fluorescent quantitative PCR to detect the remaining amount of DNA substrate and enzyme digestion efficiency, and screen effective small molecule inhibitors.

Experimental results

Screening and development of ENDOD1 inhibitors can be achieved through the following aspects: inhibiting the expression level of ENDOD1 and/or its regulatory genes; inhibiting the endonuclease activity of ENDOD1 protein, blocking its DNA repair activity, destroying its subcellular localization, Destroy post-translational modifications such as ubiquitination and phosphorylation; interfere with ENDOD1 and its regulatory protein cleavage and signal peptide maturation and other biological functions.

By inhibiting the endonuclease activity of the ENDOD1 protein, as shown in Figure 15, it is a flow chart for the screening of ENDOD1 inhibitors. The concentration of ENDOD1 protein was decreased by gradient, and the cleavage efficiency was detected by fluorescent quantitative PCR. The remaining amount of DNA substrate should be negatively correlated with the concentration of ENDOD1 protein. This method can be used as an in vitro drug screening system for ENDOD1 endonuclease.

sequence listing

<110> West China Second Hospital of Sichuan University

Chengdu Jiluo Kelin Biotechnology Co., Ltd.

<120> A drug containing the function of inhibiting endonuclease and its anti-tumor application

<160> 357

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4651

<212>DNA

<213> Human (Homo sapiens)

<400> 1

gtagtgcgct ccccgcccag cctgcagagc tcgcgccgcg gcagcccagc cgctcggccc 60

cgccgcgctc gcagaggccg ccatgggcac cgcgcgctgg ctcgcgctgg gcagcctctt 120

cgccctggct gggctgctgg aaggccggct cgtgggcgag gaggaagccg gctttggcga 180

atgtgacaag ttcttctacg ccgggacccc gcctgcgggg ctggcggccg attccacgt 240

gaagatctgt cagcgcgcgg agggtgctga gcgcttcgcc accctctaca gcacccggga 300

ccgcatcccc gtgtactccg cgttccgcgc cccgcgccct gcgcccggcg gcgccgagca 360

gcgatggctg gtggagccgc agatcgatga ccccaacagc aaccttgagg aggcgattaa 420

tgaggcagag gccatcacct ctgtgaacag cctgggaagc aagcaagcct tgaatacaga 480

ttaccttgat tctgattacc aaagaggaca gctttacccca ttctccctta gcagtgatgt 540

ccaggtggcc aatttactc tcacaaattc agccccaatg actcagtcct tccaggaacg 600

gtggtatgtg aatctccaca gcctaatgga ccgggctttg accccacagt gtggcagtgg 660

ggaagaccta tatatcctca caggcacagt gccctcagac tacagagtta aagacaaagt 720

ggcagtccct gagtttgttt ggctggcagc ctgttgtgct gtccctggag gaggctgggc 780

catgggcttt gtcaagcaca cccgggacag tgacatcata gaagatgtga tggtaaaaga 840

tcttcagaaa ctgcttccat ttaaccctca gctgtttcag aacaactgtg gtgaaactga 900

gcaagacaca gagaaaatga aaaaaatcct ggaagtggtt aaccaaatcc aggatgaaga 960

acgaatggta caatctcaaa agagttctag tcccctttct agcaccagga gcaagaggtc 1020

tactctgttg cctccagagg catctgaggg aagtagtagc tttttgggaa aactcatggg 1080

cttcattgct accccattca tcaagctttt tcaattaatt tattaccttg tggtagcaat 1140

cctgaagaac attgtctatt tcctgtggtg tgttaccaag caggtgatta atggcataga 1200

aagttgcctt taccgcctgg gctcagccac catctcatac ttcatggcca ttggggaaga 1260

gttggtgagc attccctgga aggtgctcaa ggtcgtggcc aaagtcatca gggctctcct 1320

ccggatcctt tgttgtctgc tgaaggccat ttgccgagtt ctgagcatcc ctgtccgtgt 1380

ccttgtggat gtggccactt tccctgtgta caccatgggc gctattccaa ttgtttgcaa 1440

ggacattgca ctgggccttg gtggcactgt ctcactgctc tttgacactg cttttggtac 1500

cctgggtggc ctatttcagg tggtttttag tgtctgcaag cggattggct acaaggttac 1560

ttttgacaat tctggggagt tataaactca aaaaactaat agtatccagt cacagtgaat 1620

ttgaaagctg gaatagtttg tctttacaat gggtttctgt tcactgtcag ttatcattat 1680

attttggcct ttggtgggga tgtctgcttg tttttgcaaa agaagatggc agaatttaga 1740

cttgacagag gagaaatgct cagggtgaga ttaggtgtag taatctgctg tttacctcca 1800

gttatatgtg caaactccca agccactaat aacttcagtt atgcactcta acacagacga 1860

ccacctgaaa tgcactggta tttatttctg ataattaaaa attacagggg agggaagaac 1920

tagaaaaaga acaactttag accaaaggtg tctgagaaaa ggagaaaggg agcttgttct 1980

tcccattgct ctttgtgatt taaggcaaaa cagattaaaa aaaaaatctg cagccaattt 2040

cttggcattt gcttctcttt ctcctcaact cgactgacct tggtggaatg cagataatgc 2100

ctctttgttg aaataacttt gtgggaatgt acaattttct atatctctta gctttccgtg 2160

gttctcaagg atatgtacag ttttcatttt cctccaaagt tgaattttgc tacatttttc 2220

ttttaggtaa tgataagcat tttttaaaaa atcatttta ggtaatggta agcattttat 2280

gccaaatgtg gcataacaga gtttgaattg aagggcaaag ttttcttttc tttttttttt 2340

gggccccttg aatggtataa tacagtcctc tccggtggaa agaagagaag agaaggtgga 2400

cagccctgct cttagtaggt gctgcagatc caaggacatc ttttgtccag cttggattaa 2460

cttgacgtgt atccctgcct gacaaggttg aactgaaaga tctatatgtt aagctaacat 2520

gaaaattcat attctgcaac atagtagatt tttctaatgc atgaaataag taccccagcac 2580

agtaaaaata ctctgactta tgtccctaaa tggttgtttt gatacaacta tatagaaaag 2640

agccacaaaa taaagataaa agttattgtg gccatctctg aaaaaaatat ataaaatatt 2700

taagaataat tatatcttag gaaattattt ttacagtgtg tttgaggcta caaacataac 2760

tcccccatta atacaaatta aatgtgagag ctcattctca aaattttttt gatcaagcac 2820

ttgtcatttt aaatcttgca ctaaaaaatg gtaacaagag ggaccaaact ttgcttccca 2880

caattgggat ggaatcacct ggatttttct gaatgtttta aagaattgct gagaggtaga 2940

aacagccaag tatgaaatac tgatcttggg gctaccgccc aggatcaatc agaaagttat 3000

atgcaaaaat tcggggtccc aacaaggaga gaacaatatg tcaaccattt gcatggggtc 3060

gatggatgag aaacatgagc gtagcaagag ttacattttg cagaaaaata gcggaggcac 3120

accccagagta gaacagcagc cagcaagctg ccatcctgat caatttgtga tgcaaggtta 3180

gggagtatga gcaccgcatg ggtccatgct agggagatgt gcaccaggct tagctaagaa 3240

acttaaagca gtattttacc aactcttgtc ttaggggagca taaagtttgg atgtcccttt 3300

atttcagcag tgtgaaggta aatggaaggg tgagggtttg acttggtact gattggtcaa 3360

gaatcctgct ggataaagaa aaggaatttt agaagtgacc agagggacaag tccagccaaa 3420

ctattatttg ataaggaacc caaggccctg ggaggcaaca ggctagccct tagtttcatg 3480

gctagctgag agcagattag gagccaacgt tgtttgcaca tgtcccatca cacctgagat 3540

gtcagacatg ggaagttcgt gctattattc agttgcctct ctggaccatg gcaagatttc 3600

ctcattcatc aaacagactc caggccctga caaagcagtt ggatttggca tgtgtgatga 3660

aaacaattag ccatccatgt acaacattat gcttactgca tcccatggaa acttaattcc 3720

ctcctagcac atatttgcat actgaaaggt ccgaaaaggg catccacggc agcttgagcc 3780

ccttagcacc atgtaaggag cacagcatcc aaacggcttc ttgagaacca ttggggaatg 3840

tctttctttt tcacatccaa ttgtttagtg tctatttatt cttgggtggc cagttttgaa 3900

acctaaaaag ggacaataaa gcaaaaagta tcagtaagga taggtggctg agaccactg 3960

ccctgagcta ctagtgtggc tgtgcctgtg ggtctctaga accatctgca ttggacgtga 4020

agccacagca ggtggctgga ctgctggcct gttcctaatg agctacgctg ggctttgagg 4080

tagaggttgg ggtttatgaa ccccaactct ggcttaaaga tcttgctgtg gctctgttat 4140

gttctgaggc cttgggatta gcctcttcct catttatggag ctgattttct agtctgtgga 4200

tcagctatgc ctttggacac ttctcttttc cattgtgcct tttgaatgtt gtcttctcac 4260

tcagcatcag cacttcgatc taaatgcaga ctaggtagtt gggaggagga accaaagtga 4320

accatccttc atttattcag tcattcgttc atctgtcaaa cacgtatttg gacatcaagg 4380

ttgcagagat gaacaatgca tggatttcat ctttgaggag ttcaaaacct agtggagaga 4440

acacatggta caatcgtaac acatgaagga caagtaagtg ctgcagtaaa ggtactaata 4500

acatgttcct tggaacagag gaagaaaaac cacgaaacca tggaaattag ggaagccttt 4560

acagagggtg tgacaaaact caatttgaca ttttcaagct atgtacaatg atgtgcacct 4620

tgcagatgct caataaagta attackgaca a 4651

<210> 2

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 2

aagaacuugu cacauucgcc a 21

<210> 3

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 3

gcgaauguga caaguucuuc u 21

<210> 4

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 4

agaagaacuu gucacauucg c 21

<210> 5

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 5

gaaugugaca aguucuucua c 21

<210> 6

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 6

aagguaaucu guauucaagg c 21

<210> 7

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 7

cuugaauaca gauuaccuug a 21

<210> 8

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 8

ucagaaucaa gguaaucugu a 21

<210> 9

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 9

cagauuaccu ugauucugau u 21

<210> 10

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 10

aaucagaauc aagguaaucu g 21

<210> 11

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 11

gauuaccuug auucugauua c 21

<210> 12

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 12

uuggaauca gaaucaaggu a 21

<210> 13

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 13

ccuugauucu gauuaccaaa g 21

<210> 14

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 14

uuuguaauc agaaucaagg u 21

<210> 15

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

cuugauucug auuaccaaag a 21

<210> 16

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

auuugugaga guaaaugugg c 21

<210> 17

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cacauuuacu cucacaaauu c 21

<210> 18

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 18

uuucugaaga ucuuuuacca u 21

<210> 19

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gguaaaagau cuucagaaac u 21

<210> 20

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

uaaauggaag caguuucuga a 21

<210> 21

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 21

cagaaacugc uuccauuuaa c 21

<210> 22

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 22

auuuuuuuca uuuucucugu g 21

<210> 23

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 23

cagagaaaau gaaaaaaauc c 21

<210> 24

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 24

uucuucaucc uggauuuggu u 21

<210> 25

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 25

ccaaauccag gaugaagaac g 21

<210> 26

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 26

uuuugagauu guaccauucg u 21

<210> 27

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 27

gaaugguaca aucucaaaag a 21

<210> 28

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 28

acucuuuuga gauuguacca u 21

<210> 29

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 29

gguacaaucu caaaagaguu c 21

<210> 30

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 30

aacucuuuug agauuguacc a 21

<210> 31

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 31

guacaaucuc aaaagaguuc u 21

<210> 32

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 32

aguuuuccca aaaagcuacu a 21

<210> 33

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 33

guagcuuuuu gggaaaacuc a 21

<210> 34

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 34

augaguuuuc ccaaaaagcu a 21

<210> 35

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 35

gcuuuuuggg aaaacucaug g 21

<210> 36

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 36

ugaaaaagcu ugaugaaugg g 21

<210> 37

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 37

cauucaucaa gcuuuuucaa u 21

<210> 38

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 38

aauuaauuga aaaagcuuga u 21

<210> 39

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 39

caagcuuuuu caauuaauuu a 21

<210> 40

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 40

auaaauuaau ugaaaaagcu u 21

<210> 41

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 41

gcuuuuucaa uuaauuuauu a 21

<210> 42

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 42

aauaaauuaa uugaaaaagc u 21

<210> 43

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 43

cuuuuucaau uaauuuauua c 21

<210> 44

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 44

uguucuucag gauugcuacc a 21

<210> 45

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 45

guagcaaucc ugaagaacau u 21

<210> 46

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 46

aauagacaau guucuucagg a 21

<210> 47

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 47

cugaagaaca uugucuauuu c 21

<210> 48

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 48

uuucuaugcc auuaaucacc u 21

<210> 49

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 49

gugauuaaug gcauagaaag u 21

<210> 50

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 50

acuuucuaug ccauuaauca c 21

<210> 51

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 51

gauuaauggc auagaaaguu g 21

<210> 52

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 52

accaaaagca gugucaaaga g 21

<210> 53

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 53

cuuugacacu gcuuuugga c 21

<210> 54

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 54

aaaaaccacc ugaaauaggc c 21

<210> 55

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 55

ccuauuucag gugguuuuua g 21

<210> 56

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 56

uaaaaaccac cugaaauagg c 21

<210> 57

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 57

cuauuucagg ugguuuuuag u 21

<210> 58

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 58

ucaaaaguaa ccuuguagcc a 21

<210> 59

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 59

gcuacaaggu uacuuuugac a 21

<210> 60

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 60

auugucaaaa guaaccuugu a 21

<210> 61

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 61

caagguuacuuuugacaauu c 21

<210> 62

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 62

agaauuguca aaaguaaccu u 21

<210> 63

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 63

gguuacuuuu gacaauucug g 21

<210> 64

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 64

uuauaacucc ccagaauugu c 21

<210> 65

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 65

caauucuggg gaguuauaaa c 21

<210> 66

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 66

uuuuugaguu uauaacuccc c 21

<210> 67

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 67

ggaguuauaa acucaaaaaa c 21

<210> 68

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 68

uuuuuugagu uuauaacuccc c 21

<210> 69

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 69

gaguuauaaa cucaaaaaac u 21

<210> 70

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 70

aguuuuuuga guuuauaacu c 21

<210> 71

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 71

guuauaaacu caaaaaacua a 21

<210> 72

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 72

auacuauuag uuuuuugagu u 21

<210> 73

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 73

cucaaaaaac uaauaguauc c 21

<210> 74

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 74

uaaagacaaa cuauuccagc u 21

<210> 75

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 75

cuggaauagu uugucuuuac a 21

<210> 76

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 76

uguaaagaca aacuauucca g 21

<210> 77

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 77

ggaauaguuu gucuuuacaa u 21

<210> 78

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 78

uuguaaagac aaacuauucc a 21

<210> 79

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 79

gaauaguuug ucuuuacaau g 21

<210> 80

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 80

acagaaaccc auguaaaga c 21

<210> 81

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 81

cuuuacaaug gguuucuguu c 21

<210> 82

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 82

aaauauaaug auaacugaca g 21

<210> 83

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 83

gucaguuauc auuauauuuuu g 21

<210> 84

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 84

uucuuuugca aaaacaagca g 21

<210> 85

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 85

gcuuguuuuu gcaaaagaag a 21

<210> 86

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 86

uuacuacacc uaaucucacc c 21

<210> 87

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 87

gugagauuag guguaguaau c 21

<210> 88

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 88

uauaacugga gguaaacagc a 21

<210> 89

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 89

cuguuuaccu ccaguuauau g 21

<210> 90

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 90

uaacugaagu uauuaguggc u 21

<210> 91

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 91

ccacuaauaa cuucaguuau g 21

<210> 92

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 92

auaacugaag uuauuagugg c 21

<210> 93

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 93

cacuaauaac uucaguuaug c 21

<210> 94

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 94

aaauaccagu gcauuucagg u 21

<210> 95

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 95

cugaaaugca cuggauuuua u 21

<210> 96

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 96

auaaauacca gugcauuuca g 21

<210> 97

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 97

gaaaugcacu gguauuuauu u 21

<210> 98

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 98

uaucagaaau aaauaccagu g 21

<210> 99

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 99

cuggauuuua uuucugauaa u 21

<210> 100

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 100

auuaucagaa auaaauacca g 21

<210> 101

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 101

gguauuuauu ucugauaauu a 21

<210> 102

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 102

aauuaucaga aauaaauacc a 21

<210> 103

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 103

guauuuauuu cugauaauua a 21

<210> 104

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 104

uguaauuuuu aauuaucaga a 21

<210> 105

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 105

cugauaauua aaaauuacag g 21

<210> 106

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 106

uucuuuuucu aguucuuccc u 21

<210> 107

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 107

ggaagaacua gaaaaagaac a 21

<210> 108

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 108

aaaguuguuc uuuuucuagu u 21

<210> 109

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 109

cuagaaaaag aacaacuuua g 21

<210> 110

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 110

ucuaaaguug uucuuuuuucu a 21

<210> 111

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 111

gaaaaagaac aacuuuagac c 21

<210> 112

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 112

aucuguuuug ccuuaaauca c 21

<210> 113

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 113

gauuuaaggc aaaacagauu a 21

<210> 114

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 114

uuuuuuaauc uguuuugccu u 21

<210> 115

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 115

ggcaaaacag auuaaaaaaa a 21

<210> 116

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 116

uuuuuuuaau cuguuuugcc u 21

<210> 117

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 117

gcaaaacaga uuaaaaaaaa a 21

<210> 118

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 118

uuuuuuuuaa ucuguuuugc c 21

<210> 119

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 119

caaaacagau uaaaaaaaaa a 21

<210> 120

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 120

agauuuuuuu uuuaaucugu u 21

<210> 121

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 121

cagauuaaaaaaaaaaucug c 21

<210> 122

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 122

agcaaaugcc aagaaauugg c 21

<210> 123

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 123

caauuucuug gcauuugcuu c 21

<210> 124

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 124

agaaagagaa gcaaaugcca a 21

<210> 125

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 125

ggcauuugcu ucucuuucuc c 21

<210> 126

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 126

acaaagaggc auuaucugca u 21

<210> 127

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 127

gcagauaaug ccucuuuguu g 21

<210> 128

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 128

aacaaagagg cauuaucugc a 21

<210> 129

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 129

cagauaaugc cucuuuguug a 21

<210> 130

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 130

ucaacaaaga ggcauuaucu g 21

<210> 131

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 131

gauaaugccu cuuuguugaa a 21

<210> 132

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 132

aguuauuuca acaaagaggc a 21

<210> 133

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 133

ccucuuuguu gaaauaacuu u 21

<210> 134

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 134

aaguuauuuc aacaaagagg c 21

<210> 135

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 135

cucuuuguug aaauaacuuu g 21

<210> 136

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 136

auagaaaauu guacauuccc a 21

<210> 137

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 137

ggaauguaca auuuucuaua u 21

<210> 138

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 138

uauagaaaau uguacauucc c 21

<210> 139

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 139

gaauguacaa uuuucuauau c 21

<210> 140

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 140

uaagagauau agaaaauugu a 21

<210> 141

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 141

caauuuucua uaucucuuag c 21

<210> 142

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 142

uguacauauc cuugagaacc a 21

<210> 143

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 143

guucucaagg auauguacag u 21

<210> 144

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 144

aaaacuguac auauccuuga g 21

<210> 145

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 145

caaggauaug uacaguuuuc a 21

<210> 146

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 146

augaaaacug uacauauccu u 21

<210> 147

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 147

ggauauguac aguuuucauu u 21

<210> 148

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 148

aaugaaaacu guacauaucc u 21

<210> 149

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 149

gauauguaca guuuucauuu u 21

<210> 150

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 150

aggaaaauga aaacuguaca u 21

<210> 151

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 151

guacaguuuu cauuuuccuc c 21

<210> 152

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 152

ucaacuuugg aggaaaauga a 21

<210> 153

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 153

cauuuuccuc caaaguugaa u 21

<210> 154

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 154

uagcaaaauu caacuuugga g 21

<210> 155

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 155

ccaaaguuga auuuugcuac a 21

<210> 156

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 156

aaauguagca aaauucaacu u 21

<210> 157

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 157

guugaauuuu gcuacauuuu u 21

<210> 158

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 158

accuaaaaga aaaauguagc a 21

<210> 159

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 159

cuacauuuuu cuuuuaggua a 21

<210> 160

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 160

auuaccuaaa agaaaaaugu a 21

<210> 161

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 161

cauuuuucuuuuagguaaug a 21

<210> 162

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 162

aaaaaugcuu aucauuaccu a 21

<210> 163

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 163

gguaaugaua agcauuuuuu a 21

<210> 164

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 164

aaaaaaugcu uaucauuacc u 21

<210> 165

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 165

guaaugauaa gcauuuuuua a 21

<210> 166

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 166

uuuuuaaaaa augcuuauca u 21

<210> 167

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 167

gauaagcauu uuuuaaaaaa u 21

<210> 168

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 168

augauuuuuu aaaaaaugcu u 21

<210> 169

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 169

gcauuuuuua aaaaaucauu u 21

<210> 170

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 170

aaugauuuuu uaaaaaaugc u 21

<210> 171

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 171

cauuuuuuuaa aaaaucauuu u 21

<210> 172

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 172

uuaccauuac cuaaaaauga u 21

<210> 173

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 173

cauuuuuagg uaaugguaag c 21

<210> 174

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 174

uaaaaugcuu accuuaccu a 21

<210> 175

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 175

gguaauggua agcauuuuau g 21

<210> 176

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 176

auaaaaugcu uaccauuacc u 21

<210> 177

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 177

guaauguaa gcauuuuaug c 21

<210> 178

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 178

ucuguuaugc cacauuuggc a 21

<210> 179

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 179

ccaaaugugg cauaacagag u 21

<210> 180

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 180

aauucaaacu cuguuaugcc a 21

<210> 181

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 181

gcauaacaga guuugaauug a 21

<210> 182

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 182

aaacuuugcc cuucaauuca a 21

<210> 183

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 183

gaauugaagg gcaaaguuuu c 21

<210> 184

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 184

aaagaaaaga aaacuuugcc c 21

<210> 185

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 185

gcaaaguuuu cuuuuuuuu u 21

<210> 186

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 186

aaaagaaaag aaaacuuugc c 21

<210> 187

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 187

caaaguuuuc uuuucuuuuu u 21

<210> 188

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 188

aaaaaaaaga aaagaaaacu u 21

<210> 189

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 189

guuuucuuuuuuuuuuuuuuuuu 21

<210> 190

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 190

uguauuauac cauucaaggg g 21

<210> 191

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

ccuugaaugg uauaauacag u 21

<210> 192

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 192

auagaucuuu caguucaacc u 21

<210> 193

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 193

guugaacuga aagaucuaua u 21

<210> 194

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 194

uaacauauag aucuuucagu u 21

<210> 195

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 195

cugaaagauc uauauguuaa g 21

<210> 196

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 196

uuagcuuaac auauagaucu u 21

<210> 197

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 197

gaucuauaug uuaagcuaac a 21

<210> 198

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 198

auuuucaugu uagcuuaaca u 21

<210> 199

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 199

guuaagcuaa caugaaaauu c 21

<210> 200

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 200

uaugaauuuu cauuguuagcu u 21

<210> 201

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 201

gcuaacauga aaauucauau u 21

<210> 202

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 202

auaugaauuu ucauguuagc u 21

<210> 203

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 203

cuaacaugaa aauucauauu c 21

<210> 204

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 204

uacuauguug cagaauauga a 21

<210> 205

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 205

cauauucugc aacauaguag a 21

<210> 206

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 206

agaaaaaucu acuauguugc a 21

<210> 207

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 207

caacauagua gauuuuucua a 21

<210> 208

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 208

auuagaaaaa ucuacuaugu u 21

<210> 209

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 209

cauaguagau uuuucuaaug c 21

<210> 210

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 210

augcauuaga aaaaucuacu a 21

<210> 211

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 211

guagauuuuu cuaaugcaug a 21

<210> 212

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 212

uacuuauuuc augcauuaga a 21

<210> 213

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 213

cuaaugcaug aaauaaguac c 21

<210> 214

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 214

aaccauuuag ggacauaagu c 21

<210> 215

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 215

cuuauguccc uaaauguug u 21

<210> 216

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 216

ucaaaacaac cauuuaggga c 21

<210> 217

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 217

cccuaaaugg uuguuuugau a 21

<210> 218

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 218

aucaaaacaa ccauuuaggg a 21

<210> 219

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 219

ccuaaauggu uguuuugaua c 21

<210> 220

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 220

uaucaaaaca accauuuagg g 21

<210> 221

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 221

cuaaaugguu guuuugauac a 21

<210> 222

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 222

uaguuguauc aaaacaacca u 21

<210> 223

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 223

gguuguuuug auacaacuau a 21

<210> 224

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 224

auaguuguau caaaacaacc a 21

<210> 225

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 225

guuguuuuga uacaacuaua u 21

<210> 226

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 226

uauauaguug uaucaaaaca a 21

<210> 227

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 227

guuuugauac aacuauauag a 21

<210> 228

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 228

uuuucuauau aguuguauca a 21

<210> 229

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 229

gauacaacua uauagaaaag a 21

<210> 230

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 230

uuauuuugug gcucuuuucu a 21

<210> 231

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 231

gaaaagagcc acaaaauaaa g 21

<210> 232

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 232

uuuuaucuuu auuuuguggc u 21

<210> 233

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 233

ccacaaaaua aagauaaaag u 21

<210> 234

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 234

aacuuuuauc uuuauuuugu g 21

<210> 235

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 235

caaaauaaag auaaaaguua u 21

<210> 236

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 236

uuauauauuu uuuucagaga u 21

<210> 237

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 237

cucugaaaaa aauauauaaa a 21

<210> 238

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 238

uuuuauauau uuuuuucaga g 21

<210> 239

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 239

cugaaaaaaa uauauaaaau a 21

<210> 240

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 240

uauuuuauauauuuuuuuca g 21

<210> 241

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 241

gaaaaaaaua uauaaaauau u 21

<210> 242

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 242

uguuuguagc cucaaacaca c 21

<210> 243

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 243

guguuugagg cuacaaacau a 21

<210> 244

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 244

uauuaauggg ggaguuaugu u 21

<210> 245

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 245

cauaacuccc ccauuaauac a 21

<210> 246

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 246

uuuaauuugu auuaaugggg g 21

<210> 247

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 247

cccauuaaua caaauuaaau g 21

<210> 248

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 248

auuuaauuug uauuaauggg g 21

<210> 249

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 249

ccauuaauac aaauuaaaug u 21

<210> 250

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 250

aaaaauuuug agaaugagcu c 21

<210> 251

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 251

gcucauucuc aaaauuuuuu u 21

<210> 252

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 252

aaaaaauuuu gagaaugagc u 21

<210> 253

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 253

cucauucuca aaauuuuuuuu g 21

<210> 254

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 254

ugaucaaaaa aauuuugaga a 21

<210> 255

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 255

cucaaaauuuuuuugaucaa g 21

<210> 256

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 256

ucuuguuacc auuuuuuagu g 21

<210> 257

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 257

cuaaaaaaug guaacaagag g 21

<210> 258

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 258

aacauucaga aaaauccagg u 21

<210> 259

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 259

cuggauuuuu cugaauguuu u 21

<210> 260

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 260

aaaacauuca gaaaaaucca g 21

<210> 261

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 261

ggauuuuucu gaauguuuua a 21

<210> 262

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 262

uaaaacauuc agaaaaaucc a 21

<210> 263

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 263

gauuuuucug aauguuuuaa a 21

<210> 264

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 264

aauucuuuaa aacauucaga a 21

<210> 265

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 265

cugaauguuu uaaagaauug c 21

<210> 266

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 266

aucaguauuuuuuacuuggc u 21

<210> 267

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 267

ccaaguauga aauacugauc u 21

<210> 268

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 268

auaacuuucu gauugauccu g 21

<210> 269

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 269

ggaucaauca gaaaguuaua u 21

<210> 270

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 270

uauaacuuuc ugauugaucc u 21

<210> 271

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 271

gaucaaucag aaaguuauau g 21

<210> 272

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 272

uuuugcauau aacuuucuga u 21

<210> 273

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 273

cagaaaguua uaugcaaaaa u 21

<210> 274

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 274

auuuuugcau auaacuuucu g 21

<210> 275

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 275

gaaaguuaua ugcaaaaauu c 21

<210> 276

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 276

auuguucucu ccuuguuggg a 21

<210> 277

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 277

ccaacaagga gagaacaaua u 21

<210> 278

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 278

uuuuucugca aaauguaacu c 21

<210> 279

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 279

guuacauuuu gcagaaaaau a 21

<210> 280

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 280

uuuaaguuuc uuagcuaagc c 21

<210> 281

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 281

cuuagcuaag aaacuuaaag c 21

<210> 282

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 282

aaauacugcu uuaaguuucu u 21

<210> 283

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 283

gaaacuuaaa gcaguauuuu a 21

<210> 284

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 284

aauaaaggga cauccaaacu u 21

<210> 285

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 285

guuuggaugu cccuuuauuu c 21

<210> 286

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 286

aaucaguacc aagucaaacc c 21

<210> 287

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 287

guuugauug guacugauug g 21

<210> 288

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 288

aaucuugacc aaucaguacc a 21

<210> 289

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 289

guacugauug gucaagaauc c 21

<210> 290

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 290

auuccuuuuc uuuauccagc a 21

<210> 291

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 291

cuggauaaag aaaaggaauu u 21

<210> 292

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 292

aaauuccuuu ucuuuaucca g 21

<210> 293

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 293

ggauaaagaa aaggaauuuu a 21

<210> 294

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 294

aaaauuccuu uucuuuaucc a 21

<210> 295

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 295

gauaaagaaa aggaauuuua g 21

<210> 296

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 296

acuucuaaaa uuccuuuuuuu 21

<210> 297

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 297

gaaaaggaau uuuagaagug a 21

<210> 298

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 298

uuaucaaauaauaguuuggc u 21

<210> 299

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 299

ccaaacuauuauuugauaag g 21

<210> 300

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 300

acaacguugg cuccuaaucu g 21

<210> 301

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 301

gauuaggagc caacguuguu u 21

<210> 302

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 302

aauaauagca cgaacuuccc a 21

<210> 303

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 303

ggaaguucgu gcuauuauuc a 21

<210> 304

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 304

agcauaaugu uguacaugga u 21

<210> 305

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 305

ccauguacaa cauuaugcuu a 21

<210> 306

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 306

aagcauaaug uuguacaugg a 21

<210> 307

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 307

cauguacaac auuaugcuua c 21

<210> 308

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 308

ucaguaugca aauaugugcu a 21

<210> 309

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 309

gcacauauuu gcauacugaa a 21

<210> 310

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 310

uucaguaugc aaauaugugc u 21

<210> 311

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 311

cacauauuug cauacugaaa g 21

<210> 312

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 312

ucaagaagcc guuuggaugc u 21

<210> 313

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 313

cauccaaacg gcuucuugag a 21

<210> 314

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 314

acaauuggau gugaaaaaga a 21

<210> 315

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 315

cuuuuucaca uccaauuguu u 21

<210> 316

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 316

aauagacacu aaacaauugg a 21

<210> 317

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 317

caauuguuua gugucuauuu a 21

<210> 318

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 318

uuuuagguuu caaaacuggc c 21

<210> 319

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 319

ccaguuuuga aaccuaaaaa g 21

<210> 320

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 320

uuuuuagguu ucaaaacugg c 21

<210> 321

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 321

caguuuugaa accuaaaaag g 21

<210> 322

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 322

uuuauugucc cuuuuuaggu u 21

<210> 323

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 323

ccuaaaaagg gacaauaaag c 21

<210> 324

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 324

acuuuuugcu uuauuguccc u 21

<210> 325

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 325

ggacaauaaa gcaaaaagua u 21

<210> 326

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 326

uacuuuuugc uuuauugucc c 21

<210> 327

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 327

gacaauaaag caaaaaguau c 21

<210> 328

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 328

acguguuuga cagaugaacg a 21

<210> 329

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 329

guucacugu caaacacgua u 21

<210> 330

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 330

agaugaaauc caugcauugu u 21

<210> 331

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 331

caaugcaugg auuucaucuu u 21

<210> 332

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 332

uuugaacucc ucaaagauga a 21

<210> 333

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 333

caucuuugag gaguucaaaa c 21

<210> 334

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 334

acgauuguac cauuguucu c 21

<210> 335

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 335

gaacacaugg uacaaucgua a 21

<210> 336

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 336

uuauuaguac cuuuacugca g 21

<210> 337

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 337

gcaguaaagg uacuaauaac a 21

<210> 338

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 338

auguuauuag uaccuuuacu g 21

<210> 339

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 339

guaaagguac uaauaacaug u 21

<210> 340

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 340

aauuuccaug guuucguggu u 21

<210> 341

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 341

ccacgaaacc auggaaauua g 21

<210> 342

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 342

ucaaauugag uuuugucaca c 21

<210> 343

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 343

gugacaaaac ucaauuugac a 21

<210> 344

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 344

ugucaaauug aguuuuguca c 21

<210> 345

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 345

gacaaaacuc aauuugacau u 21

<210> 346

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 346

aaugucaaau ugaguuuugu c 21

<210> 347

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 347

caaaacucaa uuugacauuu u 21

<210> 348

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 348

uugaaaaugu caaauugagu u 21

<210> 349

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 349

cucaauuuga cauuuucaag c 21

<210> 350

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 350

uguacauagc uugaaaaugu c 21

<210> 351

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 351

cauuuucaag cuauguacaa u 21

<210> 352

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 352

aguaauuacu uuauugagca u 21

<210> 353

<211> 21

<212> RNA

<213> Artificial Sequence

<400> 353

gcucaauaaa guaauuacug a 21

<210> 354

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 354

ccagcgcgag ccagcgcgcg g 21

<210> 355

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 355

cagcctcttc gccctggctg g 21

<210> 356

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 356

tgtcacattc gccaaagccg g 21

<210> 357

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 357

cccgggtgct gtagaggtg g 21

Claims (9)

1. A pharmaceutical composition for the treatment or prevention of tumors, containing ENDOD1 endonuclease inhibitory drug, said inhibitory drug can inhibit the gene expression, activity or function of ENDOD1 endonuclease, said endonuclease is The endonuclease encoded by the ENDOD1 gene, the ENDOD1 gene sequence is SEQ.ID No1, the tumor has TP53, PTIP, WRN, Fanconi, EMSY, MRE11, NBS1, BLM, ARID1A, ARID1B, WDR70, BRCA1 or/and BRCA2 gene mutation and HBV or HPV infection, the tumor is lung cancer, breast cancer, colorectal cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia or sarcoma, and the inhibitory drug is a double-stranded siRNA that inhibits the expression of ENDOD1 gene , which is 18 to 28 nucleotides in length and complementary to the nucleotide sequence of the ENDOD1 gene, wherein the nucleotide sequence of one strand of the double-stranded siRNA is selected from the following groups: 5'-CCAGCGCGAGCCAGCGCGCGG-3' 5'-CAGCCTCTTCGCCCTGGCTGG-3' 5'-TGTCACATTCGCCAAAGCCGG-3' 5'-CCCGGGTGCTGTAGAGGGTGG-3 5'-GCAAGCGGATTGGCTACAA-3' 5'-GGATGAAGAACGAATGGTA-3' 5'-GTGGCCACATTTACTCTCA-3' 5'-CCGCGCGCTGGCTCGCGCTGG-3' 5'-CCGGCTTTGGCGAATGTGACA-3' 5'-CCACCCTCTACAGCACCCGGG-3' 5'-GACTACAGAGTTAAAGACA-3' 5'-CCATGGGCTTTGTCAAGCA-3' 5'-GGTAAAAAGATCTTCAGAAA-3' 5'-TACCCTGGGTGGCCTATTT-3' 5'-TTCGCCACCCTCTACAGC-3' 5'-CCAACAGCAACCTTGAGGA-3' 5'-CAACCTTGAGGAGGCGATT-3' 5'-GGAGGCGATTAATGAGGCA-3' 5'-GTCCAGGTGGCCACATTTA-3' 5'-GCAGTCCCTGAGTTTGTTT-3' 5'-CAATGACTCAGTCCTTCCA-3' 5'-GACTCAGTCCTTCCAGGAA-3' 5'-CAGCCACCATCTCATACTT-3' 5'-TGTCTGCTGAAGGCCATTT-3' 5'-TGCTGTTTACCTCCAGTTA-3' 5'-GTCCAGCTTGGATTAACTT-3' 5'-TCAGCAGTGTGAAGGTAAA-3' 5'-CACGAAACCATGGAAATTA-3' 5'-CATCCAAACGGCTTCTTGA-3' 5'-CCAATTGTTTAGTGTCTAT-3'. 2. The pharmaceutical composition according to claim 1, said tumor has TP53, BRCA1 or/and BRCA2 gene mutation, gene expression or function defect. 3. The pharmaceutical composition of claim 1, said tumor harbors the genome or gene expression of hepatitis B virus or human papillomavirus. 4. The pharmaceutical composition as claimed in claim 1, said nucleoside sequence is selected from the following sequences: 5'-CCAGCGCGAGCCAGCGCGCGG-3' 5'-CAGCCTCTTCGCCCTGGCTGG-3' 5'-TGTCACATTCGCCAAAGCCGG-3' 5'-CCCGGGTGCTGTAGAGGGTGG-3' 5'-GCAAGCGGATTGGCTACAA-3' 5'-GGATGAAGAACGAATGGTA-3' 5'-GTGGCCACATTTACTCTCA-3'. 5. The pharmaceutical composition according to any one of claims 1-4, further comprising pharmaceutical excipients. 6. The pharmaceutical composition according to any one of claims 1-4, wherein its preparation form is an injection. 7. The pharmaceutical composition of claim 6, further comprising a liposomal nucleic acid delivery system. 8. The pharmaceutical composition of any one of claims 1-7 is used in the manufacture of medicines for treating or preventing tumors, and said tumors include lung cancer, breast cancer, colorectal cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma , leukemia or sarcoma. 9. purposes as claimed in claim 8, further comprise that said pharmaceutical composition is used in combination with another or more antineoplastic drugs or form compound preparations, and said another one or more antineoplastic drugs are selected as Platinum, camptothecin, or DNAPK inhibitors.

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