Method for specifically killing cancer cells by gene editing technology combined with dna damage repair inhibitors

Abstract

Cancer poses a significant threat to human health, and currently there is no particularly effective treatment. The primary reason is the lack of a highly effective method to specifically kill cancer cells while minimizing impact on normal cells. DNA breaks, if left unrepaired, lead to cell death—this is the fundamental principle of radiotherapy—but radiotherapy also causes DNA damage in normal cells. This invention proposes a method for specifically killing cancer cells. It utilizes gene editing technology to create DNA breaks unique to cancer cells (these DNA cut points are absent in normal cells), simultaneously combining this with DNA damage repair inhibitors to suppress DNA repair in cancer cells, leading to specific cancer cell death. This invention provides a new approach to precision cancer treatment.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and cancer treatment, specifically to a highly specific method for killing cancer cells that can effectively address the problems of cancer cell evolution and heterogeneity, as well as corresponding products and applications. Background Technology

[0002] Cancer is a serious disease that threatens human life. Currently, the main treatments for tumors include surgery, chemotherapy, and radiation therapy. Very few cancer patients can be completely cured. Surgery cannot remove all cancer cells, and other tumor treatments such as radiation therapy and chemotherapy cannot specifically kill only cancer cells. Therefore, finding a method to kill cancer cells without harming normal cells may be of great significance for the treatment of tumors.

[0003] Recent gene-editing technologies have enabled precise cutting, mutation, modification, insertion, and replacement of DNA within cells, showing great promise for applications. Existing gene-editing technologies include zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and regularly clustered short palindromic repeats (CRISPR).

[0004] For example, CRISPR gene editing technology was discovered in the bacterial immune mechanism. CRISPR (clustered regularly interspaced short palindromic repeats) are DNA sequences present in the bacterial genome, i.e., clustered regularly interspaced short palindromic repeats. There are several types of CRISPR-associated genes (Cas), Cas9 being one type, whose expression product is the Cas9 endonuclease. Taking CRISPR gene editing using Cas9 as a cutting element as an example, researchers achieve rapid and precise cutting of target DNA by introducing Cas9 and a specially designed guide RNA (gRNA) into the cell. The specific mechanism is that the gRNA binds to the target DNA site through the principle of base complementarity, guiding the Cas9 protein to locate the target DNA site and causing a break in the DNA at the binding site at a precise location. Then, utilizing the DNA damage repair mechanism naturally present in the cell, mutations, insertions, substitutions, and other modifications are made to the DNA at and near the break site, achieving the purpose of gene editing. The inventors have also been researching CRISPR-Cas9 gene editing technology and have optimized the technology in terms of safety. They have successfully induced chromosomal translocations at specific sites (see patent literature: Liu Houqi, Jiang Junfeng, et al., A safe nucleic acid molecule encoding Cas9 protein and its expression vector, patent number CN 201510092144.9, authorization announcement number CN104805099B; Method for constructing chromosomal translocation stem cells and animal models using CRISPR-Cas9 technology, patent number CN201510075127.4, authorization announcement number CN104726494B). We have also invented a new CRISPR method that effectively knocks out important LncRNAs in prostate cancer cells by inserting terminators through non-homologous DNA end joining (NHEJ) (see patent literature: Jiang Junfeng, et al., A method for knocking out transcribed elements by knocking in terminators using gene editing technology, patent application number CN201811415619.3, publication number CN111218479A). Because the most crucial step in CRISPR gene editing is the precise cutting of the target DNA site, CRISPR is often likened to a pair of "magic scissors" that can cut DNA at specific sites (see: Mali, P., Yang, L., Esvelt, KM, Aach, J., Guell, M., DiCarlo, JE, Norville, JE, and Church, GM (2013). RNA-guided human genome engineering via Cas9. Science 339, 823-826).

[0005] We know that the basic principle of radiotherapy for cancer is to break DNA through radiation. When these broken DNA fragments cannot be repaired, they activate multiple death signaling pathways, including apoptosis, leading to cell death. However, because radiotherapy inevitably causes DNA damage to normal cells, the dosage and range are strictly controlled, and cancer cells cannot be completely eliminated, with significant side effects.

[0006] Precision treatment of cancer has always been a key research area in this field. Currently, there is no literature reporting a method that combines gene editing technology with DNA damage repair inhibitors to specifically kill cancer cells. Summary of the Invention

[0007] This invention provides a method for specifically killing cancer cells by inducing DNA breaks at specific sites in cancer cells through gene editing technology, and by combining this method with DNA damage repair inhibitors to prevent DNA repair, thereby inducing cell death.

[0008] The inventors envisioned that if it were possible to cause DNA breaks in cancer cells without allowing those breaks to be repaired, it might be possible to specifically kill cancer cells. Therefore, against the backdrop of the increasing prevalence of DNA sequencing technology and the continuous development of gene editing technology, the inventors proposed a novel personalized precision treatment method for cancer: a method that uses gene editing technology combined with DNA damage repair inhibitors to specifically kill cancer cells. Figure 1 As shown:

[0009] S1. Identify at least one DNA sequence that is unique to cancer cells and differs from that of normal cells;

[0010] This can be accomplished through sequencing, such as by first performing DNA sequencing on cancer patients, and then using sequence analysis to identify several DNA sequences unique to their cancer cells that differ from those in normal cells; or by using existing technologies to identify cancer-specific sequences that have been reported.

[0011] Unique sequences include, but are not limited to, point mutations, insertion mutations, deletion mutations, and chromosomal translocations;

[0012] S2. Design and synthesize a gene editing system targeting the specific sequence determined in step S1, and introduce it into cancer cells to break or damage the specific sequence;

[0013] Gene editing systems can be zinc finger nuclease (ZFN) systems, transcription activator-like effector (TALEN) systems, or CRISPR gene editing systems, which can cause DNA damage at specific sites.

[0014] In a preferred embodiment of the invention, we use the CRISPR system: designing and synthesizing gRNA and Cas9 systems with sequences specific to cancer cells that cannot recognize DNA in any normal cells;

[0015] Zinc finger nuclease (ZFN) systems can achieve the same effects as CRISPR systems (see references: Zhao Guohua, Pu Jiali, Tang Beisha. Application of ZFN, TALEN and CRISPR / Cas9 gene editing technologies in disease research and gene therapy [J]. Chinese Journal of Medical Genetics, 2016, 33(6):6. and Rui Y, Wilson DR, Green JJ. Non-Viral Delivery To Enable Genome Editing. Trends Biotechnol. 2019 Mar; 37(3):281-293. PMID:30278987)

[0016] The transcription activator-like effector (TALEN) system can achieve the same effect as the CRISPR system (see references: Zhao Guohua, Pu Jiali, Tang Beisha. Application of ZFN, TALEN and CRISPR / Cas9 gene editing technologies in disease research and gene therapy [J]. Chinese Journal of Medical Genetics, 2016, 33(6):6. and Rui Y, Wilson DR, Green JJ. Non-Viral Delivery To Enable Genome Editing. Trends Biotechnol. 2019 Mar; 37(3):281-293. PMID:30278987).

[0017] The gene editing system can be directly synthesized or it can be a vector that can be introduced into cells to generate an effective editing system, such as plasmids, RNA, viruses (adenovirus, lentivirus), etc.

[0018] S3. Introduce drugs that inhibit DNA damage repair into cancer cells;

[0019] Drugs that inhibit DNA damage repair include known or future inhibitors that can inhibit DNA damage repair, such as inhibitors of the non-homologous endjoining (NHEJ) pathway, such as the DNA-PKcs inhibitor NU7441; or inhibitors of the homologous recombination (HR) pathway, such as the ATM inhibitor KU55933; or inhibitors that can inhibit both NHEJ and HR, such as wortmannin; including both combined use and single use.

[0020] In steps S2 and S3, cancer cells can be introduced simultaneously or separately. Simultaneous introduction can involve individual cells being introduced at the same time, or they can be introduced together as a complex (e.g., using nanotechnology to form a nanocomposite). The introduction methods can be the same or different, including injection, oral administration, topical application, inhalation, etc. It can be either conventional introduction or targeted introduction targeting specific cancer cells.

[0021] This personalized tumor treatment strategy, which uses gene editing technology to precisely break DNA mutation sites specific to cancer cells, kills cells based on the same principle as radiotherapy—causing DNA double-strand breaks (DSBs). The difference lies in that our strategy only causes DNA breaks in cancer cells. Normal cells lack the DNA sequences that the designed gene-editing system can bind to. Even if the system enters normal cells, it will not produce a killing effect. Therefore, this invention holds the promise of killing cancer cells without harming normal cells. Furthermore, the broken DNA cannot be repaired, truly achieving the goal of specifically killing cancer cells and restoring patients to health.

[0022] Furthermore, the present invention provides a method for specifically killing cancer cells using gene editing technology combined with DNA damage repair inhibitors, comprising the following steps:

[0023] S11. Cancer patients first undergo sequencing to determine their DNA sequence;

[0024] The cancers mentioned include solid malignant tumors such as liver cancer, stomach cancer, intestinal cancer, prostate cancer, breast tumors, head and neck tumors, glioblastoma, bladder tumors, pancreatic tumors, ovarian tumors, skin tumors, osteosarcoma, fibrosarcoma, etc., as well as leukemia, lymphoma, etc.

[0025] The sequencing technology mentioned above refers to all methods that can directly or indirectly identify all or part of the DNA sequence in a patient's cancer cells, such as whole genome sequencing, whole exome sequencing, first-generation sequencing, second-generation sequencing, third-generation sequencing, RNA sequencing, single-cell sequencing, and spatial transcriptome sequencing.

[0026] In one of our specific embodiments (Example 1), we detected the DNA sequence of hepatocellular carcinoma HepG2 by whole-genome sequencing.

[0027] In another specific embodiment (Example 5), we detected sequences in prostate cancer PDX by whole exome sequencing.

[0028] S12. Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0029] Various methods capable of identifying DNA sequences unique to cancer cells can be used in this step. These cancer-specific DNA sequences can be determined by comparing the cancer cell's DNA sequence with a reference genome, or by comparing it with the DNA sequence in the patient's own normal cells. Specific sequences include point mutations, insertion mutations, deletion mutations, and chromosomal translocations.

[0030] In one specific embodiment (Example 1), we compared the DNA sequence of hepatocellular carcinoma HepG2 with the normal human reference genome (Feb. 2009 (GRCh37 / h19)) and found 3,985,698 single nucleotide variants (SNVs) and 817,723 insertion or deletion mutations (indels). These mutation sites are different from the DNA of normal cells and are DNA sites specific to the HepG2 cancer cell.

[0031] S21. Design and synthesize gene editing systems that target specific sequences in cancer cells;

[0032] Techniques capable of causing DNA damage at specific sites can be used to implement this method, such as zinc finger nuclease (ZFN) systems, transcription activator-like effector (TALEN) systems, and CRISPR gene editing systems.

[0033] In one of our specific embodiments (Example 1), we selected 8 mutation sites in hepatocellular carcinoma HepG2, the mutated sequences of which are significantly different from the DNA sequences in normal cells. We designed 8 gRNA-Cas9 expression systems for these sites.

[0034] S22. Introducing a gene-editing system targeting these sequences specific to cancer cells into cancer cells; and introducing drugs that inhibit DNA damage repair into cells.

[0035] The relevant gene editing systems can be directly synthesized, or they can be vectors that can be introduced into cells and generated into effective editing systems by the cells, such as plasmids, RNA, viruses (adenovirus, lentivirus), RNA-protein complexes, etc.

[0036] The vectors for introducing drugs into cells can be viruses, nanomedicine-encapsulated expression vectors, nanomedicine-encapsulated RNA-protein complexes, etc.

[0037] The administration methods can include injection, oral administration, topical application, inhalation, etc.

[0038] In one specific embodiment (Example 1), we selected CRISPR gene editing technology in combination with a DNA damage repair inhibitor to treat hepatocellular carcinoma. Our CRISPR gene editing system uses adenovirus expression (a viral vector expressing both gRNA and Cas9).

[0039] In another specific embodiment (Example 2), we chose to treat prostate cancer using CRISPR gene editing technology in combination with DNA damage repair inhibitors. Our CRISPR gene editing system uses lentiviral expression (gRNA expressed on one viral vector and Cas9 expressed on another lentiviral vector).

[0040] S3. Introduce drugs that inhibit DNA damage repair into cells.

[0041] In one specific embodiment (Example 1), the DNA damage inhibitor used was NU7441+KU55933, and the administration method was direct addition.

[0042] In another specific embodiment (Example 2), the DNA damage inhibitor used waswortmannin, which was administered by direct addition.

[0043] In another specific embodiment (Example 4), the DNA damage inhibitor used was NU7441+KU55933, and the administration method was injection.

[0044] Through the above steps, the designed DNA sites in cancer cells are precisely cut by the gene-editing system. Due to the presence of DNA damage inhibitors, the DNA damage cannot be repaired, triggering cell death pathways and leading to cancer cell death. Since normal cells lack the DNA sequences that the designed gene-editing system can bind to, even if the system enters normal cells, it will not produce a killing effect. Therefore, it holds promise for achieving the goal of killing cancer cells without harming normal cells.

[0045] Another aspect of the present invention provides the application of a gene editing system and a DNA damage repair inhibitor in combination in the preparation of a drug for treating tumors.

[0046] The gene editing system described is designed and synthesized to target DNA sequences unique to cancer cells that differ from those in normal cells, in order to break or damage these unique DNA sequences in cancer cells.

[0047] The unique DNA sequences mentioned include, but are not limited to, point mutations, insertion mutations, deletion mutations, and chromosomal translocations.

[0048] The gene editing systems mentioned include, but are not limited to, zinc finger nuclease (ZFN) systems, transcription activator-like effector (TALEN) systems, and may also be gene editing systems that can cause DNA damage at specific sites, such as CRISPR gene editing systems.

[0049] The gene editing system can be directly synthesized or it can be a vector that can be introduced into cells to generate an effective editing system, such as plasmids, RNA, viruses (adenovirus, lentivirus), etc.

[0050] The DNA damage repair inhibitors mentioned include inhibitors that are currently known or will be developed in the future that can inhibit DNA damage repair, such as inhibitors of the non-homologous end joining (NHEJ) pathway, such as the DNA-PKcs inhibitor NU7441; or inhibitors of the homologous recombination (HR) pathway, such as the ATM inhibitor KU55933; or inhibitors that can inhibit both NHEJ and HR, such as wortmannin; including both combined use and single use.

[0051] The aforementioned anti-tumor drug can be administered via injection, oral administration, topical application, inhalation, etc. Preferably, it is administered via direct injection, intraperitoneal injection, peritumoral injection, or other suitable routes of administration.

[0052] The method and application of the present invention have at least the following four advantages:

[0053] 1. True cancer cell-specific killing.

[0054] As mentioned above, only cancer cells are killed by this strategy; normal cells do not have cleavage sites and are minimally affected.

[0055] 2. It holds promise for effectively addressing the continuous mutation and evolution of cancer cells.

[0056] One of the most challenging problems in cancer treatment is the continuous mutation and evolution of cancer cells, which leads to drug resistance even with the best targeted drugs or immunotherapies available today after a certain period of time.

[0057] Our strategy targets specific pre-existing mutations, so the key to sustained treatment effectiveness lies in the persistence of these mutations. DNA mutations are random, but due to the vastness of the genome, the probability of a mutated site recurring is very low. In one example, we performed whole-genome sequencing on single-cell clones of prostate cancer cells DU145 cultured in the lab for extended periods, as well as on standard DU145 cells. We found that after prolonged culture, most mutations were preserved, and our designed seven cleavage sites were completely retained in several single clones, suggesting that all the "scissors" will be effective. Furthermore, if we choose to create 10 "scissors," the probability that the sites corresponding to these 10 "scissors" will all become ineffective as the tumor progresses is quite low.

[0058] Even if most of these cleavage sites have mutated and these "scissors" have become ineffective, it is very easy to overcome this problem. Just redesign and synthesize new "scissors" and introduce them.

[0059] 3. It holds promise for addressing the issue of tumor heterogeneity.

[0060] Tumor heterogeneity also stems from the individual evolution of cancer cells. This heterogeneity means that only a subset of cancer cells are sensitive to drugs, a problem that current treatments struggle to overcome. However, with future advancements in sequencing technologies, such as single-cell sequencing, and bioinformatics techniques like clonal evolution analysis, it may be possible to obtain common mutation sequences shared by all cancer cells in a patient. Utilizing our strategy to specifically cleave these shared DNA sequences could potentially overcome the problems caused by tumor heterogeneity.

[0061] 4. Our strategy has good individualized treatment timeliness.

[0062] Since gene-editing systems ("scissors") can be designed simply by knowing the DNA sequence, and this customized gene-editing system and the drug mixture of DNA damage repair inhibitors can be prepared within weeks of knowing the DNA sequence, it can be said to have good timeliness for personalized cancer treatment.

[0063] Furthermore, in the future, gene editing system reagents can be pre-designed to target high-frequency mutation sites in tumors. This way, only the DNA sequence of the patient's sample needs to be sequenced to select the appropriate gene editing system reagents according to our strategy. It may not even be necessary to use PDX models to test drug sensitivity, which is expected to greatly shorten the treatment response time and bring greater benefits to patients.

[0064] This invention provides a new approach to precision treatment of cancer. Attached Figure Description

[0065] Figure 1A schematic diagram of the strategy for personalized cancer treatment according to this invention;

[0066] Figure 2 Example 1: Effect of the method of the present invention on the killing effect of HepG2 liver cancer cells, wherein... Figure 2 A represents the results of flow cytometry analysis for cell apoptosis. Figure 2 B is Figure 2 The statistical results of A;

[0067] Figure 3 Example 2: Effect of the method of the present invention on the killing effect of prostate cancer cells DU145, wherein... Figure 3 A represents the results of flow cytometry analysis for cell apoptosis. Figure 3 B is Figure 3 The statistical results of A;

[0068] Figure 4 Example 3: Effect of the method of the present invention on the killing effect of HepG2 liver cancer cells;

[0069] Figure 5 Example 4: Effect diagram of the method of the present invention on killing PDX of liver cancer;

[0070] Figure 6 Example 5: Effect diagram of the method of the present invention on killing prostate cancer PDX;

[0071] Figure 7 Example 6: Effect diagram of the method of the present invention on killing organoids of liver cancer. Detailed Implementation

[0072] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0073] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Guide (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. All technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art unless otherwise defined. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred methods and materials described herein are for illustrative purposes only.

[0074] Example 1: Specific killing of HepG2 liver cancer cells using the method of the present invention.

[0075] Step 1: Perform DNA sequencing first;

[0076] We detected the DNA sequence of HepG2 hepatocellular carcinoma through whole-genome sequencing.

[0077] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0078] By comparing the DNA sequence of hepatocellular carcinoma HepG2 cells with the normal human reference genome (Feb. 2009 (GRCh37 / h19), we identified 3,985,698 single nucleotide variants (SNVs) and 817,723 insertion or deletion mutations (indels). These mutation sites differ from those in normal cells and are unique to the HepG2 cancer cell line.

[0079] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0080] We are using the CRISPR system, and our initial assessment indicates that there are approximately 1,000 sites suitable for gRNA design.

[0081] We selected the following 8 sites for gRNA design and synthesis, and constructed adenovirus expression vectors for these gRNAs, which can also express Cas9 endonuclease.

[0082] The following are the DNA mutations and gRNA design sequences specific to HepG2 liver cancer cells:

[0083]

[0084] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in HepG2 cancer cells.

[0085] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0086] We added a mixture of adenoviruses expressing Cas9 and the eight gRNAs mentioned above (constructed using conventional techniques, see Chen ZH, Yu YP, Zuo ZH, et al. Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene. Nat Biotechnol. 2017; 35(6):543-550. doi:10.1038 / nbt.3843) and DNA damage repair inhibitors (in this example, we added NU7441, an inhibitor of DNA-PKcs in the NHEJ pathway (purchased from Selleck), and KU55933, an ATM inhibitor in the HR pathway (purchased from Selleck), to HepG2 cell culture medium, and then detected cell death.

[0087] Experimental results showed that our strategy could significantly kill HepG2 cells, such as Figure 2 As shown:

[0088] This experiment used flow cytometry to detect cell apoptosis. T4-set1 and T4-set2 are combinations of four of the eight target gRNA expression vectors, while T8-set1+2 represents the results of treating HepG2 cells with all eight target gRNA expression vectors. Figure 2 B is Figure 2 The statistical results in section A show that adding the CRISPR system, which targets DNA specific to HepG2 cells, along with two DNA damage repair inhibitors (NU7441 and KU55933) to HepG2 cells has a significant killing effect on these cells.

[0089] Example 2: Specific killing of DU145 prostate cancer cells using the method of the present invention.

[0090] Step 1: Perform DNA sequencing first;

[0091] We analyzed the DNA sequence of DU145 prostate cancer cells using whole-genome sequencing.

[0092] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0093] By comparing the DNA sequence of DU145 prostate cancer cells with that of a normal human reference genome (Feb.2009(GRCh37 / h19), we identified DNA sequence sites unique to DU145 prostate cancer cells.

[0094] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0095] We used the CRISPR system. We initially evaluated the mutation sites in DU145 cells, selected the following 7 sites for gRNA design and synthesis, and constructed lentiviral expression vectors for these gRNAs.

[0096] The following are the DNA mutations specific to DU145 prostate cancer cells and the designed gRNA sequence:

[0097]

[0098]

[0099] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in DU145 prostate cancer cells.

[0100] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0101] We added a mixture of lentiviruses expressing Cas9 and the aforementioned seven gRNAs (constructed using conventional techniques, see Platt RJ, Chen S, Zhou Y, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014; 159(2):440-455. doi:10.1016 / j.cell.2014.09.014) and a DNA damage repair inhibitor (in this example, we used wortmannin, an inhibitor that can inhibit both the NHEJ and HR pathways—purchased from Selleck, or a combination of NU7441 and KU55933) to the DU145 cell culture medium, and then detected cell death.

[0102] Experimental results showed that our strategy could significantly kill DU145 cells, see [link to study]. Figure 3 As shown:

[0103] This experiment uses flow cytometry to detect apoptosis in DU145 prostate cancer cells (containing gRNA and Cas9-recognized specific DNA mutations) and HEK293T human embryonic kidney epithelial cells (not containing the introduced gRNA and Cas9-recognized specific DNA mutations) treated with the method of this invention. Figure 3 B is Figure 3The statistical results in section A show that the CRISPR system targeting DNA specific to DU145 cells, whether combined with two DNA damage repair inhibitors (NU7441 and KU55933) or with a single DNA damage repair inhibitor (wortmannin), exhibits significant specific killing effects on DU145 cells, while showing no significant killing effect on normal human embryonic kidney epithelial cells HEK293T.

[0104] Example 3: Specific killing of Hep3B liver cancer cells using the method of the present invention.

[0105] Step 1: Perform DNA sequencing first;

[0106] We analyzed the CCLE website (https: / / depmap.org / portal / cell_line / ACH-000979?tab=mutation), a tumor database website, to obtain DNA mutation information in Hep3B liver cancer cells.

[0107] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0108] By comparing the downloaded DNA sequence information of Hep3B liver cancer cells with the normal human reference genome (Feb.2009(GRCh37 / h19), we identified the unique DNA sequence sites in Hep3B liver cancer cells.

[0109] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0110] We used the CRISPR system, selected the following four sites for gRNA design and synthesis, and constructed lentiviral expression vectors for these gRNAs. The Cas9 endonuclease was expressed by another lentivirus.

[0111] The following are the DNA mutations and gRNA design sequences specific to Hep3B liver cancer cells:

[0112]

[0113] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in Hep3B cancer cells.

[0114] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0115] We added a mixture of lentiviruses expressing Cas9 and the four gRNAs mentioned above, along with DNA damage repair inhibitors (in this example, we added NU7441, an inhibitor of DNA-PKcs in the NHEJ pathway, and KU55933, an inhibitor of ATM in the HR pathway), to the culture medium of Hep3B cells, and then examined cell death.

[0116] Experimental results showed that our strategy could significantly kill Hep3B cells, see [link to study]. Figure 4 As shown:

[0117] The CCK-8 assay was used to detect the survival of Hep3B liver cancer cells treated with the method of this invention. It was observed that introducing the CRISPR system targeting the DNA specific to Hep3B cells, along with two DNA damage repair inhibitors (NU7441 and KU55933), into Hep3B liver cancer cells resulted in a significant and specific killing effect on these cells. Other groups served as single-component controls or other combination controls.

[0118] Example 4: Specific killing of liver cancer PDX using the method of the present invention.

[0119] Step 1: Perform DNA sequencing first;

[0120] We used next-generation sequencing to identify the DNA sequence in cancer cells from a patient-derived xenograft (PDX) model derived from fresh samples from clinical liver cancer patients.

[0121] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0122] By comparing the DNA sequence information of cancer cells in liver cancer PDX with the normal human reference genome (Feb.2009(GRCh37 / h19), we identified the unique DNA sequence sites in cancer cells in liver cancer PDX.

[0123] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0124] We used the CRISPR system, selected the following 7 sites for gRNA design and synthesis, and constructed lentiviral expression vectors for these gRNAs, which simultaneously express the Cas9 endonuclease.

[0125] The following are the unique DNA mutations and gRNA design sequences in liver cancer PDX:

[0126]

[0127]

[0128] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in cancer cells in liver cancer PDX.

[0129] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0130] We attempted to treat liver cancer in PDX model mice by intratumoral injection of a lentiviral mixture expressing Cas9 and the aforementioned seven gRNAs, along with DNA damage repair inhibitors (in this example, we added NU7441, an inhibitor of DNA-PKcs in the NHEJ pathway, and KU55933, an ATM inhibitor in the HR pathway). We found that our strategy significantly inhibited tumor growth, as shown in the experimental results. Figure 5 As shown:

[0131] The effectiveness of the method of the present invention was verified in hepatocellular carcinoma PDX mice. PDX mice were treated with a CRISPR system targeting DNA specific to the cancer cells and two DNA damage repair inhibitors (NU7441 and KU55933) via intratumoral injection. Tumor size was measured every 3 days after injection, and tumor growth curves were plotted. It was observed that compared with the control group (lentivirus with only Cas9 and no gRNA + DNA inhibitors), the tumor growth rate in the experimental group was significantly slower.

[0132] Example 5: Specific killing of prostate cancer PDX using the method of the present invention.

[0133] Step 1: Perform DNA sequencing first;

[0134] We used whole-exome sequencing to identify the DNA sequences in cancer cells from a patient-derived xenograft (PDX) model derived from fresh samples from clinical prostate cancer patients.

[0135] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0136] By comparing the DNA sequence information of cancer cells in prostate cancer PDX with the normal human reference genome (Feb.2009(GRCh37 / h19), we identified the unique DNA sequence sites in cancer cells in prostate cancer PDX.

[0137] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0138] We used the CRISPR system, selected the following 5 sites for gRNA design and synthesis, and constructed lentiviral expression vectors for these gRNAs, which simultaneously express the Cas9 endonuclease.

[0139] The following are the DNA mutations specific to cancer cells in prostate cancer PDX and the gRNA design sequences:

[0140]

[0141] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in cancer cells in prostate cancer PDX.

[0142] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0143] We attempted to treat prostate cancer in PDX model mice by intratumoral injection of a lentiviral mixture expressing Cas9 and the aforementioned five gRNAs, along with DNA damage repair inhibitors (in this example, we added NU7441, an inhibitor of DNA-PKcs in the NHEJ pathway, and KU55933, an ATM inhibitor in the HR pathway). We found that our strategy significantly inhibited tumor growth. (See below.) Figure 6 As shown:

[0144] The effectiveness of the method of this invention was verified in prostate cancer PDX mice. PDX mice were treated with a CRISPR system targeting DNA specific to prostate cancer cells and two DNA damage repair inhibitors (NU7441 and KU55933) via intratumoral injection. Tumor size was measured approximately every 3 days after injection, and tumor growth curves were plotted. It was observed that compared to the control group (lentivirus with only Cas9 and no gRNA + DNA inhibitors), the tumor growth rate in the experimental group was significantly slower.

[0145] Example 6: Specific killing of liver cancer organoids using the method of the present invention.

[0146] Step 1: Perform DNA sequencing first;

[0147] We used next-generation sequencing to identify the DNA sequence of cancer cells in liver cancer organoids derived from fresh samples from clinical liver cancer patients.

[0148] Step 2: Sequence analysis can identify several DNA sequences unique to cancer cells and different from those in normal cells;

[0149] By comparing the DNA sequence information of cancer cells in liver cancer organoids with the normal human reference genome (Feb.2009(GRCh37 / h19), we identified the unique DNA sequence sites in cancer cells in liver cancer organoids.

[0150] Step 3: Then design and synthesize gene editing systems that target these sequences specific to cancer cells;

[0151] We used the CRISPR system, selected the following three sites for gRNA design and synthesis, and constructed lentiviral expression vectors for these gRNAs, which simultaneously expressed the Cas9 endonuclease.

[0152] The following are the DNA mutations and gRNA design sequences specific to cancer cells in liver cancer organoids:

[0153]

[0154] These gRNAs cannot recognize DNA in any normal cells, but they can recognize DNA sequences in cancer cells in liver cancer organoids.

[0155] Step 4: Introduce gene editing systems that target these sequences specific to cancer cells, along with drugs that inhibit DNA damage repair, into the cells.

[0156] By adding a lentiviral mixture expressing Cas9 and the three gRNAs mentioned above, along with DNA damage repair inhibitors (in this example, we added NU7441, an inhibitor of DNA-PKcs in the NHEJ pathway, and KU55933, an ATM inhibitor in the HR pathway), to the culture medium for liver cancer organoids, we found that our strategy could significantly inhibit the growth of these liver cancer organoids. (See...) Figure 7 As shown:

[0157] A CRISPR system targeting the cancer cell-specific DNA of the liver cancer organoids and two DNA damage repair inhibitors (NU7441 and KU55933) were added to the culture medium of the liver cancer organoids, and the growth of the organoids was observed. It was observed that this strategy significantly inhibited the growth of the liver cancer organoids. The effectiveness of the method of this invention was verified in liver cancer organoids.

[0158] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application. sequence list <110> Naval Medical University of the Chinese People's Liberation Army <120> A method that uses gene editing technology combined with DNA damage repair inhibitors to specifically kill cancer cells. <130> manual <160> 35 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <400> 1 ggcgggctca tggaaggtgt 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 ttgaactatc atctgaagag 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 cttggaaggg tagcaggact 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 ggtccaggta agctgcacca 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 gggcgcacgg cggggctagc 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 tggtggaatg gtatggaatg 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <400> 7 gaaaggaatc aatccgagaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <400> 8 aataagtaga ataaacatta 20 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <400> 9 attgtgcaca aggacatcg 19 <210> 10 <211> 19 <212> DNA <213> Artificial sequence <400> 10 tcttcaggaa ttacggaaa 19 <210> 11 <211> 19 <212> DNA <213> Artificial sequence <400> 11 gagctacatc aaccttagg 19 <210> 12 <211> 19 <212> DNA <213> Artificial sequence <400> 12 accgaggagg caaaacttc 19 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 ttggggtgga gttgcccggc 20 <210> 14 <211> 20 <212> DNA <213> Artificial sequence <400> 14 ggtgagtagg tgcagccatt 20 <210> 15 <211> 20 <212> DNA <213> Artificial sequence <400> 15 ttggtcttcc caaattcttc 20 <210> 16 <211> 19 <212> DNA <213> Artificial sequence <400> 16 attgtgcaca aggacatcg 19 <210> 17 <211> 20 <212> DNA <213> Artificial sequence <400> 17 aatccggcaa acaaagcagc 20 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <400> 18 cgcggcgggc gggcccatct 20 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <400> 19 accctataaa tgtaaaatgt 20 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <400> 20 agaggaggaa ataaaagagg 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <400> twenty one cacctgtgca tttggcaggc 20 <210> twenty two <211> 20 <212> DNA <213> Artificial sequence <400> twenty two gctcaagtgg aacttacatg 20 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <400> twenty three ccagtccagg gctttccgat 20 <210> twenty four <211> 20 <212> DNA <213> Artificial sequence <400> twenty four aatgaacata tctgaaatgc 20 <210> 25 <211> 20 <212> DNA <213> Artificial sequence <400> 25 gggcaggggt ggggaatcat 20 <210> 26 <211> 19 <212> DNA <213> Artificial sequence <400> 26 tctctgtcca gtccagggt 19 <210> 27 <211> 20 <212> DNA <213> Artificial sequence <400> 27 gttgcgcgtg gcttacagac 20 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <400> 28 tggctggacc ctgggcaccc 20 <210> 29 <211> 20 <212> DNA <213> Artificial sequence <400> 29 agaagaggcg gtggagttag 20 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <400> 30 accagatttg gagtggtatg 20 <210> 31 <211> 20 <212> DNA <213> Artificial sequence <400> 31 aatcctgggc aacttcacgc 20 <210> 32 <211> 20 <212> DNA <213> Artificial sequence <400> 32 cctctcgctg ctgcccctct 20 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <400> 33 acagagtgat ttaagataca 20 <210> 34 <211> 20 <212> DNA <213> Artificial sequence <400> 34 tcaggaaaca acaggtggag 20 <210> 35 <211> 20 <212> DNA <213> Artificial sequence <400> 35 gatatataga atcttgtacc 20

Claims

1. The application of a gene editing system combined with a DNA damage repair inhibitor in the preparation of a tumor treatment drug, wherein the gene editing system is designed and synthesized to target DNA sequences unique to cancer cells and different from those in normal cells, thereby causing breakage or damage to these unique DNA sequences in cancer cells. in, The tumor is selected from liver cancer, prostate cancer, pancreatic tumors, leukemia, or colorectal cancer. The DNA damage repair inhibitors include, but are not limited to, any one or a combination of the following: inhibitors of the non-homologous end joining pathway and inhibitors of the homologous recombination pathway. The gene editing system described is either a transcription activator-like effector (TALEN) system or a CRISPR gene editing system.

2. The application of the gene editing system and DNA damage repair inhibitor described in claim 1 in the preparation of a drug for treating tumors, wherein the unique DNA sequence is a DNA sequence unique to cancer cells and distinguishable from normal cells, including but not limited to point mutations, insertion mutations, deletion mutations, and chromosomal translocations.

3. The application of the gene editing system and DNA damage repair inhibitor as described in claim 1 in the preparation of a drug for treating tumors, wherein the gene editing system is directly synthesized or is a carrier that can be generated by cells to form an effective editing system after being introduced into cells.

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