CN115998872B - A drug containing a compound that inhibits endonuclease function and its anti-tumor use - Google Patents

Abstract

The present invention discloses an inhibitory drug and SSA enhancer for ENDOD1 endonuclease, and uses of the inhibitory drug and SSA enhancer for ENDOD1 endonuclease in manufacturing drugs for treating or preventing human tumors and hepatitis B virus infection diseases. The endonuclease is an endonuclease encoded by the 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 mutations of tumor suppressor genes, gene expression or functional defects, and has no toxicity to non-tumor cells with normal genes or functions.

Description

Medicine containing endonuclease inhibiting function and anti-tumor application thereof

The application is a divisional application of the application application with the application number of CN202010295776.6, the application date of 2020.04.15, the publication number of CN111671904A and the publication date of 2020.09.18.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a function of inhibiting endo-1 endonuclease in preparing a medicine for treating or preventing human tumors.

Background

The malignant tumor has high resistance to chemical drugs and is easy to generate drug resistance, and the aims of temporarily controlling tumor growth and prolonging the survival time of patients are difficult to achieve, so the clinical requirement for developing new generation tumor targeted therapy is particularly urgent, and the key point is to find drugs which can effectively kill tumor cells but have relatively small damage to normal non-tumor cells. One strategy for drug development to achieve this goal is synthetic lethality (SYNTHETIC LETHALITY), i.e., the discovery of a specific defect in a tumor cell and the determination that disruption of another cell function in that defective state results in death of the tumor cell (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 is at any time threatened by DNA damage factors, which leads to mutation of oncogenes and cancer suppressor genes and uncontrolled cell proliferation, and promotes tumorigenesis. In this process, the DNA damage response and DNA repair mechanism play a key role in inhibiting tumor-related genome mutation, eliminating cells with abnormal genome structure or replication defect, ensuring genome stability and organ tissue non-cancerous (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 cancer suppressor genes including PTEN, CDKN2A, TP53, APC, VHL, p INK4A, p ARF, LKB1, FBXW7, NF1, NF2 and WT-1, etc., the mutation of which causes glioma, neurofibromatosis, lung cancer, gastric cancer, liver cancer, melanoma, carcinoma of large intestine, nasopharyngeal carcinoma, renal 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.). particularly TP53 gene, the mutation of which is widely present in 50-80% of tumors, and which can be destroyed by Human Papilloma Virus (HPV), hepatitis B Virus (HBV) and other viral factors (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, DNA damage response is mainly regulated by ATM and ATR kinase, CHK1 and CHK2 are activated through signal transduction paths such as gamma H2AX, 53BP1, MDC1 and CDC25, the progress of cell cycle is controlled, and the activation of DNA damage repair mechanism is promoted. Known DNA damage repair mechanisms include non-identical end links (NHEJ), homologous recombination repair (HR), single-stranded DNA fusion (SSA), nucleotide Excision Repair (NER), base Excision Repair (BER), single-stranded DNA fragmentation 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.). that inactivation of these mechanisms leads to the occurrence of skin cancer, breast cancer, ovarian cancer, liver cancer, cholangiocellular carcinoma, colorectal cancer, prostate cancer, leukemia, etc.).

During the cell proliferation process, DNA replication generates a large amount of DNA damage, which is liable to cause gene mutation. These lesions are mainly eliminated by repair mechanisms that rely on homologous sequences, such as HR, SSA. HR is regulated primarily by factors such as BRCA1, BRCA2, MRE11, NBS1, BLM, 53BP1, RAD51, RPA, WDR70, and Vanconi (FANC), while SSA depends on repair factors associated with RAD52, MRE11, RAD54B, and RPA (see: haber, J.E., DNA Repair: THE SEARCH for homology.Bioess, 2018.40 (5): p.e 1700229.).

The SSA mechanism is extremely conserved in the evolution process and mainly occurs in homologous sequences near DNA breakpoints, and the process depends on RAD52 and other repair factors to combine with the DNA breakpoints to form a nucleoprotein complex synapse, invade and fuse the homologous sequences near the DNA breakpoints. RAD52 is a key molecule of SSA pathway, can be directly bound to the broken end of processed DNA, especially single-stranded DNA product, protects processed DNA from degradation of nuclease, promotes fusion of homologous sequences on 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 can also promote interchain exchange at the later stage of homologous recombination repair, RAD52 can also be bound to single-stranded DNA formed by replication fork blocking, maintains stability of replication fork, prevents degradation of newly replicated DNA strand, plays an important role in DNA stability during replication (see ：Malacaria,E.,et al.,Rad52 prevents excessive replication fork reversal and protects from nascent strand degradation.Nat Commun,2019.10(1):p.1412.). is considered as a repair mode of low fidelity or even toxicity due to possible loss of homologous sequences; thus, disturbance of SSA pathway regulation may lead to defects of DNA response and damage repair, leading to instability of genome).

Disclosure of Invention

The invention aims to provide an endo-1 endonuclease inhibitory drug or an SSA enhancer and application thereof in preparing a drug for preventing or treating tumors, in particular to application in treating tumors with DNA homologous recombination defects, DNA damage response defects, DNA repair defects, TP53 and other cancer suppressor genes, gene expression or functional defects.

The endo D1 endonuclease is used as a novel broad-spectrum antitumor drug target, and is a protein coded by an endo D1 gene, wherein the mRNA sequence of the endo D1 is SEQ ID No.1 (corresponding NCBI reference number NM_ 015036.3), and the coded amino acid sequence of the protein is a sequence corresponding to NCBI accession number NP_ 055851.1.

An endo d1 endonuclease inhibitory drug herein may also be referred to as an endo d1 endonuclease inhibitor.

In one embodiment, the invention provides a method of treating or preventing a tumor comprising administering to a tumor patient an effective amount of an endo d1 endonuclease inhibitory drug that inhibits the function of endo d1 endonuclease, which endonuclease is an endonuclease encoded by an endo d1 endonuclease gene, or an SSA enhancer. The SSA enhancer may enhance the function of single stranded DNA fusion repair (SINGLE STRAND ANNEALING, SSA).

The above-described method for treating a tumor according to the present invention contains a defect in homologous recombination of DNA, a defect in DNA damage response, a defect in DNA repair, a defect in gene expression or function of an oncogene such as TP53, etc. Specifically lung cancer, breast cancer, carcinoma of large intestine, gastric cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma, or/and cholangiocarcinoma.

The endo-1 endonuclease inhibitory drug or SSA enhancer of the present invention may be any substance that has an inhibitory effect on endo-1 endo-nucleases and the SSA enhancer may be any substance that enhances single strand DNA fusion repair (SSA), including, but not limited to, antibodies, organic or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, and inorganic substances.

The methods of treatment of the present invention described above further comprise the use of the compositions in combination with one or more other anti-tumor treatments. The other one or more anti-tumor therapeutic methods include surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, targeted therapy, adjuvant therapy, and/or immunotherapy.

In some embodiments, the invention provides a pharmaceutical composition for treating or preventing a tumor, comprising an endo d1 endonuclease inhibitory drug or SSA enhancer, said endo d1 inhibitory drug being capable of inhibiting gene expression, activity or function of an endo d1 endonuclease, said endonuclease being an endonuclease encoded by an endo d1 endonuclease gene. The SSA enhancer may up-regulate the function of single stranded DNA fusion repair (SSA). The tumor has DNA homologous recombination defect, DNA damage response defect, DNA repair defect, TP53 and other cancer suppressor gene mutation, gene expression or function defect. In particular lung cancer, breast cancer, colorectal cancer, gastric cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, neuroblastoma, glioma, sarcoma or/and cholangiocarcinoma.

The aforementioned pharmaceutical composition of the present invention, the endo 1 endonuclease inhibitory drug may be any substance having an inhibitory effect on endo 1 endonuclease, and the SSA enhancer may be any substance up-regulating single strand DNA fusion repair (SSA) function, and specifically includes, but is not limited to, substances selected from the group consisting of antibodies, synthetic organic compounds, natural organic compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, and inorganic substances.

In some embodiments, the use of an endo d1 endonuclease inhibitory drug or SSA enhancer of the invention in the manufacture of a medicament for the treatment and/or prevention of a tumor, said endo d1 inhibitory drug being capable of inhibiting the gene expression, activity or function of an endo d1 endonuclease, said endonuclease being an endonuclease encoded by an endo d1 endonuclease gene. The SSA enhancer may up-regulate the function of single stranded DNA fusion repair (SSA).

The use of the present invention as described above, wherein the tumor has a defect in DNA homologous recombination, a defect in DNA damage response, a defect in DNA repair, a defect in gene expression or function of an oncogene such as TP53, etc. The tumor includes, but is 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, or/and cholangiocarcinoma.

The use of the present invention as described above, the endo 1 endonuclease inhibitory drug may be any substance having an inhibitory effect on gene expression, activity or function of endo 1 endonuclease, and the SSA enhancer may be any substance up-regulating the function of single strand DNA fusion repair (SSA), specifically including but not limited to antibodies, organic or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, inorganic substances, and the like.

The use of the invention as described above further comprises the use of said endo d1 endonuclease inhibitory drug or SSA enhancer in combination with another anti-tumor drug or drugs or radiotherapy or as a composite composition.

The use of the invention as described above, the further anti-tumour agent or agents are selected from the group consisting of chemotherapeutic agents, polypeptide agents, antibody agents, TKI (kinase inhibitors), small nucleic acids and protein agents. Preferably, the chemotherapeutic agent is selected from the group consisting of platinum, mitomycin C, camptothecin, PARP inhibitors, DNAPK inhibitors, radioisotopes, vinca alkaloids, anti-tumor alkylating agents, monoclonal antibodies, and antimetabolites.

The use of the present invention as described above refers to the simultaneous or sequential administration of an endo-1 endonuclease inhibitory drug or SSA enhancer with another anti-tumor agent or agents.

The use of the invention described above, wherein the tumor with defective DNA response comprises a tumor with a ATM, ATR, CHK, CHK2, DNAPK gene mutation or functional defect, or with CDC25A, CDC25B, CDC C, cyclin E, cyclin B1, cyclin D1 over-expression characteristics.

In some embodiments, a method of the invention for treating or preventing a tumor with a mutation, gene expression, or functional defect of DNA damage repair comprises administering to a subject an effective amount of an ENDOD1 endonuclease inhibitory drug that inhibits gene expression, activity, or function of an ENDOD1 endonuclease or an SSA enhancer that upregulates single strand DNA fusion repair (SSA) function. Preferably, the DNA damage repair defects include homologous recombination repair defects, base excision repair defects, nucleotide excision defects, single strand DNA break repair defects, and defects with gene mutations or gene expression and function associated with the repair mechanism. The endo 1 endonuclease inhibitory drug is any substance capable of inhibiting endo 1 endonuclease function and the SSA enhancer may be any substance that upregulates single-stranded DNA fusion repair (SSA) function.

In some embodiments, a pharmaceutical composition of the invention for treating or preventing a tumor with a mutation, gene expression, or functional defect of an oncogene comprises an ENDOD1 endonuclease-inhibiting drug that inhibits gene expression, activity, or function of an ENDOD1 endonuclease, or an SSA enhancer that upregulates single-stranded DNA fusion repair (SSA) function. Preferably, the cancer suppressor gene expression or functional defect comprises a gene mutation or gene expression and functional defect of a gene or protein such as TP53, PTEN, CDKN2A, APC, p16.sup.INK4A, p ARF, LKB1, FBXW, and the like.

The aforementioned pharmaceutical composition, the endo-1 endonuclease inhibitory drug may be any substance having an inhibitory effect on the activity or function of endo-1 endonuclease, and specifically includes, but is not limited to, antibodies, organic or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, inorganic substances, and the like.

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

In another embodiment, the invention provides a method of treating and preventing hepatitis B virus infection comprising administering to a subject an effective amount of an endo 1 endonuclease inhibitory drug or SSA enhancer, wherein the endonuclease is an endonuclease encoded by an endo 1 endonuclease gene.

The methods of treatment or pharmaceutical compositions described above also include use in combination with or in combination with another one 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 endonucleon encoded by an endonucleon gene of ENDOD1, the ENDOD1 endonuclease inhibitory drug can be any substance with an inhibitory effect on the activity or function of the ENDOD1 endonuclease, and the SSA enhancer can be any substance which up-regulates the function of single-stranded DNA fusion repair (SSA), and particularly comprises, but is not limited to, antibodies, organic compounds or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, inorganic substances and the like.

In another embodiment, the invention provides a pharmaceutical composition for the treatment and prevention of hepatitis B virus infection comprising an effective dose of an endo 1 endonuclease inhibitory drug or SSA enhancer, and a pharmaceutical adjuvant, wherein the endonuclease is an endonuclease encoded by the endo 1 endonuclease gene. Preferably, the endo 1 endonuclease inhibitory drug may be any substance having an inhibitory effect on the activity or function of endo 1 endonuclease, and the SSA enhancer may be any substance up-regulating the function of single strand DNA fusion repair (SSA), including, but not limited to, antibodies, organic or natural compounds, small nucleic acids, small ribonucleic acids, proteins, polypeptides, antisense acids, and inorganic substances.

The aforementioned pharmaceutical composition of the present invention, the endo d1 endonuclease inhibitory drug or SSA enhancer comprises a small molecule compound, an antibody, a polypeptide or an antisense compound.

In yet another embodiment, the use of an endo-1 endonuclease inhibitory drug or SSA enhancer in the manufacture of a medicament for the treatment and prevention of hepatitis b virus infection, said endonuclease being an endonuclease encoded by an endo-1 endonuclease gene. It may further comprise the simultaneous or sequential combination of an endo 1 endonuclease inhibitory drug or SSA enhancer with other antiviral therapies.

The method, the effect of the endo-1 endonuclease inhibitory drug is to inhibit the expression level of the endo-1 endonuclease and/or the regulatory genes thereof, inhibit the endonuclease activity of the endo-1 endonuclease, block the DNA repair activity of the endo-1 endonuclease, destroy the subcellular localization of the endo-1 endonuclease, destroy the protein degradation, ubiquitination and phosphorylation post-translational modification of the endo-1 endonuclease or interfere with the shearing of the endo-1 endonuclease and the regulatory proteins thereof and the mature biological functions of the signal peptide.

In the method, the SSA enhancer can be used for up-regulating a single-stranded DNA fusion repair (SSA) function, enhancing recruitment of RAD52, MRE11 and RAD54B at DNA breaking sites, enhancing combination of RAD52 with DNA breaking point ends and single-stranded DNA, enhancing combination of RAD52 with RPA70 and RPA32, or enhancing fusion of homologous sequences on the single-stranded DNA by the RAD 52.

The invention also provides a method for screening anti-tumor drugs or anti-hepatitis B infection drugs, which comprises the steps of contacting a screening target substance with cells expressed by the endo D1 endonuclease and selecting substances with inhibition effect on the activity, function or expression of the endo D1 endonuclease.

The invention also provides a method for screening anti-tumor drugs or anti-hepatitis B infection drugs, which comprises the steps of contacting a screening target substance with target cells and selecting a substance with an up-regulating effect on the activity, function or expression of a single-stranded DNA fusion (SSA) repair factor.

Homologous genes of human endo D1 endonuclease (NCBI gene ID, 23052) are widely present in higher eukaryotes, but not in lower eukaryotes such as yeast, drosophila and nematode.

The human endo D1 endonuclease gene encodes a protein product of 500 amino acid residues (AA) belonging to the His-Cys endonuclease family. There are 5 subtypes of endo 1 endonuclease proteins, which are produced due to post-translational cleavage, signal peptide cleavage, and ubiquitination modification of endo 1 endo nucleases.

Human endo-1 endonucleases localize to the nuclear membrane and nucleus in normally growing cells. After DNA damage, ENDOD1 endonuclease can enter DNA breakpoint to form aggregation point, tightly bind to chromatin, recruit to around DNA breakpoint 2.5 Kb.

Human endo D1 endonuclease participates in DNA repair, promoting non-homologous end joining (NHEJ) and Homologous Recombination (HR) repair functions, and more importantly, inhibiting single strand DNA fusion (SSA) functions. Endo 1 endonuclease inhibition of SSA function requires the participation of protein (RAD 52), endo 1 endonuclease can also promote aggregation of NHEJ factors (γh2ax and 53BP1, etc.) and HR factors (BLM, MRE11, etc.) at the site of injury. The function of endood 1 endonuclease to regulate DNA damage repair depends on its endonuclease activity.

Endo D1 endonuclease has endonuclease activity, and can cleave DNA molecules with structural damage, such as ultraviolet damage, DNA double strand breaks (5 'and 3' overlapping), oxidative damage (hydrogen peroxide treatment), and the like.

The endo d1 endonuclease gene expression or function inhibition is lethal in combination with the defect of multiple DNA damage response functions, including homologous recombination-related proteins, including, but not limited to, CHK1, ATM, CHK2, DNAPK, etc.

The inhibition of endo 1 endonuclease gene expression or function is lethal in combination with defects in a variety of DNA damage repair functions, including BRCA1, BRCA2, MRE11, ARID1A, ARID1B, CTIP, EXO, FANC, WDR70, and the like.

The simultaneous inhibition of endo-1 endonuclease and DNA damage repair function (e.g., BRCA 1) may lead to more serious DNA repair defects.

The endo-1 endonuclease function inhibition is combined with TP53, PTEN and other cancer suppressor genes to kill.

Endo-1 endonuclease is a good tumor specific therapeutic target, the reason for this is:

1) The inhibition of endo 1 endonuclease gene expression or function has no effect on proliferation of normal non-tumor cells (e.g., RPE-1, MRC-5, and L02).

2) The endo D1 endonuclease gene expression or functional inhibition can effectively kill tumor cells from various tissue sources, accounting for 76% (19/25) of the tested tumor cell lines.

3) The endo D1 endonuclease gene expression or function inhibition can effectively kill tumor cells with DNA repair gene mutation or function defect.

4) The endo D1 endonuclease gene expression or function inhibition can effectively kill tumor cells with TP53 cancer suppressor gene mutation or function defect.

5) The endo D1 endonuclease gene expression or function inhibition can synergistically enhance the anti-tumor activity of conventional chemoradiotherapy, and the drug treatment under the same condition has no obvious cytotoxic effect on normal cells.

6) In addition to inhibiting the expression level of the endo 1 endonuclease gene, specific elimination of the nuclease active region of the endo 1 endonuclease can also be effective in killing tumor cells.

One example demonstrates that tumors with defective gene mutations, gene expression or function of oncogenes such as DNA homologous recombination, DNA damage response, DNA repair, TP53, etc., but not killing normal non-tumor cells, can be specifically treated or prevented by administering a sufficient dose of an ENDOD1 endonuclease inhibitory drug to a subject. The same principle applies to agents that increase SSA activity (SSA enhancers), since it is predicted that ENDOD1 inhibitory drugs can effectively enhance SSA activity, whereas tumor killing is dependent on an increase in SSA activity, it is inferred that other SSA enhancers should have the same therapeutic effect.

The present invention has found that, to maintain the survival of tumor cells, endoD1 endonuclease expression or activity cannot be simultaneously lost with the functions of oncogenes TP53, PTEN, etc., and that single-chain fusion repair (SSA) activity increase cannot simultaneously lose the functions of oncogenes TP53, PTEN, etc. Silencing the ENDOD1 endonuclease expressed gene, or enhancing single strand fusion repair (SSA), can significantly lead to rapid death of defective tumor cells such as TP53, PTEN, etc. Consistent with cytologic results, a mouse model of TP53 deficient tumors would be completely inhibited by the in vivo silencing ENDOD1 endonuclease gene. Tumor defective in the tumor suppressor gene such as TP53 and PTEN includes any defect that reduces, slows down, damages and prevents the expression of TP53 gene or the reduction of protein level, and performs the normal function of the suppressor gene, including deletion, point mutation, frameshift mutation, reduced gene expression (methylation at gene locus), and the like. These mutations are mutations that can be detected by any normal sequencing assay. Without being limited by any theory of certainty, inhibiting ENDOD1 endonuclease protein or enhancing single-stranded fusion repair (SSA) function may enhance the death of tumor cells in cancer that are tumor suppressor gene-deficient in TP53, PTEN, etc.

The invention also discovers that to maintain the survival of tumor cells, the endoD1 endonuclease activity and the functions of homologous recombination repair genes (such as BRCA1, BRCA2, WDR70, FANC and the like) cannot be simultaneously lost, and the increase of single-chain fusion repair (SSA) activity cannot simultaneously lose homologous recombination repair. Silencing the ENDOD1 endonuclease protein expressed gene can significantly lead to rapid death of homologous recombination defective tumor cells. Consistent with cytology results, a mouse model of homologous recombination deficient tumors would be completely inhibited by the in vivo silencing ENDOD1 endonuclease gene. Homologous recombination-deficient tumors include any defect that reduces, slows down, damages and prevents the expression of homologous recombination repair genes or protein levels from decreasing, and performs the normal repair function of homologous recombination, including deletion, point mutation, frameshift mutation, reduced gene expression (methylation at the gene locus), and the like. These mutations are mutations that can be detected by any normal sequencing assay. Without being limited by any theory of certainty, inhibiting ENDOD1 endonuclease protein or enhancing single-stranded fusion repair (SSA) function may enhance death of homologous recombination-deficient tumor cells.

Human tumor cells sensitive to inhibition of ENDOD1 endonuclease function carry the genome of integrated Hepatitis B Virus (HBV) or express HBx, and these cells disrupt the function of homologous recombination repair factor (CRL 4WDR 70) (see ：Ren,L.,et al.,The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B.Hepatology,2019.69). silencing ENDOD1 endonuclease protein expressed gene can significantly lead to rapid death of HBV positive or HBx expressing cells, while inhibiting ENDOD1 endonuclease function has no equivalent toxic effect on hepatocytes negative for hepatitis b virus that do not express HBx gene. Consistent with cytology results, tumor models of HBV positive cells can be completely inhibited by in vivo silencing ENDOD1 endonuclease gene expression can effectively reduce HBV antigen titer and viral load in mouse serum.

The treatment range of the endo 1 endonuclease inhibitory drug or SSA enhancer comprises diseases such as 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, melanoma, endometrial cancer, glioma, sarcoma, cholangiocarcinoma and the like, and symptoms of the physiological index abnormality, respiratory system, circulatory system, nervous system, digestive system, hematopoietic system and motor system caused by tumor growth.

Endo 1 endonuclease inhibitory drugs or SSA enhancers can treat tumors with homologous recombination defects in the tumor types described above.

Endo 1 endonuclease inhibitory drugs or SSA enhancers can treat tumors of the above tumor types that have mutations or functional defects in the oncogenes such as TP53, PTEN, etc.

Endo 1 endonuclease inhibitory drugs or SSA enhancers can treat radiation and chemotherapy insensitive tumors in the tumor types described above.

Endo 1 endonuclease inhibitory drugs or SSA enhancers can treat tumors of the above tumor types that have significant endo 1 endonuclease gene and single strand fusion repair (SSA) factor expression.

The range of tumors for which endo 1 endonuclease inhibitory drugs or SSA enhancers are useful include, but are not limited to, breast, ovarian, prostate, gastric and osteosarcoma tumors that harbor homologous recombination repair defects (BRCA 1, BRCA2, PTIP, ARID1A, ARID B, fanconi gene, WDR70, etc.).

The range of tumors for which endo 1 endonuclease inhibitory drugs or SSA enhancers are useful include, but are not limited to, tumors such as colorectal cancer with base excision repair defects (MLH 1, MSH2 mutations or functional defects).

Tumors for which endo d1 endonuclease inhibitory drugs or SSA enhancers are useful include, but are not limited to, tumors such as skin cancers and melanomas with nucleotide excision repair defects (XPA, XPB, XPC, XPD, XPE and XPF mutations or functional defects).

The range of tumors for which endo-1 endonuclease inhibitory drugs or SSA enhancers are useful include, but are not limited to, viral infections with HBV positive or HBx expression, liver cancer and cholangiocarcinomas, and liver and gall diseases caused by HBV viral infection.

The endo D1 endonuclease inhibitor or SSA enhancer is suitable for a range of tumors, as well as other types of tumors such as lung cancer, cervical cancer, lymphoma, leukemia, prostate cancer, melanoma, endometrial cancer, glioma, etc., which have DNA damage response and repair defects, including but not limited to tumors with DNA damage response, DNA repair gene mutations 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 with cancer suppressing gene mutations such as TP53, PTEN, CDKN2A, APC, p16INK4A, p ARF, LKB1, FBXW, VHL, WT-1, etc.

Another object of the present invention is to reduce or eradicate HBV-infected cells or hepatitis cells expressing the HBx gene, reduce serum viral antigen titer and viral load in HBV carrier patients, and reduce infectivity. The method comprises administering to the patient an effective amount of an endo d1 endonuclease inhibitory formulation or SSA enhancer.

As used herein, the aforementioned endo-1 endonuclease inhibitory drugs include any agent that can reduce, decrease, hinder, impair and prevent the activity of endo-1 endo-endonuclease in cells relative to a control solvent, or any agent that can reduce or decrease the gene expression level and protein level of endo-1 endo-endonuclease. In particular, ENDOD1 endonuclease proteins comprise distinct regulatory regions of protein activity, and thus related drugs should also include agents that inhibit the activity of this region, including signal peptide processing, protein cleavage, subcellular localization of the nucleus, golgi and nuclear membranes, and ENDOD1 endonuclease degradation and ubiquitination, among others. In particular, these agents also include agents that reduce, slow, damage and prevent the endo 1 endonuclease active protein region.

The endo 1 endonuclease inhibitory drug acts as a target for inhibiting the function of endo 1 endonuclease, including reducing or completely eliminating endo 1 endonuclease gene expression and/or protein levels, or inhibiting endo 1 endonuclease enzyme activity, interfering with DNA damage repair functions, blocking protein cleavage, processing and maturation, disrupting subcellular localization, disrupting ubiquitination regulation, or interfering with biological functions such as protein cleavage and maturation of endo 1 endonuclease. These agents also include agents that reduce, slow, damage and prevent the endo d1 endonuclease active protein region.

Here, the SSA enhancer includes any agent capable of up-regulating, increasing the activity of single-stranded fusion repair (SSA) relative to a control solvent in a cell, or any agent capable of up-regulating, decreasing or lowering the expression level and protein level of single-stranded fusion repair (SSA) genes. In particular, SSA enhancers include distinct regulatory regions of protein activity, and thus related drugs should also include agents that enhance the activity of that region, including subcellular localization of protein maturation, cell nucleus, chromatin and DNA breakpoints, as well as phosphorylation, acetylation, degradation, ubiquitination of SSA regulatory factors, and the like. Specifically, these agents also include agents that can affect the single-stranded DNA fusion repair (SSA) active protein regions of RAD52, MRE11, and RAD54B, etc. The endo 1 endonuclease inhibitory drugs or SSA enhancers may be any form of small molecules, antibodies and antibody fragments, gene editing, polypeptides, and antisense compositions.

In particular, endo D1 endonuclease inhibition drugs may be a class of molecules targeting endo D1 endonuclease mRNA and DNA, which act to reduce and prevent endo D1 endonuclease protein expression. Specifically, the endo-1 endonuclease-related agent may interfere with the nucleic acid sequence of the DNA or RNA of the endo-1 endonuclease, e.g., siRNA, shRNA, dsRNA, miRNA, amiRNA, an antisense oligonucleotide, or gene editing or gene knockout using CRISPR/CAS9 or derivatization methods, to reduce the expression level of the endo-1 endonuclease or its regulatory genes. Such drugs may be in modified or unmodified form. Such modified drugs may include modified nucleotides, sugar groups, modified backbone carriers and any prior modifications. For example, modifications include, but are not limited to, R2' O-Me modifications, nucleotide 2' -fluoro (2 ' -F) modifications, substitution of nucleotide analogs, backbone phosphorylation modifications, and the like.

The present invention also provides an siRNA or shRNA inhibiting expression of an ENDOD1 gene, which is a single-stranded or double-stranded RNA molecule of 12 to 100 nucleotides, wherein one strand comprises 12 to 24 continuous or discontinuous sequences that can be matched with a certain stretch of nucleotides of the ENDOD1 gene, preferably, the siRNA inhibiting expression of the ENDOD1 gene is a single-stranded or double-stranded RNA of 18 to 28 nucleotides in length, more preferably, the siRNA inhibiting expression of the ENDOD1 gene, the nucleotide sequence of which is selected from the group consisting of SEQ ID No.2-No.353 sequences.

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

5’-CCAGCGCGAGCCAGCGCGCGG-3’

5’-CAGCCTCTTCGCCCTGGCTGG-3’

5’-TGTCACATTCGCCAAAGCCGG-3’

5’-CCCGGGTGCTGTAGAGGGTGG-3’

The endo D1 endonuclease inhibitory drugs or SSA enhancers described above, including compositions containing them, are useful in the treatment of tumors and related diseases. Endo 1 endonuclease inhibitory drugs or SSA enhancers may be used in combination with other anti-tumor therapies (e.g., anticancer drugs, surgery, transplantation, and radiation therapy) that have synergistic effects. Further, the synergistic use with other drugs includes all agents and modes of administration that can increase the anticancer effect of the combination. Such agents that produce a synergistic effect include any cancer treatment modality, including but not limited to agents, therapeutic combinations, drug techniques (e.g., surgery, radiation therapy, etc.). Specific anti-cancer therapies include, but are not limited to, surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, and immunotherapy in combination and alone.

The combined medicine can be taken by two medicines respectively at intervals or simultaneously, or a medicine composition is formed by the endo D1 endonuclease inhibitory medicine or SSA enhancer and other targeted anticancer medicines.

The combination or composition of the endo-1 endonuclease inhibitory drug or SSA enhancer further includes combination with a DNA toxic anticancer drug such as platins (including but not limited to cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, TRIPLATIN, LIPOPLATIN), camptothecins, mitomycins, and the like. The combined administration can be carried out together or at intervals.

The combination or composition of the endo D1 endonuclease inhibitory drug or SSA enhancer further comprises a PARP inhibitor, a DNAPK inhibitor, an HDAC inhibitor, a ATMi inhibitor, a vinca alkaloid, an antitumor alkaloid, a monoclonal antibody and an antimetabolite. The combined administration can be carried out together or at intervals.

The combination or composition of the endo-1 endonuclease inhibitory drug or SSA enhancer described above further includes combination with radiation therapy, including but not limited to ⁶⁷Cu,⁶⁷Ga,⁹⁰Y,¹³¹I,¹⁷⁷LU,¹⁸⁶Re,¹⁸⁸Re,α_Particle emitter,²¹¹At,²¹³Bi,²²⁵Ac,Auger-electron emitter,¹²⁵I,²¹²Pb,and ¹¹¹In., which may be administered concurrently with the radiation therapy or at intervals.

Further, examples of antineoplastic alkylating agents include, but are not limited to, nitrogen mustard, cyclophosphamide, methyl dioxyethylamine or moustine (HN 2), uramustine or uracil, mustard, lysosarcoma, chloramphenicol, isophosphamide, bendamustine, nitrosoureas, carmustine, lomustine, streptozotocin, alkyl sulfonates, butyl dimesylate, thiotepa, promazine, hexamethylmelamine, triazine, dacarbazine, mitozolomide, and temozolomide. The combined administration can be carried out together or 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-Il31、ibritumomab tiuxetan、alemtuzumab, gemtuzumab, trastuzumab and rituximab, vinca alkaloids and derivative analogs. The combined administration can be carried out together or at intervals.

Further combinations or compositions of the aforementioned endo 1 endonuclease inhibitory drugs or SSA enhancers include, but are not limited to, antimetabolite agents. For example, agents include, but are not limited to, fluorouracil, cladribine, capecitabine, 6-mercaptopurine, pemetrexed, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarbine, clofarabine, cytarabine, decitabine, deazaaminopterin, 5-fluorouracil deoxynucleoside, 6-thioguanine.

Further combinations or compositions of the aforementioned endo 1 endonuclease inhibitory drugs or SSA enhancers include, but are not limited to, cellular immunotherapy, antibody therapy or cytokine therapy. Endo 1 endonuclease inhibitory drugs act synergistically with immunotherapy. Examples of cellular immunotherapy include, but are not limited to, dendritic cell therapy and Sipuleucl-T. Examples of antibody therapies include, but are not limited to, necitumumab、dinutuximab、nivolumab、Blinatumab、pembrolizumab、ramucirumab、obinutuzumab、adotrastuzumab emtansine、pertuzumab、brentuximab、ipilimumab、ofatumumab、catumaxomab、bevacizumab、cetuximab、tositumomab-Il31、ibritumomab tiuxetan、alemtuzumab. cytokine therapies, examples of which include, but are not limited to, interferons (e.g., IFNa, IFNp, IFNy, IFNX) and interleukins. In some embodiments, the immunotherapy comprises 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 combined administration can be carried out together or at intervals.

Further, other examples of anti-cancer therapies include, but are not limited to, diformate acetate (e.g., ZYTIGA), ABVD, ABVE, ABVE-PC, AC-T, ADE, ado-trastuzumab antracetam (e.g., KADCYLA), afatinib diformylate (e.g., GILOTRIF), aldesleukin (e.g., PROLEUKIN), alemtuzumab (e.g., CAMPATH), anastrozole (e.g., ARIMIDEX), arsenic trioxide (e.g., TRISENOX), asparaginase (e.g., ERWINAZE), axitinib (e.g., INLYTA), azacytidine (e.g., MYLOSAR, VIDAZA), BEACOPP, belinostat (e.g., BELEODAQ), bendamustine hydrochloride (e.g., TREANDA) BEP, bevacizumab (e.g., AVASTIN), bicalutamide (e.g., CASODEX), blomycin (e.g., BLNOXAXA-E), blonamab (e.g., BLINCYTO), bortezomib (e.g., VELCADE), bo Su Tini (e.g., BOSULIF), pertuzumab (e.g., ADCETRIS), busulfan (e.g., BUSULFEX, MYLERAN), cabazitaxel (e.g., JEVTANA), cabazitaxel malate (e.g., COMETRIQ), CAF, capecitabine (e.g., xelda), CAPOX, carboplatin (e.g., PARAPLAT, PARAPLATIN), carboplatin paclitaxel, carfilzomib (e.g., KYPROLIS), carbomycin (e.g., BECENUM, BICNU, CARMUBRIS), carbomycin implants (e.g., GLIADEL WAFER, GLIADEL), ceritinib (e.g., ZYKADIA), cetuximab (e.g., ERBITUX), chloramphenicol (e.g., AMBOCHLORIN, AMBOCLORIN, LEUKERAN, LINFOLIZIN), chloramphenicol prednisone, CHOP, carboplatin, cisplatin (e.g., PLATINOL, PLATINOL-AQ), chlorofluorobutane (e.g., CLOFAREX, CLOLAR), CMF, COPP, COPP-AB V, crizotinib (e.g., XALKORI), CVP, cyclophosphamide (e.g., CLAFEN, CYTOXAN, NEOSAR), cytoside (CYTOSAR-U, TARABINE PFS), dabrafenib (e.g., TAFINLAR), and combinations thereof, Dacarbazine (e.g., DTIC-DOME), actinomycin (e.g.,), (e.g., ONTAK), denosumab (e.g., procia, XGEVA), (e.g.,), docetaxel (e.g., TAXOTERE), (e.g., DOXIL, DOX-SL, EV ACET, LIPODOX), (e.g., XT ANDI), (e.,), EPOCH, (e.g., TARCEVA), etoposide (e.g.,), (e.g., ETOPOPHOS), everolimus (e.g.,), exemestane (e.g., FEC, (e.g.,), fluorouracil (e.g., x), irinotecan, irinotecan in combination with bevacizumab, irinotecan in combination with cetuximab, oxaliplatin in combination with chemotherapy, oxaliplatin, FU-LV, fulvestrant (e.g., FASLODEX), gefitinib (e.g., IRESSA), gefitinib hydrochloride (e.g., gemzaar), gefitinib in combination with cisplatin, gefitinib in combination with oxaliplatin (e.g.,), hyper-CVAD, temozolomab (e.g., ZEVALIN), (e.g.), ICE (e.g., a), ICE (e.g.,), (e.g., GLEEVEC), imiquimod (e.g., ALDARA), ipilimumab (e.g., YER VOY), irinotecan hydrochloride (e.g., CAMPTOSAR), ixabepilone (e.g., IXEMPRA), IXEMPRA (e.g., IXEMPRA), letrozole (e.g., FEMARA), IXEMPRA (e.g., IXEMPRA), IXEMPRA (e.g., IXEMPRA), methotrexa (e.g., IXEMPRA-AQ), mitomycin C (e.g., IXEMPRA), IXEMPRA, MOPP, IXEMPRA (e.g., IXEMPRA), IXEMPRA (e.g., IXEMPRA), nivolumab (e.g., IXEMPRA), obinutuzumab (e.g., IXEMPRA), IXEMPRA, ofatumumab (e.g., IXEMPRA), OFF, olapanib (e.g., IXEMPRA), IXEMPRA, oxaliplatin (e.g., ELOXATIN), paclitaxel (e.g., TAXOL), IXEMPRA), paclitaxel nanoparticle agents (e.g., IXEMPRA), PAD, palbociclib (e.g., IXEMPRA), pamidronate disodium (e.g., arida), panitumumab (e.g., IXEMPRA), panobinostat (e.g., IXEMPRA), IXEMPRA (e.g., IXEMPRA), pessase (e.g., IXEMPRA), alfa-2b polyethylene glycol interferon (e.g., PEG-INTRON, SYLATRON), pembrolizumab (e.g., 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., MATULANE), radium 223dichloride (e.g., XOFIGO), raloxifene hydrochloride (e.g., EVISTA, KEOXIFENE), ramucirumab (e.g., CYRAMZA), R-CHOP, recombinant human papilloma virus bivalent vaccine (e.g., CERVARIX), recombinant human papilloma virus (e.g., HPV) monovalent vaccine (e.g., GARD AS IL 9), nonavalent vaccine (e.g., HPV) tetravalent vaccine (g., GARDASIL), alfa-2b recombinant leukocyte interferon (e.g., INTRON A), regorafenib (e.g., GARDASIL), rituximab (e.g., RITUXANN), romide (e.g., 2), 2 (e.g., GARDASIL), 2 (e.g., 39352), e.g., 2-methyl, 393 (e.g., GARDASIL), oxamide (e.g., 393-GARDASIL), oxamic acid (e.g., 393-393), etc. (e.g., 393-methyl), oxamic acid (e.g., 393-393, GARDASIL, 393, etc., FARESTON), tositumomab, and radioiodine (I131) plus tositumomab (e.g., BEXXAR), TPF, trametinib (e.g., MEKINIST), trastuzumab (e.g., HERCEPTIN), VAMP, vandetanib (e.g., CAPRELSA), VEIP, vemurafenib (e.g., ZELBORAF), vinblastine sulfate (e.g., VELBAN, VELSAR), VINCRISTINE SULFATE (e.g., VINCAS AR PFS), VINCRISTINE SULFATE LIPOSOME (e.g., MARQIBO), vinorelbine tartrate (g., navlbine), vismodegib (e.g., ERIVEDGE), vorinostat (e.g., ZOLINZA), xelairi, XELOX, ziv-aflibercept (e.g., ZALTRAP), zoledronic acid (e.g., ZOMETA), or any combination thereof.

Further, the anticancer therapy may be selected from epigenetic and transcriptional regulatory factors. Examples include, but are not limited to, DNA class a transferase inhibitors, histone acetylation inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotics (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors, protein stabilization modulators (e.g., protease inhibitors), hsp90 inhibitors, glucocorticoids, trans-retinoic acid compounds, and other differentiation-promoting factors.

In particular, endo 1 endonuclease inhibitory drugs or SSA enhancers may be independently involved in any of the anti-cancer therapies. Such anti-cancer therapies include, but are not limited to, surgery, radiation therapy, transplantation (stem cell transplantation, bone marrow suppression), immunotherapy and chemotherapy.

Tumors for which particular of the aforementioned endo d1 endonuclease-inhibiting or SSA-potentiating therapeutic regimens are applicable include, but are not limited to, lung cancer (e.g., bronchogenic carcinoma, small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma), kidney cancer (e.g., nephroblastoma, also known as wilms' tumor, renal cell carcinoma), acoustic neuroma, adenocarcinoma, adrenal cancer, anal cancer, hemangioma (e.g., lymphoangioma, lymphangiocndothcliosarcoma, angiosarcoma) adnexa cancer, benign monoclonal gammaglobulina, gall bladder cancer, breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast medullary carcinoma), brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma), bronchogenic cancer, carcinoid tumor, cervical cancer (e.g., cervical cancer), gall bladder cancer, spinal tumor, craniopharyopharyoma, colorectal cancer (e.g., colon cancer), Rectal cancer, colorectal adenocarcinoma), connective tissue cancer, epithelial cancer, ependymoma, endothelial sarcoma (e.g., kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., esophageal adenocarcinoma, barrett's esophageal adenocarcinoma), ewing's sarcoma, ocular cancer (e.g., intraocular melanoma, retinoblastoma), eosinophilia, gallbladder cancer, gastric cancer (e.g., gastric adenocarcinoma), gastrointestinal stromal tumor (GIST), germ cell carcinoma, head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal carcinoma), Pharyngeal cancer, nasopharyngeal cancer, and throat cancer), heavy chain diseases (such as alpha chain disease, gamma chain disease, etc, μchain disease, angioblastoma, hypopharyngeal carcinoma, inflammatory myofibroma, immunocytoamyloidosis, liver cancer (such as hepatocellular carcinoma (HCC), malignant hepatoma), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), muscle cancer, myelodysplastic syndrome (MDS), mesothelioma, myelodysplasia (MPD) (e.g., polycythemia Vera (PV), primary thrombocythemia (ET), non-primary myeloid metaplasia (AMM), myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic Myelogenous Leukemia (CML), chronic Neutrophilic Leukemia (CNL), hypereosinophilic syndrome (HES), neuroblastoma, neurofibromas (e.g., multiple neurofibromatosis (Nr) type 1 or type 2, neurosphingomatosis), neuroendocrine carcinoma (e.g., GEP-NET), benign tumors), osteosarcoma (e.g., ovarian cancer, such as bone cyst), ovarian cancer (e.g., adenoma) Ovarian embryo cancer, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer, intraductal Papillary Myxoma (IPMN), islet cell tumor, penile carcinoma (e.g., scrotum Paget disease), pineal gland tumor, primitive neuroectodermal tumor, plasmacytoma, paraneoplastic syndrome, intraepithelial tumor, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous Cell Carcinoma (SCC), keratoacanthoma (KA), melanoma, basal Cell Carcinoma (BCC)), small intestine cancer (e.g., appendiceal carcinoma), soft tissue sarcoma (e.g., malignant Fibrous Histiocytoma (MFH), liposarcoma, malignant Peripheral Nerve Sheath Tumor (MPNST), Chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, small intestine cancer, sweat gland carcinoma, synovial carcinoma, testicular carcinoma (e.g., seminoma, testicular embryo carcinoma), thyroid carcinoma (e.g., papillary Thyroid Carcinoma (PTC), medullary thyroid carcinoma, urethra carcinoma, vaginal carcinoma, vulvar carcinoma (e.g., paget's disease of vulva).

In particular, the treatment of cancer with endo d1 endonuclease inhibitory drugs or SSA enhancers, "treatment" refers to reversing, alleviating, delaying the onset of, or progression of cancer. In particular, treatment may be performed after one or more signs or symptoms of the disease have progressed, or have been observed, or in asymptomatic and signaled disease. For example, the susceptible person can be treated prior to the appearance of symptoms (e.g., based on a history of symptoms and/or a history of pathogen exposure). Treatment may also be continued after the symptoms have disappeared, for example, to delay and/or prevent recurrence.

In particular, "administering" an endo-1 endonuclease inhibitory drug or SSA enhancer in the treatment of cancer refers to implanting, orally administering, absorbing, injecting, inhaling, or in any other manner introducing a DO inhibitory drug or SSA enhancer as described herein, or a composition thereof, into or out of a subject.

In the context of the present invention, the term "administering" refers to implanting, orally administering, absorbing, injecting, inhaling, or in any other way introducing into or out of the body of a subject an endo 1 endonuclease inhibitory drug or SSA enhancer as described herein, or a combination thereof.

The term "single-stranded DNA fusion repair", SINGLE STRAND ANNEALING (SSA), refers to a repair that relies primarily on RAD52 mediation, requiring single-stranded DNA and homologous sequences. The regulation and control channels also comprise RAD52, MRE11, RAD54B, RPA and other factors.

The term "inhibit" refers to the ability of a compound to reduce, slow, stop and/or arrest the activity of a particular biological process within a cell relative to a vector. By "control" or "prevent" is meant that the activity is inhibited, hindered, controlled or prevented by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%,60%, 65%, 70%,75%, 80%, 85%,90%, 95%, or 100% compared to the control activity. Specifically, "inhibit, hinder, control or prevent" means that the expression of the inhibitor target is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%,60%, 65%, 70%,75%, 80%, 85%,90%, 95% or 100% as compared to the control. Specifically, "inhibit, hinder, control or prevent" means that the targeted activity (e.g., the biological enzyme activity) of the inhibitor is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%,60%, 65%, 70%,75%, 80%, 85%,90%, 95%, or 100% as compared to the control.

An "effective dose" refers to a dose capable of eliciting a desired biological response. Such reactions can be understood by conventional techniques in the art. The effective dosage of the ingredients of the present invention may vary depending on the desired biological endpoint, the pharmacokinetics of the compound, the therapeutic environment, the mode of administration, the age and health of the subject. Effective dosages include, but are not limited to, dosages necessary to slow, reduce, inhibit, ameliorate, reverse one or more symptoms associated with cancer. In particular, an "effective dose" refers to a dose of an agent (e.g., an ENDOD1 inhibitory drug or SSA enhancer) that results in a tumor cell species that reduces ENDOD1 expression or/and activity. An effective dosage range for "reducing ENDOD1 expression or/and activity" includes any number between 2-fold and 100-fold, and any number between 100% and 1%. In particular, an effective dose in the treatment of cancer refers to a dose that achieves a lack of expression of the ENDOD1 protein or/and its protein activity in tumor cells.

If two or more inhibitors are administered to a subject, the effective amounts may be co-selected amounts. When the first inhibitor is used with the second and optionally with the third inhibitor, the selective amount of the first inhibitor may be different. When two or more inhibitors are used simultaneously, the effective amount of each inhibitor may be the same or the same as when used alone. The expected effect can be achieved at lower doses, and the effective amount of each drug can be less than when used alone. In particular, where a subject is better able to tolerate one or more inhibitors, and higher doses may provide greater therapeutic benefit, the effective amount of each drug may be greater than when used alone. In particular, the effective amount of one compound may vary from about 0.001mg/kg to about 1000mg/kg depending on the day or days of administration (depending on the mode of administration) of the one or more administrations. In certain embodiments, the effective dose varies from about 0.001mg/kg to about 1000mg/kg, from about 0.01mg/kg to about 750 mg/kg. From about 0.1mg/kg to about 500mg/kg, from about 1.0mg/kg to about 250mg/kg, from about 10.0mg/kg to about 150mg/kg. One of ordinary skill in the art will be able to empirically determine the appropriate therapeutically effective amount.

"Subject" or "patient" or "subject" includes, but is not limited to, humans and animals capable of suffering from cancer, or any disease that is directly or indirectly related to cancer. The subject includes a mammal. Such as humans, dogs, cattle, horses, pigs, sheep, goats, cats, rats, rabbits, mice, and transgenic non-human animals. In particular, "subject" or "patient" or "subject" includes companion animals such as dogs, cats, rabbits, and rats. In particular, "subject" or "patient" or "subject" includes livestock, such as cattle, pigs, sheep, goats, and rabbits. In particular, "subject" or "patient" or "subject" includes a purebred or displayed animal, such as horses, pigs, cattle, and rabbits. In particular, a "subject" or "patient" or "subject to be treated" is a human, e.g., a human having or likely to be at risk of cancer.

The compounds described in this patent may be administered to a subject in any order. The first therapeutic agent, e.g., an ENDOD1 inhibitor, can be administered to the subject concomitantly with or subsequent to (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 later) administration of the second therapeutic agent (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 later). Thus, the ENDOD1 inhibitor may be administered separately, sequentially or simultaneously with a second therapeutic agent (e.g., a chemotherapeutic agent as described herein).

The compounds described herein may be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal. Interaction specification. Rectal, intravaginal, intraperitoneal, topical (e.g., powder, ointment, salve and/or drops), mucosal, nasal, buccal, sublingual, intratracheal instillation, bronchial instillation and/or inhalation, and/or oral spray, nasal spray and/or aerosol. In particular, routes contemplated include oral, intravenous (e.g., systemic intravenous), regional administration via blood and/or lymph supply, and/or direct administration to a lesion or suspected lesion site. In particular, 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 is able to tolerate oral administration). The particular amount of compound required (to achieve an effective amount) will vary from subject to subject, e.g., depending upon the species, age and health of the subject, the severity of side effects or disorders of the individual's biological system, the nature of the particular compound, the mode of administration, and the like. The desired dose may be administered three times daily, twice daily, once daily, every other day, every third day, weekly, every second week, every third week, or every fourth week. In particular, the desired dose may be administered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations). In particular, an effective amount of a compound administered one or more times per day to a 70kg adult human may comprise from about 0.0001mg to about 3000mg, from about 0.0001mg to about 2000mg, from about 0.0001mg to about 1000mg. About 0.001mg to about 1000mg, about 0.01mg to about 1000mg, about 0.1mg to about 1000mg, about 1mg to about 100mg, about 10mg to about 1000mg, or about 100mg to about 1000mg of the compound per unit dosage form.

In particular, the compounds provided herein may be delivered at a dosage level sufficient to deliver from about 0.001mg/kg to about 100mg/kg, from about 0.01mg/kg to about 50mg/kg, preferably from about 0.1mg/kg to about 40mg/kg, preferably from about 0.5mg/kg to about 30mg/kg, from about 0.01mg/kg to about 10mg/kg, from about 0.1mg/kg to about 10mg/kg, more preferably from about 1mg/kg to about 25mg/kg of the subject's body weight, once or more per day, to achieve the desired therapeutic effect. It is noted that the dosage ranges as described herein provide guidance for administration of the provided pharmaceutical ingredients to adults. The particular amount administered to a child or adolescent may be determined by a physician or skilled artisan and may be less than or equal to the amount administered to an adult.

In the present embodiment, the term "endoD1 endonuclease" refers to the gene of human endoD1 endonuclease (NCBI gene ID, 23052), mRNA (NM-015036.3) and protein (NP-055851.1). .

In the present embodiment, the endo 1 endonuclease inhibitory drug may be administered by parenteral route such as oral administration or injection, or may be administered by biological medium such as lentivirus, adenovirus, etc.

In another aspect, it is a further object of the present invention to determine methods and strategies for cell biology screening of endo 1 endonuclease inhibitory drugs, including but not limited to methods of cell biology described herein, including ion-beam or chemotherapy drug treatment, using immunofluorescent or fluorescent fusion proteins to determine the aggregation capacity of functional proteins in cells at DNA breakpoints, such as reduced signals for endo 1 endonuclease, MRE11, gH2AX, 53BP1 and single stranded DNA production, enhanced aggregation signals for RAD52, ATM-pSerine 1981, BLM, etc.

In yet another aspect, a further object of the invention is to identify screening methods and strategies for endo d1 inhibitory drugs, including the in vitro endonuclease activity assay methods, screening methods for cellular level SSA dysfunction, and screening methods for inhibitors of endo d1 gene expression described herein. Specific assay signals include, but are not limited to, fluorescence, biotin, absorbance, chemiluminescence, electrical signals, radiological imaging, and the like, and derivatization assays include competitive inhibition of enzymatic activity, flow cytometry, scintillation proximity detection (SPA), fluorescence polarization detection (FPA), surface plasmon resonance techniques, and the like.

Drawings

FIG. 1, human endo D1 endonuclease gene and protein sequence characterization

(A-B) the coding sequences of mRNA (NM-015036.3) and protein (NP-055851.1) of the human endoD1 endonuclease gene (23052), (C) the protein domain comprising a signal peptide, an endonuclease and active sites (residues C190, C224 and C269), transmembrane region I-III, (D) the structure of the endoD1 endonuclease gene comprising 2 exons and 1 intron sequence, (E) the phylogenetic tree of the endoD1 endonuclease gene drawn by Genetree software, the homologous genes of which are present in higher eukaryotes but not in lower eukaryotes such as yeast, drosophila and nematodes;

FIG. 2, characteristics of human endo-1 endonuclease and post-translational modification

(A) The CRISPR/CAS9 gene of the ENDOD1 endonuclease gene was edited, the gRNA target is shown as A1, and the underlined nucleotide is PAM sequence. A2 shows the construction of the CAS9-gRNA gene editing plasmid used, and (B) 3 small interfering RNA sequences (B1) and silencing effect (B2) used for silencing the endoD1 endonuclease gene, si001 is the siRNA commonly used in this study. And (C) immunoWestern blotting to determine the protein product of the endoD1 endonuclease in RPE-1 cells, which has a major band size of about 55Kd (full length, a). And (D) an RPE-1 cell line with the gene knockout (2F 4) or mutation (2F 6) of the endoD1 endonuclease obtained by editing the CAS9-gRNA gene. 2F4 was a complete knockout and no endo 1 endonuclease protein band was detected by western blotting. 2F6 is protein processing mutation, only expresses a band and b and c bands are deleted, and (E) immunoblotting detection of siRNA (si 001) gene silencing RPE-1 cells. The protein subtype e of the ENDOD1 endonuclease is generated by ubiquitination modification, wherein the modification mode comprises polyubiquitination modification of lysine 48 (K48) and lysine 63 (K63), the signal peptide position (residue 22) of the ENDOD1 endonuclease predicted by bioinformatics software (PrediSci) is a possible protease cleavage site, and the low-order region (discrete) of the ENDOD1 endonuclease is predicted by software (H) NetSurfP-2.0, residues 300-325 are found to be potential protease cleavage sites, and b, c and d subtypes of the ENDOD1 endonuclease are generated together with the cleavage activity of the residue LEG-RL sequence;

FIG. 3 binding of human endo-1 endonuclease to DNA damage site

(A) Immunofluorescence showed that endonucleases were primarily localized to the cytoplasm and nuclear membrane in normally growing cells. After DNA damage (ion radiation exposure), the ENDOD1 endonuclease can enter the DNA breakpoint at 40 minutes to form an ENDOD1 endonuclease aggregation point and persist for 2 hours after exposure. The method comprises the steps of (A) performing a nuclear DNA transfection with DAPI, (B) performing immunofluorescence to show that endo D1 endonuclease can enter a DNA breakpoint to form an aggregation point after treatment of Camptothecine (CPT) and Hydroxyurea (HU), (C) performing immunofluorescence to show that the endo D1 endonuclease can co-locate with gamma H2AX at the DNA injury aggregation point after irradiation of ion rays, as shown by an arrow, (D) performing a chromatin immunoprecipitation experiment to prove that the endo D1 endonuclease is combined at 0.5, 1 and 2.5Kb from the DNA breakpoint, (E) performing a chromatin immunoprecipitation experiment of a single-stranded DNA binding factor (RPA 32) to prove that the RPA32 has no obvious defect after silencing of the endo D1 endonuclease, but can eliminate the combination of the RPA at the DNA breakpoint under the condition of co-silencing of homologous recombination repair factor (WDR 70), and (F) performing a chromatin separation experiment to prove that a, B, C, D forms of the endo D1 endonuclease can be tightly combined with chromatin;

FIG. 4, human endo D1 endonuclease involved in DNA repair

(A) After silencing the endoD1 endonuclease gene in RPE-1 cells, DNA break points were determined by I-Sce1 induction of DNA breaks on pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids. The endoD1 endonuclease gene silencing slightly inhibited non-homologous END joining (NHEJ) and Homologous Recombination (HR) repair functions, more importantly promoted single strand DNA fusion (SSA), as shown in FIG. A, tested DNA fragmentation repair efficiency in RPE-1 wild-type, 2F4 and 2F6 cells, and (C) the doxycycline-induced wild-type endoD1 endonuclease plasmid (Wt) was transfected into 2F4 cells with the endoD1 endonuclease gene knockdown to correct the repair abnormalities, whereas the endoD1 endonuclease plasmid (ND) with the endoD endonuclease inactivation introduced (ΔEND: endonuclease deleted) had no correction function. Demonstrating that the endo D1 endonuclease regulates DNA repair depending on the endonuclease activity thereof, and (D) the RPE-1 cells silence the endo D1 endonuclease and/or SSA functional gene (RAD 52), and the DNA fragmentation repair efficiency is tested. RAD52 silencing can reverse the hyperfunction of SSA caused by the defect of the ENDOD1 endonuclease, and (E-F) immunofluorescence shows that the RPE-1 cell knockout (E) or silencing (F) the ENDOD1 endonuclease gene can improve the aggregation of the RAD52 to the DNA breakpoint, which indicates that the ENDOD1 endonuclease can inhibit the SSA repair efficiency by reducing the recruitment of the RAD52 to the DNA damage site. Both 2F4 and 2F6 had the defect of RAD52 hyperactivity compared to wild-type RPE-1. E1, F1 immunofluorescent staining pictures, white circles indicate the positions of the nuclei. E2, F2. RAD52 counts of aggregation at the site of injury. Cellular DNA was transfected with DAPI. The statistical methods described herein all use a two-tailed T test to verify the P value, P.ltoreq.0.05, if not specifically stated. NS, no statistical difference;

FIG. 5 regulation and control mechanism of endonucleases for other DNA repair factors

(A) silencing the aggregation points of the ENDOD1 endonuclease gene expression inhibiting IR-induced gamma H2AX and 53BP1, (B) knocking out or mutating the aggregation points of the ENDOD1 endonuclease RPE-1 cells (2F 4 and 2F 6) failing to form IR-induced FK2, (C) silencing the aggregation points of the ENDOD1 endonuclease gene expression enhancing IR-induced ATM autophosphorylation (pS 1981), and (D-E) silencing the aggregation points of the ENDOD1 endonuclease gene expression inhibiting IR-induced BLM (D) and MRE11 (E);

FIG. 6 cleavage effect of endonucleases for damaged DNA

(A) BL21 bacteria purified endo 1 endonuclease-STREP tag protein. Including the Full Length (FL) of the ENDOD1 endonuclease, residues 22-233 and 1-344. SN: cell lysate; pu: purified endo 1 endonuclease composition; B) 0.5. Mu.g of purified endo 1 endonuclease domain fragment (residues 22-344) was added with 0.37. Mu.g of DNA double-stranded fragment or DNA/RNA hybrid and incubated in cleavage buffer (100mM NaCl,50mM Tris-HCl,10mM MgCl2,100. Mu.g/ml BSA, pH 7.9). DNA double strand comprising three forms of blunt end (30 bp), 3 '(28 bp) and 5' -overhang (28 bp) and (C) 0.5. Mu.g of purified endoD1 endonuclease protein was added to 0.2. Mu.g of each treated pEGFP-C3 plasmid DNA and subjected to in vitro digestion reaction (37 ℃ C., 16 hours) in digestion buffer. Endo D1 endonucleases can cleave plasmids with structural lesions such as UV-lesions, DNA double strand breaks (5 'and 3' overlapping), oxidative lesions (hydrogen peroxide treatment). HTRA2 purified protein served as a control without nuclease activity;

FIG. 7, endoD1 endonuclease gene knockout or silencing did not affect proliferation of normal non-tumor cells

(A) Normal somatic (RPE-1, MRC-5, GES-1 and L02) cells silence the endoD1 endonuclease gene (siEN) and determine its proliferation profile;

FIG. 8, endoD1 endonuclease gene silencing inhibiting proliferation of tumor cells

The proliferation rate was measured by transfection of endo 1 endonuclease specific siRNA (si 001) into the tumor cells shown in (A-B). Tumor cells exhibit both the characteristics of sensitivity (a) and insensitivity (B). Calculating cell proliferation fold of experimental end point by MDA-MB-231, NCI-H1299 and HL60 cells, (C) carrying out crystal violet staining on part of the test cells at the experimental end point, wherein the dark color shows the remained living cells, (D) carrying out immunoblotting to show the silencing effect of siENDOD1 in insensitive cells (HO 8910-PM, SW480, GES-1 and L02), (E) silencing ENDOD1 in SKOV3 cells, simultaneously carrying out doxycycline induced overexpression of ENDOD1 wild type resisting siENDOD1 or nuclease inactivation (ND), detecting cell proliferation fold, and (F) simultaneously transfecting siRNA of ENDOD1 endonuclease and RAD52 in SKOV3 cells, and measuring cell proliferation fold;

FIG. 9, combined lethal effects of endonucleases and DNA damage response and homologous recombination repair genes

(A) The proliferation capacity of RPE-1 and 2F4 cells was measured after treatment with DNA damage response factor inhibitors or gene silencing. The living cells are stained by crystal violet, and (B) the proliferation capacity and the combined lethal effect of the endo D1 endonuclease after being co-silenced with homologous recombination repair genes such as BRCA1, BRCA2, BLM, MRE11, CTIP, EXO1, ARID1A/RID1B, WDR70 and the like are measured, and (C) the quantitative graph of the combined lethal effect of the endo D1 endonuclease after being co-silenced with the DNA repair genes is provided. The vertical axis shows the proliferation multiple of the cells at the experimental end point (12 days), 3 different ENDOD1 endonuclease siRNAs and BRCA1 genes are co-silenced to have combined lethal effect on RPE-1 cells, and (E) the silenced BRCA1, FANCD2 or FANCC genes in the cells (2F 4) are knocked out, and the combined lethal effect is measured;

FIG. 10, combined lethal effect of endonucleases and oncogenes of endoD1

(A) silencing TP53 gene in RPE-1 cells or 2F4 cells silencing the ENDOD1 endonuclease, carrying out crystal violet staining on surviving cells after 12 days, (B) silencing the ENDOD1 endonuclease and TP53 on a tumor cell line (HO 8910-PM) with high level of TP53 at the same time, measuring proliferation times and crystal violet staining of experimental end points, (C) simultaneously transfecting a wild type TP53 expression plasmid (oeTP) in cells with pure and mutant TP53 gene silencing the ENDOD1 gene (NCI-H1975), carrying out gene back-repair, measuring proliferation times and crystal violet staining of experimental end points, and (D) silencing cancer suppressor gene PTEN in RPE-1 and 2F4 cells, measuring proliferation times and crystal violet staining of experimental end points;

FIG. 11, analysis of correlation of tumor cell gene mutations with endoD1 endonuclease gene silencing cytotoxicity

The protein level of the ENDOD1 endonuclease in normal human tissues shown in the immunoblotting experiment detection and the tumor cell line, (B) the protein level of the ENDOD1 endonuclease in normal human tissues shown in Human Protein Atlas database, (C) the expression level of the ENDOD1 endonuclease in human tumor tissues of various sources in Human Protein Atlas database, and (D) the inhibition effect of the gene silencing of the ENDOD1 endonuclease on tumor cells with TP53 and other gene mutations (such as BRCA1, BRCA2 and the like) is obviously higher than that of non-mutated tumor cells. The difference between groups is verified by using a double-tail T test, and P is less than or equal to 0.05 and is considered to be a significant difference;

FIG. 12 sensitization to chemotherapy by endo D1 endonuclease gene silencing

Normal (RPE-1) and tumor (a 549) cells were treated with targeting inhibitors such as CPT, cisplatin, DNAPK, etc. after silencing endo 1 endonuclease, and their proliferation curves were determined;

FIG. 13 therapeutic effect of endoD1 endonuclease gene silencing on mouse tumor-bearing models

(A) A nude mouse subcutaneous tumor model of SKOV3, HCC1937 and OVCAR-8TR cells was established, control or ENDOD1 endonuclease specific siRNA (si 001) was injected twice weekly in vivo, and tumor volumes were determined. Silencing the inhibition rate (T/C%) of the ENDOD1 gene to 3 tumor models;

FIG. 14, combination lethal effect of endo D1 endonuclease function inhibition and active hepatitis B Virus on cells

(A) The combined lethal effect of endo D1 endonuclease and WDR70 on HBV negative liver cells (L02), and (B) silencing endo D1 endonuclease gene in HBV positive (T43) or negative cells (L02), and determining the proliferation capacity thereof. Establishing T43 (a nude mouse subcutaneous tumor model, injecting control or endoD1 endonuclease siRNA twice a week, measuring tumor volume (C1), dissecting experimental end points and separating control and silencing endoD1 tumor (C2), a group of experimental end point tumor dissected samples, (E) determining the titer of HBV antigen (HBsAg) in serum at the indicated time point by a nude mouse vein inoculation HepG2.2.15, ELISA method, (F) determining the titer of HBV viral DNA in serum at the indicated time point by a fluorescent quantitative PCR determination method by using a nude mouse vein inoculation HepG2.2.15;

FIG. 15 in vitro titration of endo D1 endonuclease activity

The competitive inhibition method is used for screening the ENDOD1 endonuclease activity small molecule inhibitor flow chart, which comprises the steps of adding ENDOD1 protein, linear double-stranded DNA substrate and small molecule inhibitor library into a 384-hole PCR skirt plate, performing enzyme digestion reaction, adding fluorescent quantitative PCR premix containing primers, performing fluorescent quantitative PCR to detect the residual quantity of the DNA substrate and enzyme digestion efficiency, and screening effective small molecule inhibitor.

Detailed Description

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

In the process of researching a DNA damage response mechanism, the invention unexpectedly discovers that the endonuclease coded by the ENDOD1 endonuclease gene has the function of inhibiting RAD52 mediated SSA repair and has less influence on other DNA repair mechanisms. The literature reports that endo-1 endonuclease is considered an oncogene, silencing the gene function can promote tumor proliferation (see ：Qiu,J.,et al.,Identification of endonuclease domain-containing 1as a novel tumor suppressor in prostate cancer.BMC Cancer,2017.17(1):p.360.). but our study surprisingly found that inhibition of endo-1 endonuclease function can be shown to have strong synthetic lethal effects with defects in DNA damage response (e.g., ATM, CHK 1), DNA repair defects (e.g., BRCA1, BRCA2, MRE11, FANC, positive for hepatitis b virus, etc.), and can also be synthetic lethal with cells harboring oncogene mutations (e.g., TP53, PTEN), and can effectively kill tumor cells harboring these defects; in contrast, in non-tumor cells with normal functions of cancer suppressor genes such as DNA damage response, DNA repair mechanism and TP53, the inhibition of the function of the endo D1 endonuclease can prevent the proliferation of the cells even if the endo D1 endonuclease gene is completely knocked out, and the inhibition of the expression or the function of the endo D1 endonuclease can make the tumor cells with or without the defects more sensitive to cytotoxicity treatment (radiotherapy or chemotherapy) and have synergistic sensitization effect, and the same drug treatment has no toxic effect on normal cells.

The types and sources of the cell lines in the following examples are shown in Table 1, and the sources are ATCC, china academy of sciences typical culture Collection Committee cell banks or the department of Cooperation of the biological research institute of Chengdu national academy of sciences. The ENDOD1 endonuclease gene edited RPE-1 cells (2F 4 and 2F 6) were constructed by the university of Sichuan Hua Xidi, two hospitals Liu Cong laboratory. Nude mice (BALB/c-Nude strain, SPF grade, female, from university of Sichuan laboratory animal center) were used for the experiments in the present invention.

The bacterial strains used in the examples are the commonly used strains DH5a and BL 21. Plasmids such as pET28a, pLVX-IRES-ZsGreen1, pcDNA3, pLVX-Tet3G, pLVX-TRE3G and the like used for cloning and cell expression of the endoD1 endonuclease molecules are manufactured by Liu Cong laboratories of the university of Sichuan Hua Xidi. Plasmids for the I-Sce1DNA fragmentation induction system are provided by the university of Zhejiang Chen Jun. The CRISPR/CAS9 gene editing system used was constructed by Beijing African Shang Lide Biotechnology Co. Small interfering RNAs (sirnas) were synthesized by Shanghai Ruibo biotechnology limited. The molecular cloning primer is synthesized by catalpa in Chengdu Optimu Biotechnology Co.

Lipofectamine 3000 (Invitrogen, L3000015), rabbit anti-human ENDOD1 endonuclease antibody (Abcam,Ab121293)、RAD52(LSBio,LS-C176555/122111)、ATM-pSerine1981(Cell Signaling,526S)、FK2(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 inhibitors (Ku 55933, selleck, S1092), MRE11 inhibitors (Mirin, selleck, S8096), CHK2 inhibitors (BML-277, selleck, S8632), DNAPK inhibitors (NU 7026, selleck, S2893), CGK733 (Selleck, S7136), ATR inhibitors (Caffeine, sigma, C1778-5 VL), 4-OHT ((Z) -4-hydroxytrianiline) and other various agents were all used for the experiments in the present invention. The dosage of the common reagent is IR,8Gy;CPT,0.0125uM;HU,0.2mM, cisplatin, 2ug/mL, 4-OHT,300nM. The transfection plasmid dose was 0.25ug/10 ten thousand cells, and the transfection siRNA dose was 0.2ug/10 ten thousand cells.

The information on the genomic 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 Kochia Biotechnology Co., ltd.), hepatitis B Virus (HBV) nucleic acid amplification (PCR) fluorescent quantitative detection kit (S20030059, heidachia Biotechnology Co., ltd.) and other various reagents are all used for the experiment in the present invention, and are not used for the other purpose.

Table 1, cell lines used and culture conditions

The names, IDs, genomes and chromosome locations of the genes involved in the present invention are shown in Table 2.

TABLE 2 Gene list

Gene	NCBI Gene ID	Genomic location
			ENDOD1	23052	11q21
TP53	7157	17p13.1
			RAD52	5893	12p13.33
MRE11	4361	11q21
			RAD54B	25788	8q22.1
RPA2(RPA32)	6118	1p35.3
			RPA1(RPA70)	6117	17p13.3
ATM	472	11q22.3
			ATR	545	3q23
CHEK1(CHK1)	1111	11q24.2
			CHEK2(CHK2)	11200	22q12.1
DNAPK	5591	8q11.21
			MLH1	4292	3p22.2
MSH2	4436	2p21-p16.3
			XRCC1	7515	19q13.31
XRCC3	7517	14q32.33
			BRCA1	672	17q21.31
BRCA2	675	13q13.1
			PAXIP1(PTIP)	22976	7q36.2
WRN	7486	8p12
			FANCA	2175	16q24.3
FANCD2	2177	3p25.3
			EMSY	56946	11q13.5
ARID1A	8289	1p36.11
			ARID1B	57492	6q25.3
WDR70	55100	5p13.2
			PTEN	5728	10q23.31
CDKN2A(p16,p14)	1029	9p21.3
			APC	324	5q22.2
STK11	6794	19p13.3
			FBXW7	55294	4q31.3
WT-1	7490	11p13

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

IRIF (ionizing radiation-reduced foci), points of radiation-induced foci, i.e.sites of DNA damage;

AA (amino acid) amino acids (residues);

kb (kilobase pair);

bp (base pair)

Ul, microliters;

nM;

uM: micromolar;

Kd: kilodaltons;

Input, internal control;

ChIP, chromatin immunoprecipitation;

Ub is ubiquitin;

siScramble random siRNA controls;

NHEJ-non-homologous terminal linkage;

HR: homologous recombination;

SSA, single-stranded DNA fusion;

ctrl, control;

example 1, human endo 1 endonuclease gene and protein characterization.

The experimental method comprises searching the gene and protein sequence of the endo D1 endonuclease by NCBI database, and drawing the evolution tree of the endo D1 endonuclease gene in different species and the protein functional domain of the endo D1 endonuclease by using bioinformatics software. Editing the endo-1 endonuclease gene by using CRISPR/CAS9 technology to obtain an endo-1 endonuclease gene knockout or mutation cell strain, and silencing the endo-1 endonuclease gene by using siRNA technology to knockdown the expression of the endo-1 endonuclease gene. Extracting protein of RPE-1 cells, separating the protein by SDS-PAGE electrophoresis, transferring to a polyvinylidene fluoride membrane, and detecting the protein level of the endo 1 endonuclease by adopting specific rabbit anti-human endo 1 endonuclease and goat anti-rabbit HRP secondary antibody.

CRISPR/CAS9 gene editing

The gRNA target sequence located at exon one of the endood 1 endonuclease gene was designed to be 5'-CAGCCTCTTCGCCCTGGCTGG-3' and cloned into pCAG1-CAS9-U6-sgRNA vector, transfected into RPE-1 cells using lipo3000 transfection kit, and after 48 hours 3. Mu.g/mL puromycin was added to screen for transfected positive cells. Positive cell monoclonals were isolated and the endo 1 endonuclease gene knockout or mutant cell strains were screened by western immunoblotting.

SiRNA interference technology

Designing and synthesizing an RNA interference (RNAi) target of the endo d1 endonuclease gene:

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

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

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

Intracellular silencing of the endo D1 endonuclease gene was performed by dissolving the endo D1 endonuclease siRNA powder and the control siRNA powder (Guangzhou Ruibo) in ultrapure water (Invitrogen, 10977-015) to prepare stock solutions (5 nM) and storing at-40 ℃. The transfection mixture per well of the 24-well plate was prepared by sequentially adding 5ul of siRNA (5 nM) and 3ul Lipofectin 3000 (Invitrogen, L300015) to 30ul of serum-free medium opti-MEM, mixing, standing for 15min, and dripping into the 24-well plate. 470ul of complete medium was added to the 24 well plate cells in advance. After 24 hours of transfection, 1ml of complete medium was changed. Samples were collected after 48 hours, cells were dissolved in SDS SAMPLE buffer, and silencing efficiency was examined by western blotting.

Western immunoblotting

Equal amounts of cells (about 50 ten thousand) were collected, washed twice with PBS, centrifuged at 500x g each, and cell pellet was collected. Cells were lysed by adding 100. Mu.l SDS loading buffer (50 mM Tris-HCl [ pH6.8],2% SDS,0.1% bromophenol blue, 100% glycerol, 5% beta-mercaptoethanol) and treated in a metal bath at 100℃for 5 min. Proteins were separated by polyacrylamide gel electrophoresis (150 v/55 min after 80 v/15 min) and western blotted onto a polyvinylidene fluoride membrane (150 ma/3 hr). The membrane was blocked in blocking buffer (10 mM Tris-HCl [ pH7.5],150mM NaCl,0.1%Tween,5%BSA) for 1 hour, rabbit anti-human ENDOD1 endonuclease antibody (1:1000) was added, incubated for 4 hours at room temperature, and washed 3 times with washing buffer (10 mM Tris-HCl [ pH7.5],150mM NaCl,0.1%Tween) for ten minutes each. The membrane was incubated in blocking buffer containing goat anti-rabbit HRP secondary antibody (1:3000) for 1 hour and washed 3 times for ten minutes each. And (3) developing by using ECL luminous liquid, and acquiring images by using a BIO-RAD gel imaging system (chemidoc XRS).

Experimental results:

1. The mRNA sequence of the human ENDOD1 endonuclease gene is shown in FIG. 1A with a total of 1503 nucleotides in the coding region (ORF). The endo D1 endonuclease protein sequence is shown in FIG. 1B and is 501 amino acids in total. The endoD1 endonuclease protein domain is shown in FIG. 1C and comprises a signal peptide (1-22 amino acid residues (AA)), an endonuclease domain (49-257 AA), and 3 transmembrane sequences (I: 345-365 AA, II:411-435AA and III:455-482 AA). Cysteines C190, C224, and C269 are 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 endo 1 endonuclease genes in different species, found that its homologous genes were present in higher eukaryotes such as coral, fish, vertebrate, and human, but not in lower eukaryotes such as yeast, drosophila, and nematode (FIG. 1E).

2. Characteristics and post-translational modifications of human endo-1 endonucleases.

ImmunoWestern blot determination of protein product of endo-1 endonuclease in RPE-1 cells (FIG. 2C), major band electrophoresis size of about 55Kd. Protein electrophoresis showed that the endo-1 endonuclease has 4 modification or cleavage subtypes, 47Kd (residues 23-500, b), 42Kd (residues 23-340, c), 35Kd (residues 23-290, d) and >90Kd of covalently modified form (e), respectively. And a covalently modified form of >90Kd (e). After CISPR/CAS9 gene editing (FIG. 2A) or siRNA gene silencing (FIG. 2B), the various electrophoretic forms of the ENDOD1 endonuclease disappeared or decreased (FIGS. 2D-E). The ubiquitin (ubiquitin) or its K48R, K R mutant with signal peptide deleted ENDOD1 endonuclease expression plasmid (Flag-. DELTA.N22-ENDOD 1 endonuclease) was transferred into RPE-1 cells, and the ENDOD1 endonuclease subtype e was found to be generated due to ubiquitination covalent modification (FIG. 2F). Analysis of the protein structural features of endo D1 endonuclease by bioinformatics software such as PrediSci and NetSurfP-2.0 revealed that residues 300-325 are protein low-order regions and also potential protease cleavage sites (FIG. 2G-H), which can co-produce the b, c, d subtypes of endo D1 endonuclease protein with cleavage of the LEG-RL signal peptide sequence near residue 22.

Example 2, human endo-1 endonuclease was involved in DNA repair.

Experimental methods subcellular localization and distribution of endo d1 endonuclease proteins were characterized using immunofluorescence, chromatin co-immunoprecipitation (ChIP) and nucleoplasmic isolation methods. The ChIP experiment was performed in DiVA cells, with 4-OHT inducing expression of the AsiSI-ER endonuclease, producing a DNA break at chromosome Chr1:89,458,595-89,458,603. After 4-OHT induction for 4 hours, the corresponding protein was precipitated using specific antibodies (anti-endo 1 endonuclease or anti-Flag). The level of enrichment of the protein at the DNA cleavage site was determined by PCR amplification. Cell component separation experiments (cytosol/nuclear membrane/chromatin) methods 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)), step increase 0.1% triton-X100 to lyse nuclear membrane and golgi).

Immunofluorescence

Cells were fixed by conventional cell culture on glass plates, fixed with 4% pfa for 10 min, washed with PBS and plated with 0.3% triton/PBS. Primary antibodies were diluted in blocking buffer (2% bsa,0.3% triton/PBS) in proportion and cells were incubated at 37 ℃ for 3 min and washed 2 times with PBS. The secondary antibodies were diluted in blocking buffer (2% bsa,0.3% triton/PBS) in proportion and the cells were incubated at 37 ℃ for 30 min before PBS washing 2 times. Images were taken after blocking with DAPI-containing anti-quenching dye (Vector Laboratories) under an orthofluorescent microscope (olympus, BX 51). Counting the number of foci in the nuclei (DAPI staining), visualizing the counting results by GRAPHPAD PRISM (v8.4.0.671) software, and using a two-tailed T test, the difference between groups was considered to be a significant difference in P.ltoreq.0.05.

Chromatin co-immunoprecipitation (ChIP)

Approximately 500 ten thousand cells were collected and crosslinked in 1% formaldehyde for 15min, 2.5M glycine was added to terminate crosslinking, PBS was washed twice and resuspended in 300ul of ChIP lysate (50mM HEPES[pH 7.4];140mM NaCl;1%Triton X100;0.1%NaDeoxycholate;protease inhibitors), 4℃was lysed for 30 min, 10000x G was centrifuged and the pellet was washed once in 1ml of ChIP lysate and resuspended in 300ul of ChIP lysate, biorupter sonicator disrupted DNA at about 500bp, 21000x G was centrifuged to discard the pellet, protein G beads (invitrogen) were added after 1h incubation of antibody, or anti-Flag M2 beads (Sigma) were directly added and incubated overnight at 4 ℃. The beads were washed 2 times with ChIP lysate, chIP high salt lysate (50mM HEPES[pH 7.4; 500mM NaCl;1%Triton X100;0.1%NaDeoxycholate), chIP wash (10 mM Tris [ pH 8.0];250mM LiCl;0.5%NP-40;0.5%NaDeoxycholate;1mM EDTA), TE buffer (10 mM Tris-HCl [ pH 8.0];1mM EDTA), then added with ChIP elution buffer (50 mM Tris [ pH 8.0];1% SDS;10mM EDTA) and reverse-crosslinked at 65℃for 1 hour, and the eluted liquid was purified and recovered in 30ul of water using DNA purification kit (Omega) to obtain 5ul for quantitative analysis.

Nuclear mass separation

About 200 ten thousand cells were collected, washed twice with PBS, resuspended in 200. Mu.L of 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), incubated on ice for 5 minutes and centrifuged at 1500: 1500x g for 5 minutes, and the supernatant and pellet were collected, respectively. The supernatant was taken as cell membrane and cytoplasmic protein, and 50. Mu.L of 5X SDS loading buffer was directly added to extract the protein. The pellet was washed once in 1mL of Triton-free lysate A, centrifuged at 1500x g min and resuspended in 200. Mu.L of lysate A, incubated on ice for 5-10 min and centrifuged at 1500x g min. The supernatant was used as the nuclear membrane fraction, and 50. Mu.L of 5X SDS loading buffer was directly added to extract the protein. The pellet was washed once in 1mL of Triton-free lysate A, centrifuged at 1500x g min and resuspended in 200. Mu.L of lysate B (3mM EDTA,0.2mM EGTA,1mM dithiothreitol,protease inhibitor), incubated on ice for 10min, and centrifuged at 2000x g for 5 min. The supernatant fraction was soluble nuclear protein, and 50. Mu.L of 5X SDS loading buffer was directly added to extract the protein. The pellet was washed once in 1mL of lysate B, centrifuged at 13000x g for 1 min, the supernatant discarded and the pellet was dissolved in 100. Mu.L of SDS loading buffer. All samples were treated in a 100 ℃ metal bath for 5 minutes and examined using western blotting. Total cell Total protein, loading of 2.5% of Total, membrane, golgi, 10% of Total, chromatin% of chromatin protein, 10% of Total.

Experimental results:

In order to analyze the biological functions of the endo-1 endonuclease, a series of biological experiments prove that the endo-1 endonuclease is aggregated and activated at DNA damage sites, participates in DNA repair and ensures the stability of genetic materials.

1. Human endo-1 endonuclease binding directly to DNA damage site

When DNA is damaged, proteins involved in DNA repair are activated and aggregate to the site of the damage, which may be visualized by immunofluorescent staining methods, showing altered localization of various signaling and repair factors after DNA damage. Immunofluorescence showed that endonucleases were primarily localized to the cytoplasm and nuclear membrane in normally growing cells. After DNA damage (ion radiation exposure), the ENDOD1 endonuclease can enter the DNA breakpoint within 40 minutes to form an ENDOD1 endonuclease aggregation point and persist for 2 hours after exposure (fig. 3A). Endo d1 endonuclease can also form aggregation sites after DNA replication toxicants (CPT and HU) treatment, indicating that endo d1 endonuclease is also involved in the clearance of DNA replication errors (fig. 3B). Furthermore, the aggregation point of ENDOD1 endonuclease at DNA breaks can be co-localized with classical marker protein (γh2ax) at DNA damage site (fig. 3C), suggesting that ENDOD1 endonuclease is actively recruited to DNA break point as repair factor after DNA damage.

2. Direct binding of endo-1 endonuclease to DNA breakpoint end

In the ChIP experiment, ENDOD1 endonuclease was bound at 0.5Kb, 1Kb and 2.5Kb from the DNA breakpoint (fig. 3D), further demonstrating direct aggregation of ENDOD1 endonuclease to the end of DNA damage site. ChIP experiments have also shown that one of the functions of ENDOD1 endonuclease at DNA breakpoints is to participate in DNA end back-cutting (end resection) in that, although ENDOD1 endonuclease silencing alone does not attenuate enrichment of single-stranded DNA binding protein (RPA 32) near the breakpoint, in the case of other end back-cutting regulatory factor (e.g., WDR 70) defects, ENDOD1 endonuclease silencing can exacerbate enrichment of RPA32 to DNA breakpoints, resulting in complete suppression of recruitment of RPA32 (fig. 3E). These experiments demonstrate that simultaneous deletion of homologous recombination repair and endo 1 endonuclease function results in serious DNA repair defects.

By experiments with the separation of cytoplasmic-nuclear membrane-chromatin components, it was demonstrated that a, b, c, d forms of endo d1 endonuclease protein can bind tightly to chromatin, with the distribution of endo d1 endonuclease over chromatin accounting for 25% of the total protein (fig. 3F).

Taken together, these results indicate that the endo d1 endonuclease protein responds positively after cellular DNA damage and recruits to DNA breakpoints, involved in the regulation of DNA repair responses.

Example 3, human endo-1 endonuclease inhibits SSA repair function.

The relative efficiency of repair of endo-1 endonuclease-deficient cells (2F 4) was determined by functional complementation experiments (complementation) using either wild-type (pLVX-TRE 3G-ENDOD1 endonuclease, i.e., ENDOD1 endonuclease) or mutant-type endo-1 endonuclease plasmid (pLVX-TRE 3G-ENDOD1 endonuclease inactivation, i.e., ND; pLVX-TRE3G-ENDOD1 endonuclease deletion, i.e., ΔEND) that was expressed by doxycycline-induced expression using I-Sce1 endonuclease site-directed induction DNA cleavage (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).

Determination of DNA fragmentation repair efficiency

The pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids (16 ℃ C., 16 hours) were digested in vitro with I-Sce1 endonuclease (NEB), and the DNA was precipitated by adding 2 volumes of absolute ethanol and 1/20 volumes of 3M sodium acetate. The recovered DNA was adjusted to a concentration of 1. Mu.g/. Mu.L. Cells were first treated by siRNA or other, 24 hours later, linearized pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids were separately transfected (Lipo 3000) into the differently treated cells, cells were collected after further culture for 24 hours (about 100 tens of thousands), total DNA of the cells was recovered using high purity DNA extraction kit (Roche), real-time fluorescent quantitative PCR (BIO-RAD) was performed, and the efficiency of the different repair pathways was analyzed.

EnDOD1 endonuclease and construction and expression of mutant plasmid thereof

The endo 1 endonuclease gene and its corresponding mutants ND and DeltaEND were obtained by means of DNA synthesis (Shanghai Biotechnology Co., ltd.) and subcloned into pLVX-TRE3G vector via EcoR1 site after PCR amplification. The corresponding plasmid was co-transfected (Lipo 3000) with pLVX-Tet3G into cells and expression of the corresponding gene was induced using doxycycline (200 ng/mL) after culturing for 24 hours.

Experimental results:

DNA fragmentation damage is repaired by three pathways, mainly non-homologous end joining (NHEJ), homologous Recombination (HR) and single-stranded DNA fusion (SSA), and is regulated by a variety of repair factors, where the extent of DNA end backcut to form single-stranded DNA is an important regulatory event in the selection of these repair pathways. Based on the findings in example 2, further experiments demonstrate that ENDOD1 endonuclease recruits directly to DNA breakpoints and regulates DNA end backcuts, an important molecule for regulating repair pathway selection.

1. Endo 1 endonuclease inhibits SSA repair pathways

The repair efficiency of three pathways of DNA breakpoints can be determined by in vitro I-Sce1 cleavage of pcDNA3-NHEJ, pcDNA3-HR, pcDNA3-SSA plasmids to form linearized plasmids with site-specific DNA breaks and transfection into RPE-1 cells silencing the ENDOD1 endonuclease gene. It was found that ENDOD1 endonuclease gene silencing slightly inhibited non-homologous end joining (NHEJ) and Homologous Recombination (HR) repair functions, more importantly promoted single strand DNA fusion (SSA) functions (fig. 4A). Similarly, I-Sce1 was tested for efficiency of repair of induced DNA breaks in RPE-1 wild-type, 2F4 and 2F6 cells, and it was also found that the efficiency of repair of SSA in the endoD1 endonuclease-deficient 2F4 and 2F6 cell lines was significantly increased over wild-type RPE-1 cells (FIG. 4B). These results demonstrate that the ENDOD1 endonuclease factor can inhibit the repair pathway of SSA at the site of DNA damage.

2. Endood 1 endonuclease effectively inhibits SSA repair pathways by its endonuclease function

Transfection of wild-type endo D1 endonuclease plasmid (Wt) which is expressed by doxycycline induction in 2F4 cells with the endo D1 endonuclease gene knockdown reduced SSA levels to normal cells, whereas the endo D1 endonuclease mutant plasmids (ND and ΔEND) which had been introduced with endonuclease inactivation did not have corresponding correction functions (FIG. 4C), indicating that endo D1 endonuclease was dependent on its endonuclease activity to inhibit SSA repair pathways.

3. Endonucleases inhibit SSA function by modulating RAD52

RAD52 is an important protein regulating SSA repair, and to analyze the specific function of the endo-1 endonuclease involved in SSA pathway, silencing the endo-1 endonuclease and/or SSA functional gene (RAD 52) in RPE-1 cells, testing the repair function of both after I-Sce1 induced DNA fragmentation, it was found that RAD52 silencing could completely reverse the SSA hyperactivity caused by endo-1 endonuclease deficiency (FIG. 4D), indicating that the function of the endo-1 endonuclease to inhibit SSA depends on RAD52.

Immunofluorescence experiments showed higher levels of IR-induced RAD52 aggregation point formation at RPE-1 cell expression of endo d1 endonuclease gene knockout (fig. 4E) or silencing (fig. 4F), further demonstrating the repair function of endo d1 endonuclease to inhibit RAD52 at DNA breakpoint. In comparison with wild-type RPE-1, both 2F4 and 2F6 had the defect of RAD52 hyperactivity, indicating that in addition to the main band (55 Kd) of the ENDOD1 endonuclease protein, its posttranslational processing and subtypes b, c, d are involved in the regulation of RAD52 and SSA.

4. Regulation and control of other DNA repair factors by endonucleases

Silencing ENDOD1 endonuclease gene expression in RPE-1 cells IR treatment was performed for 48 hours to determine the level of aggregation of various DNA repair factors at the site of injury. It was found that the aggregation levels of γH2AX/53BP1 (FIG. 5A), FK2 (FIG. 5B), BLM (FIG. 5D) and MRE11 (FIG. 5E) were lower in RPE-1 cells that were silenced or edited with the ENDOD1 endonuclease than in wild-type cells, whereas the aggregation capacity of ATM autophosphorylation was instead increased in defective cells (FIG. 5C). These results demonstrate that ENDOD1 endonuclease, together with MRE11, FK2, BLM, γh2ax and 53BP1, regulate DNA repair and antagonize ATM autophosphorylation function.

It follows that the primary function of ENDOD1 endonuclease is to participate in DNA fragmentation repair, and that MRE11, γh2ax and 53BP1 act synergistically, inhibiting the RAD 52-regulated SSA pathway, and that the process is dependent on its endonuclease activity.

Example 4, ENDOD1 endonuclease has endonuclease activity.

The experimental method comprises the following steps:

Expression and purification of endo D1 endonuclease-STREP protein

The full length of the endo D1 endonuclease gene and mutants thereof are subcloned into pGEX4T1-2strep vector after PCR amplification and transformed into BL21 E.coli strain. Bacteria harboring the ENDOD1 endonuclease plasmid were cultured at 37 ℃ to the logarithmic growth phase (od=0.6), protein expression was induced by adding 1mM IPTG, and after further culture at 18 ℃ for 16 hours, the bacteria were collected by centrifugation. The cells were resuspended in NETN lysate (100mM NaCl,20mM Tris-Cl [ pH 8.0],0.5mM EDTA 0.5%Nonidet P-40), disrupted by sonication, centrifuged at 21000 and x g, and the supernatant was collected. Adding Strep-Tactin XT magnetic beads (iba) pre-washed by NETN lysate into the supernatant, incubating for 2 hours at 4 ℃, discarding the supernatant, washing the magnetic beads 5 times by using NETN lysate, and adding SDS loading buffer to extract purified proteins. Purified proteins were detected by western blotting.

DNA in vitro cleavage reaction

The DNA double-stranded fragment (28-30 bp) or DNA/RNA hybrid strand (30 bp) was synthesized by Shanghai Biotechnology Co. The pEGFP plasmid was cut with restriction enzymes ApaI, bamHI and SmaI to obtain a plasmid with a DNA double strand break (5 ', 3' overlapping and blunt ends), and treated with ultraviolet light (1200J/m ²) and hydrogen peroxide (0.03%, 1 h) to obtain a damaged plasmid. The treated DNA was recovered using a DNA purification kit and subjected to an enzyme digestion reaction in a total volume of 20. Mu.l of 50mM Tris-HCl [ pH 7.9], 100mM NaCl, 10mM MgCl2, 100. Mu.g/ml BSA, 0.5. Mu.g of ENDOD1 protein, 0.2. Mu.g (plasmid) or 0.4. Mu.g (synthetic double-stranded DNA fragment) of substrate, and after digestion at 37℃for 16 hours (plasmid) or 4 hours (synthetic double-stranded DNA fragment), agarose Gel or sequencing Gel electrophoresis was performed, and the DNA was stained with Gel-Stain dye and images were collected.

Experimental results endo d1 endonuclease has a classical endonuclease domain to verify its endonuclease activity, we expressed and purified the endo d1 endonuclease-STREP protein, including the full length endo d1 endonuclease (FL) and the polypeptide fragments (1-344, 22-344), using bacteria (fig. 6A) and examined its cleavage reaction against different types of DNA. The in vitro digestion reaction (37 ℃ C., 2 hours) was performed in digestion buffer of 0.5 micrograms of endo 1 endonuclease domain fragment (residues 22-344) with 0.37 micrograms of DNA double stranded fragment (blunt end, 3 'and 5' -overhang) or DNA/RNA hybrid. The ENDOD1 fragment can cleave these nucleic acid substrates to form smaller fragments (fig. 6B). 0.5. Mu.g of purified ENDOD1 endonuclease (residues 1-344 and Full Length (FL)) added 0.2. Mu.g of various pEGFP-C3 plasmid DNA, and the results of in vitro digestion reactions in buffer (37 ℃ C., 16 hours) indicated that the ENDOD1 endonuclease could cleave DNA molecules with structural lesions such as UV lesions, DNA double strand breaks (5 'and 3' overlapping), oxidative lesions (hydrogen peroxide treatment) without cleavage effects on the intact plasmid (FIG. 6C).

Example 5, endo 1 endonuclease gene function inhibition specifically attacks tumor cells but does not affect proliferation of normal non-tumor cells.

Experimental methods the endo 1 endonuclease gene was knocked out or silenced in normal or tumor cells and the cells were serially passaged or silenced to determine their proliferation profile, with a majority of cell experimental cycles of 21 days. Surviving cell numbers were calculated at the indicated time points and experimental endpoints and recorded as concurrent diagrams. The proliferation rate was calculated from the cell count results as proliferation rate = (N _t-N₀)/N_t x 100%, where N _t is the absolute value of the experimental end point cell count and N ₀ is the initial cell seeding number. All measured cell line control groups were tested for double tail t-test from experimental end point proliferation rate, P <0.05 considered significant statistical differences.

Crystal violet staining:

10g of crystal violet (Solarbio, C8470) powder was dissolved in methanol and formulated as a 10% stock solution. The cells were fixed with methanol for 10 minutes at the end of the experiment and air dried. The crystal violet storage solution is diluted with PBS according to a ratio of 1:10 to prepare working solution. And (5) dyeing the cell sample after fixation for 10min by using a working solution, cleaning by using clear water, and airing. The stored image is observed and scanned.

Experimental results:

DNA damage response is an important mechanism for eukaryotic cells to maintain genomic stability, which leads to cell death when the damage is not normally repaired. Example 3 it has been demonstrated that silencing of the endo 1 endonuclease gene results in RAD52 modulating SSA pathway abnormalities, affecting DNA repair, possibly affecting cell survival. Therefore, we use cell proliferation experiments to determine the effect of endo d1 endonuclease on proliferation capacity of different cell types, and found that endo d1 endonuclease gene silencing has a specific inhibition effect on tumor cells, but does not affect survival of normal cells, indicating that endo d1 endonuclease function inhibition has a potential tumor treatment effect. The specific results are as follows:

1. Endo 1 endonuclease gene knockout or silencing does not affect proliferation of normal non-tumor cells

Normal somatic cells (RPE-1, MRC-5, GES-1 and L02) were assayed for 21 days after silencing or knocking out the expression of the endo 1 endonuclease gene (FIGS. 7A-B). It can be seen that there is no statistical difference (NS) in proliferation rates of endo d1 endonuclease wild type and non-tumor cells with suppressed gene function.

2. Endo 1 endonuclease gene silencing inhibits proliferation of tumor cells

Various tumor cells showed complete or partial inhibition of proliferation, even cell death, with statistical differences after silencing of endo 1 endonuclease as shown in fig. 8A, and a small portion of tumors were not affected by proliferation after endo 1 endonuclease silencing (fig. 8A-C). Furthermore, it could be demonstrated that in cells insensitive to endo 1 endonucleases (HO 8910-PM, SW480, GES-1 and L02), endo 1 gene expression could be effectively silenced (fig. 8D), indicating that these cells do not show resistance to siENDOD due to difficulties in gene transfection, but show birth differences due to other genetic mechanisms.

3. Reduced endo-1 endonuclease function and toxicity to tumor cells are dependent on endonuclease activity

Silencing of ENDOD1 endonuclease (si 001) in SKOV3 cells while transfection of doxycycline-induced and si001 resistant ENDOD1 endonuclease wild-type or endonuclease mutant (ND) found that ENDOD1 endonuclease wild-type plasmids could rescue si001 cytotoxicity whereas ENDOD1 endonuclease plasmids lacking endonuclease function did not rescue capacity (fig. 8E), suggesting that ENDOD1 endonuclease toxicity to tumor cells required functional ENDOD1 endonuclease activity.

4. Reduced endo-1 endonuclease function and toxicity to tumor cells are dependent on increased SSA activity

Simultaneous silencing of RAD52 gene expression was found in SKOV3 cells to reduce toxicity of ENDOD1 silencing to SKOV3 (FIG. 8F). It was demonstrated that the toxicity of endo 1 endonuclease to tumor cells was dependent on the function of RAD52, and that hyperactivity of SSA function could mediate the toxicity of endo 1 endonuclease to tumor cells.

Thus, example 5 demonstrates that tumors can be specifically treated or prevented without killing normal non-tumor cells by administering a sufficient dose of an endo d1 endonuclease inhibitory drug to a subject. The same principle is also applicable to agents that increase SSA activity (SSA enhancers), since it is predicted that ENDOD1 inhibitory drugs can effectively enhance SSA activity, killing tumors depends on an increase in SSA (e.g., RAD 52) activity, and increasing SSA repair levels can also achieve a similar cytotoxic effect as inhibiting ENDOD1 endonuclease function or gene expression. It is therefore concluded that other SSA enhancers should have the same therapeutic effect as well.

Example 6, combined lethal effect of endo-1 endonuclease and DNA damage response and homologous recombination repair genes.

Experimental methods the endo-1 endonuclease was co-silenced with other functional genes in RPE-1 and 2F4 cells, or used in combination with enzyme inhibitors, and the proliferation potency of the cells was measured using cell counting or crystal violet staining methods.

Experimental results:

In order to research the mechanism of inhibiting proliferation of tumor cells by the gene deletion specificity of the endo D1 endonuclease, the invention utilizes gene silencing or enzyme inhibitor to interfere with the function of DNA injury response or repair factors and simulate the DNA response defect in tumor cells, and discovers that the gene silencing of the endo D1 endonuclease and the defect of most DNA injury response factors including ATM, CHK1 and homologous recombination repair factors are combined and killed, and the specific results are as follows:

1. Endo 1 endonuclease gene silencing and DNA damage response factor combined mortality.

Inhibitors of DNA damage response factor addition to RPE-1 and 2F4 cells include ATM kinase inhibitors (Ku 55933,20 uM), PIKK inhibitors (CGK 733,1 ug/ml), ATR inhibitors (Caffeine, 10 ug/ml), DNAPK kinase inhibitors (NU 7026,1 uM), CHK2 kinase inhibitors (BML-277, 1 uM), MRE11 nuclease inhibitors (Mirin uM), or silencing CHK1 expression with siRNA. Viable cell staining was performed 12 days later and it was found that ENDOD1 endonuclease gene silencing was lethal in combination with ATM inhibitors, MRE inhibitors and CHK1 silencing on RPE-1 cells (fig. 9A). Suggesting that ENDOD1 endonuclease function inhibition may produce selective therapeutic effects against tumors of ATM, MRE11 and CHK1 gene mutations or dysfunctions.

Thus, a tumor with a defect in DNA response, including a tumor with a ATM, ATR, CHK, CHK2, DNAPK gene mutation, gene expression, or functional defect, or with CDC25A, CDC25B, CDC C, cyclin E, cyclin B1, cyclin D1 overexpression characteristics, may be treated or prevented by administering to a subject a sufficient amount of an ENDOD1 endonuclease inhibitory drug ENDOD1 or SSA enhancer.

2. Endo 1 endonuclease gene silencing and homologous recombination repair factor combined death.

Silencing ENDOD1 endonuclease gene expression in RPE-1 cells while silencing DNA repair genes such as BRCA1, BRCA2, BLM, MRE11, CTIP, EXO1, MSH1, ARID1A/RID1B, and WDR70 were assayed for their proliferative capacity (fig. 9B-C). Co-silencing of the endo-1 endonuclease with BRCA1, BRCA2, CTIP, EXO1, ARID1A/RID1B and WDR70 genes showed combined mortality, but no significant combined lethal effect with BLM. In addition, 3 specific ENDOD1 endonuclease sirnas could produce a combined lethal effect with siBRCA a (fig. 9D), and ENDOD1 endonuclease knockdown 2F4 cells also combined lethal with BRCA1 and FANCD2/FANCC gene silencing (fig. 9E). The combined lethal effect of endo-1 endonuclease and homologous recombination repair factor was further demonstrated. These evidence suggest that inhibition of endo 1 endonuclease gene expression or protein function may be selective for tumors harboring mutations or dysfunctions of homologous recombination genes such as BRCA1, BRCA2, FANC, CTIP, EXO1, ARID1A/RID1B, and WDR 70.

Thus, it is possible to treat or prevent tumors with defects in DNA damage repair, including homologous recombination repair defects, base excision repair defects, nucleotide excision defects, single strand DNA fragmentation repair defects, and tumors with mutations in genes or functional defects associated with these repair mechanisms by administering to a subject a sufficient dose of an endoD1 endonuclease inhibitory drug or SSA enhancer. These tumors include, but are not limited to, gene mutations, gene expression or functional defects such as DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, BRCA1, BRCA2, PTIP, WRN, fanconi, EMSY, ARID1A, ARID B and WDR70, or oncogene overexpression such as CDK12 which inhibits DNA repair functions, or liver cancer or cholangiocarcinoma with hepatitis B virus infection and viral gene expression.

Example 7, combined lethal effect of endonucleases and oncogenes.

Experimental methods the ability of cells to proliferate was determined by co-silencing the endo-1 endonuclease with the TP53 functional gene in RPE-1 or tumor cells. Transferring TP53 expression plasmid into tumor cell, silencing gene expression of endo D1 endonuclease, and measuring the rescuing effect of TP53 gene to endo D1 endonuclease cytotoxicity.

Experimental results:

1. Combination lethal effect of endo-1 endonuclease and cancer suppressor gene TP53

Numerous studies have shown that TP53, PTEN, etc. are important oncogenes with mutations or deactivations in 60-80% of human tumors. To further understand the mechanism by which endo 1 endonuclease gene silencing specifically inhibits tumor cell proliferation, cell survival was determined after silencing the endo 1 endonuclease and TP53 gene in RPE-1 wild-type, or silencing TP53 in endo 1 endonuclease gene knockout (2F 4) cells. It was found that endo d1 endonuclease dysfunction resulted in cells that were highly sensitive to loss of TP53 gene function (fig. 10A). Likewise, simultaneous silencing of ENDOD1 endonuclease and TP53 was found in a TP53 high level cell line (HO 8910-PM) and cells were found to be non-viable and proliferative (fig. 10B). Indicating that intact endo d1 endonuclease gene function is an essential factor for maintaining viability in tumor cells with TP53 mutations or loss of function.

2. Restoration of TP53 function in TP53 mutant cells inhibits the cytotoxic effects of silencing the ENDOD1 endonuclease

Further, expression of wild-type TP53 by plasmid transfection, a cell line with homozygous mutation of the TP53 gene and being sensitive to silencing by the endo D1 endonuclease gene (NCI-H1975) was found to become insensitive, indicating that restoring TP53 function effectively abrogates the cytotoxic effects of endo D1 endonuclease function inhibition on tumor cells that lost TP53 function (FIG. 10C). These experiments further demonstrate that the loss of TP53 gene function directly results in sensitivity of tumor cells to inhibition of endo D1 endonuclease function, and also suggest that endo D1 endonuclease function inhibition may have a selective therapeutic effect against tumors that are mutated or dysfunctional in the TP53 gene.

3. Combined lethal effect of other cancer suppressor genes and endo 1 endonuclease gene silencing

Silencing of PTEN oncogenes in RPE-1 and 2F4 cells 2F4 was found to have a relatively high sensitivity to PTEN silencing (fig. 10D). It was demonstrated that inhibition of oncogenes other than TP53 may also be combined with inhibition of endo 1 endonuclease function to kill.

Thus, it is possible to treat or prevent tumors harboring mutations in the oncogene, gene expression or functional defects of TP53, PTEN, etc., by administering a sufficient dose of an endoD1 endonuclease-inhibiting drug or SSA enhancer to a subject.

Example 8, analysis of correlation of tumor cell gene mutations with endo d1 endonuclease gene silencing cytotoxicity.

The experimental method comprises the following steps:

Immunoblots were used to detect protein levels of endo 1 endonucleases from human normal and tumor cell lines. Protein abundance of ENDOD1 endonuclease in normal and tumor tissues was collected from Human Protein Atlas databases.

Calculation of proliferation inhibition Rate

The inhibition rate of tumor cell proliferation shown in Table 3 was calculated as = (1-Nt/Nc)%, where Nt represents the experimental end point drug group cell count and Nc represents the experimental end point control group cell count.

Experimental results:

1. Immunoblots detected the ubiquitous expression of endo D1 endonuclease proteins in normal (RPE-1,) and tumor cell lines of different tissue origins (SKOV-3, NCI-H1975, NCI-H1299, HL60, MRC-5, RPE-1, MDA-MB-231, U2OS, MDA-MB-468, raji, OVCAR-8 TR) (FIG. 11A).

2. Human Protein Atlas database shows that endo d1 endonuclease exhibits high levels of protein expression in most normal tissues (fig. 11B), and that endo d1 endonuclease also has significant levels of protein expression in more than half of tumor tissues (fig. 11C), including glioma, thyroid cancer, rectal cancer, head and neck cancer, gastric cancer, pancreatic cancer, lung cancer, renal cancer, prostate cancer, breast cancer, endometrial cancer, ovarian cancer, melanoma, skin cancer, and the like. Endo-1 endonuclease is expressed in lower proportions in individual tumors (e.g., testicular and lymphomas).

3. Table 3 shows the cytotoxicity of the ENDOD1 endonuclease on the 25 tumor cells tested and the mutations in the genes carried by the cells. Statistical analysis showed that tumor cells harboring TP53 or other gene mutations (BRCA 1, BRCA2, or harboring HBV, HPV viruses, etc.) were much more sensitive to endoD1 endonuclease gene silencing than tumor cells harboring no such mutations (FIG. 11D). Further analysis of Pearson correlation coefficients between TP53 mutations and other types of tumor gene mutations and the inhibition of the silencing of the ENDOD1 gene using SPSS software revealed a high correlation (Pearson Correlation:0.708, p < 0.0001) between the two, indicating that tumor cells harboring these variations were far more sensitive to silencing of the ENDOD1 gene than non-mutated cells.

Table 3, the relevant mutational characteristics of various human normal and tumor cell lines and the inhibition of cell proliferation following gene silencing by the ENDOD1 endonuclease. (wt: wild type; mu: mutant; null: total deletion mutant; homo: pure and mutant)

Example 9, endo 1 endonuclease function inhibits sensitization to tumor chemotherapy.

The experimental method comprises the following steps:

1 ten thousand cells (RPE-1 or A549) were seeded in 24-well plates, and the ENDOD1 endonuclease gene silencing experiments were performed 24 hours later and continued drug treatment was performed (drug concentration see FIG. 12). Cell proliferation fold was calculated at the end of the experiment (14 days).

Experimental results:

Normal cells (RPE-1) silenced with the ENDOD1 siRNA were insensitive to chemotherapeutic drugs, ENDOD1 gene silencing, or combination treatment (FIG. 12, left), but tumor cells (A549) were more sensitive to chemotherapeutic drugs, and sensitivity to Camptothecins (CPT), cisplatin, DNAPK inhibitors (Nu 7062) were greatly improved after silencing of the ENDOD1 endonuclease (FIG. 12, right).

Thus, administration of a sufficient dose of endo D1 endonuclease inhibitory drug to a subject may sensitize a low dose of chemotherapy for the treatment of tumors that do not respond clinically to radiation or chemotherapy alone. Potential combination therapies include those selected from the group consisting of platinum, mitomycin C, camptothecins, PARP inhibitors, DNAPK inhibitors, radioisotopes, vinca alkaloids, antitumor alkylating agents, monoclonal antibodies, antimetabolites. The treatment methods can be used simultaneously or sequentially, and show a synergistic effect, so that the treatment effect is superior to that of a method for independently using a medicament for inhibiting the function of the ENDOD1 or an anti-tumor medicament.

Example 10 therapeutic effect of endo 1 endonuclease gene silencing on mouse tumor-bearing models.

The experimental method comprises the following steps:

nude mouse tumor-bearing model

Preclinical animal tumor-bearing experiments were performed at the animal center of the Hua Xidi two hospitals at university of Sichuan (SPF grade). The week-old athymic male nude mice were randomly divided into 3 groups after one week of isolation observation (purchased from the Nanjing model animal research center). Each mouse was inoculated with tumor cells in the armpits and groin in an amount of 4X10 ⁶.

In vivo gene silencing

The inoculum tumor volume was about 100mm ³ time to endo 1 endonuclease in vivo gene silencing. The preparation method comprises dissolving in ultrapure water (Invitrogen, 10977-015) in vivo siRNA powder of endo D1 endonuclease and control siRNA powder (Guangzhou Ruibo), preparing into working solution (1 ug/ul), and storing at-40deg.C. Preparing 10% glucose with ultrapure water, filtering, and storing at-40deg.C. In vivo gene silencing is administered by tail vein injection with frequency of 2 times/week. The injection should be injected immediately after preparation. Each mouse injection is specifically prepared by mixing 50ul of siRNA with 50ul of 10% glucose to obtain 1 solution, mixing 25ul of ultrapure water with 50ul of 10% glucose and 25ul of in vivo transfection reagent (Engreen, 18668-11-1) to obtain 2 solution, standing for 2min, mixing 1 solution and 2 solution, and standing for 15min.

Tumor measurement and statistical analysis

Tumor size was measured starting from the day of initiation of dosing with a frequency of 3-5 days. The length and width of the tumor body surface are measured by using a vernier caliper. The data are arranged after the experiment is finished, and the drug effect is calculated and evaluated according to the following standard according to the technical guidelines of non-clinical research of cytotoxic anti-tumor drugs, wherein the tumor volume calculation formula is V=1/2×a×b2. Wherein a and b respectively represent the length and width of the tumor body surface. The tumor efficacy evaluation formula is relative tumor proliferation rate T/C% = TRTV/CRTV x 100%. T/C% <40% can be used to judge the effectiveness of the treatment. The lower the T/C% value, the more pronounced the efficacy. Wherein TRTV represents RTV of the treatment group and CRTV represents RTV of the negative control group. RTV represents relative tumor volume (relative tumor volume, RTV), calculated as RTV=Vt/V0. Where V0 is the initial tumor volume, and the tumor volume at the Vt observation time point. Vt in this experiment corresponds to the tumor volume of mice at the set experimental endpoint.

In this example, the difference between the experimental end point control group and the experimental group was compared by the T-test two-tailed test, and a P >0.05 showed a significant difference between the two groups. The experimental period was set to 21 days. According to ethical requirements, mice should be sacrificed for a tumor volume of 2000mm ³ or more when the end point of the 21-day experiment is not reached, and the sample is terminated for further experiments.

Experimental results:

The growth of the mice tumors in the endo 1 endonuclease silencing experimental group is obviously inhibited compared with the control group. Animal models effectively demonstrate that ENDOD1 endonuclease gene silencing can produce significant inhibition effects on various TP53 defects (SKOV 3, HCC1937, OVCAR-8 TR), BRCA1 defects (HCC 1937, BRCA1 pure and mutant) (fig. 13A), with inhibition rates (T/C%) SKOV3,15.2%, HCC1937,23.7%, OVCAR-8TR,25.7% (T/C% 40% indicates that the drug has significant efficacy on tumors), respectively (fig. 13B).

Thus, it is possible to treat tumors with corresponding genetic, and protein functional defects by administering to a subject a sufficient dose of an endo 1 endonuclease inhibitory drug.

Example 11, endo 1 endonuclease function inhibits the therapeutic action on active hepatitis b virus.

Previous findings indicate that hepatitis b virus encoding oncogene (HBx) inhibits homologous recombination repair by disrupting CRL4WDR70 ubiquitin enzyme (see ：Ren,L.,et al.,The Antiresection Activity of the X Protein Encoded by Hepatitis Virus B.Hepatology,2019.69). this example demonstrates that ENDOD1 endonuclease function inhibition can control HBV infection index by determining the sensitivity of HBV positive cells to ENDOD1 endonuclease gene silencing.

The experimental method comprises the following steps:

Cell proliferation assay:

5 ten thousand cells were seeded in 24-well plates and gene silencing experiments were initiated after 24 hours. The experimental period was set to 21 days and passaging was performed every 3 days. The ENDOD1 endonuclease siRNA transfection experiments were performed 24 hours after each passage. And (3) accurately counting by using a cell counter during passage, recording, and drawing a proliferation curve at the experimental end point.

HBV positive cell animal model experiment:

The evaluation of the therapeutic effect on patients with hepatitis B virus can be carried out by adopting detection indexes for detecting the viral copy number and surface antigen (HBsAG) in serum.

3 Athymic female nude mice of 6 weeks old were inoculated with HepG2.2.15 cells in the armpit and groin, and HBV mice infection model was established with cell inoculation number of 8x10 ⁶. Blood is taken from tail vein at different time points after cells are inoculated, HBV copy number change condition in blood is monitored by using a Hepatitis B Virus (HBV) nucleic acid amplification (PCR) fluorescence quantitative detection kit, and the in vivo silencing of the ENDOD1 endonuclease gene is started when the copy number rises to about 4000UI/ml which is the highest value in serum.

The preparation method comprises dissolving in ultrapure water (Invitrogen, 10977-015) in vivo siRNA powder of endo D1 endonuclease and control siRNA powder (Guangzhou Ruibo), preparing into working solution (1 ug/ul), and storing at-40deg.C. Preparing 10% glucose with ultrapure water, filtering, and storing at-40deg.C. In vivo gene silencing is administered by tail vein injection with frequency of 2 times/week. The injection should be injected immediately after preparation.

Each mouse injection is specifically prepared by mixing 50ul of siRNA with 50ul of 10% glucose to obtain 1 solution, mixing 25ul of ultrapure water with 50ul of 10% glucose and 25ul of in vivo transfection reagent (Engreen, 18668-11-1) to obtain 2 solution, standing for 2min, mixing 1 solution and 2 solution, and standing for 15min.

1. 2, 3, 4, 5, 7, 9, 15, 20 Days tail vein blood was taken, HBV genome copy number in serum was monitored, and HBsAg titer changes were detected by ELISA method. The serum copy number and HBsAg titers were monitored continuously for five days after drug withdrawal on day 16. Results time points were plotted as titer. Unless otherwise specified, all group-to-group differential calculations for the cell and zoology experiments in this example used a t-test two-tailed test. P >0.05 showed a significant difference between the two groups.

Experimental results:

1. Simultaneous silencing of combined lethal effects of endo D1 endonuclease and WDR70

Cell proliferation curves were determined by silencing ENDOD1 endonuclease and/or WDR70 in normal hepatocytes (L02). ENDOD1 gene silencing was found to be not significantly toxic to L02 cells, but could inhibit proliferation of cells in combination with siWDR a (fig. 14A). It was demonstrated that inhibition of endo-1 endonuclease has a combined lethal effect with WDR70 function.

2. Silencing the cytotoxic effects of endo-1 endonucleases on HBV positive cells

Silencing ENDOD1 endonuclease gene expression in hepatocytes (T43) integrated with HBV genome the ability of cell clone formation was measured (fig. 14B) and it was found that ENDOD1 gene silencing was effective in clearing T43 cells, whereas clone formation of normal hepatocytes (L02) under the same treatment was completely unaffected. Nude mice were inoculated subcutaneously with T43 to create a tumor model, and it was found that the gene silencing of endoD1 was effective in inhibiting the growth of T43 tumors (FIG. 14C), indicating that inhibition of endoD1 endonuclease function was effective in clearing HBV positive cells with defective CRL4WDR70 function.

3. Silencing endo d1 endonuclease to reduce HBV infection index

The method of nude tail vein inoculation HepG2.2.15, ELISA determines the titer of HBV antigen (HBsAg) in serum at the indicated time points, and the method of fluorescent quantitative PCR determines the titer of HBV viral DNA in serum. Experiments show that in vivo silencing of the endo D1 endonuclease can reduce HBV-DNA content and HBsAg antigen OD value in serum of a model mouse from about 1.5 (when the HBsAg antigen content in the serum reaches the highest value, the virus enters a stable high-level active replication period in a HepG2.2.15 cell liver cancer mouse model) to 0.05, and the value is close to or lower than an index of clinical negativity (> 0.05, and when the OD value is the detection minimum threshold). Within 5 days of cessation of endo 1 endonuclease in vivo silencing, HBV copy number and HBsAg expression in mouse serum is always within a stable negative range. The results of the cytology and animal experiments show that the in vivo silencing of the endo D1 endonuclease can permanently clear HBV virus infected host cells, and the downregulation of the function of the endo D1 endonuclease is suggested to clear HBV carried by hepatitis B virus infected persons or liver cells with HBx expression, thereby realizing the real clinical cure of the hepatitis B virus.

Thus, hepatitis B virus serum antigen index and viral titer can be reduced, and infection with hepatitis B virus can be eradicated or controlled by administering a sufficient dose of an endoD1 endonuclease inhibitory drug to a subject. The treatment methods may also be used in combination or in combination with a variety of antiviral treatments, such as antiviral metabolic drugs, gene therapy, DNA therapy, RNA therapy, targeted therapy, adjuvant therapy, immunotherapy, etc., which may be used simultaneously or sequentially to provide a therapeutic effect superior to those of agents that inhibit ENDOD1 function alone or antiviral agents alone.

Example 12 screening procedure for endo 1 endonuclease inhibitors.

Experimental method

Small molecule inhibitors that inhibit endo-1 endonuclease activity are screened by competitive inhibition methods. To 384-well PCR skirt plates (Thermo, AB 2384B) were added 0.1 μg of bacteria-expressed ENDOD1 protein, 100ng of linear double-stranded DNA substrate (sequence:

ATTCTCAGCCTGAAAGCCAGGTTCTAGAGGATGATTCTGGTTCTCACTTCAGTATGCTATCTCGACACCTTCCTAATCTCCAGACGCACAAAGAAAATCCTGTGTTGGATGTTGTGTCCAATCCTGAACAAACAGCTGGAGAAGAACGAGGAGACGGTAATAGTGGGTTCAATGAACATTTGAAAGAA) And a small molecule inhibitor library or DMSO control, in Cut Smart buffer at 37 ℃ for 16 hours. An equal volume of 2x fluorescent quantitative PCR premix (Promega, comprising specific primers with DNA substrates as templates, the sequence being F:5'-ATTCTCAGCCTGAAAGCCAGG-3'; R: 5'-TTCTTTCAAATGTTCATTGAA-3') is added to each well by using a mechanical multichannel pipette (eppendorf), and fluorescent quantitative PCR is performed to detect the residual amount of the DNA substrates and the digestion efficiency, and effective small molecule inhibitors are screened.

Experimental results

Screening and developing inhibitors of endo d1 can be accomplished by inhibiting the expression levels of endo d1 and/or its regulatory genes, inhibiting endo endonuclease activity of endo d1 protein, blocking DNA repair activity, disrupting subcellular localization, disrupting post-translational modifications such as ubiquitination and phosphorylation, interfering with biological functions such as cleavage of endo d1 and its regulatory proteins and signal peptide maturation, etc.

By inhibiting endo-endonuclease activity of endo d1 protein, as shown in fig. 15, a flow chart for endo d1 inhibitor screening was presented. The concentration of the endoD1 protein is reduced by gradient, the digestion efficiency is detected by utilizing fluorescent quantitative PCR, the residual quantity of the DNA substrate is inversely related to the concentration of the endoD1 protein, and the method can be used as an in-vitro drug screening system of the endoD1 endonuclease.

Claims (6)

1. Use of an ENDOD1 endonuclease inhibitor in the manufacture of a drug for treating and/or preventing tumors, wherein the inhibitor can inhibit the gene expression, activity or function of ENDOD1 endonuclease, the endonuclease is an endonuclease encoded by the ENDOD1 gene, the endonuclease inhibitor is a double-stranded siRNA that inhibits the expression of the ENDOD1 gene, and the tumor has DNA homologous recombination, DNA damage response, DNA damage repair or/and TP53 tumor suppressor gene mutation, gene expression or function defects, The nucleotide sequence of one strand of the double-stranded siRNA for inhibiting ENDOD1 gene expression is selected from: siENDOD1-001: 5’-GCAAGCGGATTGGCTACAA-3’ or its complementary sequence, siENDOD1-002: 5’-GGATGAAGAACGAATGGTA-3’ or its complementary sequence and siENDOD1-003: 5'-GTGGCCACATTTACTCTCA-3' or its complementary sequence, The tumor is lung cancer, breast cancer, liver cancer, ovarian cancer, cervical cancer, lymphoma, leukemia or/and sarcoma, wherein the lung cancer and ovarian cancer are TP53 mutations. 2. The use according to claim 1, wherein the tumor containing a DNA homologous recombination gene mutation, gene expression or functional defect includes a tumor with a BRCA1, BRCA2, WRN, Fanconi, EMSY, MRE11, NBS1, BLM, PTIP, ARID1A, ARID1B and WDR70 gene mutation, gene expression or functional defect. 3. The use according to claim 1, wherein the tumor comprises a tumor characterized by hepatitis B virus infection. 4. The use according to claim 1, further comprising using the ENDOD1 endonuclease inhibitor in combination with one or more other anti-tumor drugs or forming a composite composition for use. 5. The use according to claim 4, wherein the one or more other anti-tumor drugs are selected from chemotherapeutic drugs. 6. The use according to claim 5, wherein the chemotherapeutic drug is selected from platinum, mitomycin C, camptothecin, PARP inhibitors, DNAPK inhibitors, TKI, radioisotopes, vinca alkaloids, antitumor alkylating drugs and antimetabolites.