CN113755495B - Use of gene editing technology in the treatment of cancer - Google Patents

Abstract

The present invention relates to the use of gene editing technology in the treatment of cancer. According to the invention, the gRNA is designed aiming at the VEGF target, VEGF gene is specifically knocked out in the breast cancer cell, the knocking-out effect is better through protein detection, and VEGF protein is well inhibited. In addition, after the breast cancer cells with the VEGF knocked-out genes are treated by combining with the PD-1 monoclonal antibody, the breast cancer tumors are better inhibited, and a better treatment effect is achieved.

Description

Use of gene editing technology in the treatment of cancer

Technical Field

The invention relates to the field of biology, in particular to application of a gene editing technology in treating cancer.

Background

The latest global cancer burden data published by the international agency for research on cancer (IARC) of the world health organization in 2020 shows that the number of new breast cancers reaches 226 ten thousand, and the new breast cancers formally replace lung cancers and become the first cancer with the global incidence rate! In China, the incidence of breast cancer is the first of all female malignant tumors. According to the data of the '2015 annual report of Chinese tumor registration', the incidence rate of breast cancer of women in China is 42.55/10 ten thousand, and the number of breast cancer patients in China is up to 250 ten thousand predicted by 2021 years. Breast cancer is the first killer of female health and is not negligible. The overall incidence of breast cancer is approximately 0.7%. About 30 million people find the breast cancer in China every year, and the number of breast cancer patients in China is increased year by year, and the annual incidence rate is increased by about 1 to 2 percent. Breast cancer is the most common malignancy in women, accounting for approximately 17%, and breast cancer accounts for the first of female malignancies in some major cities. The etiology of breast cancer is currently unclear. Current studies indicate that estrogen is directly associated with the onset of breast cancer. The probability of breast cancer before the age of 20 years is low and the incidence rate gradually increases after the age of 20 years. The highest age is around 45 to 50 years. When one of the first-degree relatives has breast cancer, the incidence rate is 2 to 3 times that of the average person.

With the development of medical science, the emergence of new medicines and new treatment modes, the current treatment modes of breast cancer mainly comprise surgical treatment for removing lesions, radiotherapy, chemotherapy, endocrine treatment and the like.

The primary lesion excision surgery is to excise the breast from the side with cancer cells, which is more effective in preventing the spread of cancer cells. The operation can be adopted to treat early or locally middle and late breast cancer, and even some patients with early breast cancer can be cured by the operation treatment.

Chemotherapy, one of the common treatments for breast cancer, includes three cases: first, chemotherapy is required when the patient's tumor undergoes lymph node metastasis or reaches a middle or advanced stage of breast cancer; secondly, the aim of radical cure can be achieved by reducing certain larger breast cancer tumors through chemotherapy and then performing operation, so that the method becomes 'new auxiliary chemotherapy'; thirdly, some patients find the residues of tumor cells after resection operation, and the survival rate can be improved by chemotherapy, so the method becomes the postoperative chemotherapy. However, it should be noted that chemotherapy may cause some side effects to human body, such as decreased appetite, nausea and vomiting, but can be significantly improved after symptomatic treatment.

Radiation therapy is a supplement to surgery or treatment of advanced, recurrent cases by local irradiation to inactivate or kill the cells. Common side effects of radiotherapy include local skin changes, thermal burns, peeling, skin hardening, color changes, etc., or effects on surrounding tissues, such as rib inflammation, lung fibrosis, etc., due to chest irradiation.

Endocrine therapy achieves the treatment purpose by changing the level of female hormone in a human body or changing the action of the hormone, is an auxiliary means for treating breast cancer, cannot be used independently, and is generally used for patients after operation, and the life cycle of the patients is prolonged by taking medicines for a long time by the patients. The side effects of endocrine therapy are also smaller and controllable compared to chemotherapy. However, when the endocrine therapy is performed on a breast cancer patient, the dosage needs to be set under the diagnosis and guidance of a doctor, and the breast cancer patient cannot be used blindly and is harmful to life and health.

In recent years, monoclonal antibody drugs have been important drugs for treating HER2 positive breast cancer. Trastuzumab: is the first humanized monoclonal antibody aiming at HER-2 globally, which is specifically combined with the extracellular IV region of HER2 receptor and inhibits the activation of HER2 receptor. Is suitable for the adjuvant and neoadjuvant treatment of early/late HER2 positive breast cancer. Pertuzumab: is the 2 nd recombinant humanized monoclonal antibody aiming at HER-2 target, which is specifically combined with the extracellular II region of the HER2 receptor and inhibits the activation of the HER2 receptor. Is suitable for the combined treatment of the HER2 positive early-stage breast cancer patient with high recurrence risk by combining trastuzumab and chemotherapy.

The CRISPR/Cas9 system consists of a single-stranded sgRNA and an endonuclease-active Cas9 protein. The introduction of Cas9 protein mRNA and sgRNA containing specific target sequences into animal zygotes or somatic cells, which can achieve easy repair of genes or defective genes in animals, can achieve sequence editing at any sites of the genome by random INDEL effect (insertional repair (HDR) in the presence of sequences having homology arms in the cell due to self-repair function of the genome of the cell by non-homologous end joining (NHEJ) when Cas9 enzyme cleaves the genome, cancer has been one of the most important diseases facing human, and one of the current major research fields, and the advantages of Cas/9 design and simple CRISPR manipulation system, researchers have innovatively applied the CRISPR system to cancer research. The application of the CRISPR system not only enriches the technical means of cancer research, but also provides a new view and a new idea of cancer research.

Currently, there are few and limited options for breast cancer and treatment using CRISPR/Cas technology and CRISPR in combination with other drugs, and therefore, there is an urgent need to develop new treatment modalities to enrich the selection categories.

Disclosure of Invention

The invention provides a kit for treating breast cancer, which comprises an inhibitor for specifically inhibiting VEGF and a PD-1 monoclonal antibody.

Further, wherein the VEGF inhibitor is a gRNA targeting a VEGF gene, wherein the gRNA has the sequence TCTACCTCCACCATGCCAAG (SEQ ID NO: 1).

Further, the invention provides a VEGF knock-out gRNA1: Tctacctccaccatgccaag, the corresponding sgRNA of which is synthesized by adopting the following primer annealing, F: CACCGTCTACCTCCACCATGCCAAG and R: AAACCTTGGCATGGTGGAGGTAGAC.

In another aspect, the present application provides a method of treating breast cancer, the method comprising the steps of: introducing the gRNA into a diseased site in a subject in need thereof.

In certain embodiments, the introducing comprises injecting.

In certain embodiments, the introducing comprises tumor in situ injection.

The invention further provides a PD-1 monoclonal antibody. Wherein the variable region of the light chain of the antibody (SEQ ID NO: 2)

DIVITQRPALMAASPGEKDTITCYMVMIWHCPMNGWYQQKSGISPKPWIYKHYEYIHGVPARFSGSGSGTSYSLTITSMEASDAATYYCWCDEDCNFEFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 3)

EVQLEESATELARPGASVKASCKASGYIFSAFWRWWIKQRPGQGLEWIGIGCYYGRSNHDCTRTCGKATLTADKSSSTAYMQLSSLRSEDSAVYYCAGMTENNQTWGLGTTLAVSS is added. The antibody is prepared by screening hybridomas after immunizing a mouse according to the fact that PD-1 antigen is used as a target, has the affinity of 8.5nM to PD-1 protein and has better binding specificity, and the monoclonal antibody is an IgG1 subtype.

In certain embodiments, the PD-1 monoclonal antibody comprises a Fab, Fab ', F (ab)2, Fv fragment, F (ab') 2, scFv, di-scFv, and/or dAb.

In certain embodiments, the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies. For example, the antibody may be a monoclonal antibody.

Further, the fully human anti-PD-1 monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains. Such mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The invention includes antibodies, and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue of the germline sequence from which the antibody is derived, or to the corresponding residue of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residue (such sequence alterations are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a large number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, all framework and/or CDR residues within the VH and/or VL domains are derived by mutation back to residues in the original germline sequence from which the antibody was derived.

In other embodiments, one or more framework and/or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, the antibodies of the invention can comprise any combination of two or more germline mutations within the framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residues of a particular germline sequence and certain other residues that differ from the original germline sequence are retained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, improved binding specificity, increased binding affinity, improved or enhanced antagonistic biological properties, reduced immunogenicity, and the like. Antibodies and antigen-binding fragments obtained in this general manner are included in the present invention.

In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody that specifically binds to PD-1 in combination with a VEGF inhibitor and a pharmaceutically acceptable carrier. Wherein the VEGF inhibitor is a specific VEGF receptor antagonist of SEQ ID NO: 1 gRNA.

In a related aspect, the invention features a composition that also contains a combination of other therapeutic agents.

"treatment" as used herein generally refers to the desire to alter the natural course of the treated individual, and may be prophylactic or clinical intervention in the course of a clinical condition. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and palliating or improving prognosis. In some cases, antibodies (e.g., anti-PD-1 antibodies) can be used to delay disease progression or slow disease progression.

In the present application, a method of administering a dose of a compound (e.g., an anti-cancer therapeutic agent) or a pharmaceutical composition (e.g., a pharmaceutical composition comprising an anti-cancer therapeutic agent) to a subject (e.g., a patient). Administration can be by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as, if desired for local treatment, intralesional administration. Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

Advantageous effects

According to the invention, the gRNA is designed aiming at the VEGF target, VEGF genes are specifically knocked out in breast cancer cells, the knocking-out effect is better through protein detection, and VEGF proteins are well inhibited. In addition, after the breast cancer cells subjected to VEGF gene knockout are treated by combining with the PD-1 monoclonal antibody, the breast cancer tumor is better inhibited, and a better treatment effect is achieved.

Drawings

FIG. 1 LentiCRISPR V2 vector diagram

FIG. 2 is a diagram showing VEGF expression detection results of western blot

FIG. 3 is a graph showing the identification of cell proliferation potency

FIG. 4 clonogenic results of cells after knockdown

FIG. 5 tumor weight results of tumor-bearing nude mice after treatment

Detailed Description

The invention will be further described with reference to specific examples, but the scope of protection of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of specific sgrnas

According to the gene sequence of human VEGF, the sgRNA sequence is designed and prepared by utilizing the design rule of gRNA and adopting an artificial-on-line mode,

in order to enhance the specificity of gene knockout and specifically knockout the coding region which destroys VEGF, the experiment adopts a 2hitKO strategy to carry out gene knockout, namely, one sgRNA is respectively designed at the appropriate position of the upstream and downstream of the coding region, and then the two sgRNAs are assembled into the same Cas9 expression vector. 2 sgRNA sequences are designed aiming at VEGF genes, BsmBI enzyme cutting sites are added at the 5' ends of a sense strand and an antisense strand of the sequences, and the designed sgR NA primers are shown in a table 1.

TABLE 1 sgRNA primer sequences for specific VEGF gene knock-out

Primer name	Primer sequence (5 '-3')	bp
			VEGF-sgRNA1-F	CACCGTCTACCTCCACCATGCCAAG	25
VEGF-sgRNA1-R	AAACCTTGGCATGGTGGAGGTAGAC	25
			VEGF-sgRNA control-F	CACCGGTGGTCCCAGGCTGCACCCA	25
VEGF-sgRNA control-R	AAACTGGGTGCAGCCTGGGACCACC	25

And (3) entrusting the sgRNA sequence to Shanghai for biological synthesis. After synthesis, primers were diluted 100 μ M, diluted 10-fold, annealed, 50 μ l system: 20 mul of primer (100 mul of each upstream and downstream), 25 mul of water, 50 mul of NEB buffer 2(10 x), 95 ℃ and 5min, then closing the PCR instrument, and naturally cooling for 40min to obtain the corresponding sgRNA.

EXAMPLE 2 construction and transfection of expression vectors

The 2 sgrnas prepared by annealing in example 1 were each digested with bsmbi, and then ligated in mixture with the lentiCRISPR V2 vector purified by digestion with bsmbi (the lentiCRISPR V2 vector contains the U6 promoter for directing gene transcription, Cas9 nuclease, ampicillin, etc., as shown in fig. 1). T4 DNA ligase was used for ligation at 16 ℃ for 16 h. The ligation products were transformed into 100. mu.l of DH 5. alpha. competent bacteria, positive clones were selected by ampicillin resistance, and after single clones were picked, they were sequenced from the warfarin gene. The construction is successful through gene sequence identification.

Human ovarian cancer cell line SK-OV-3 (product number EY-X0733, Shanghai-Haiyan Biotech Co., Ltd.) was cultured in RPMI1640 whole culture medium containing 10% fetal bovine serum, and subcultured in a 37-constant-temperature 5% CO2 saturated humidity incubator, with liquid change/passage 1 time every 2 days.

The lentiviral packaging plasmid was transfected into 293T cells at a ratio of plasmid of interest: pSPAX 2: pMD2. G.sub.4: 3: 1. DMEM medium containing the virus was harvested 72h after transfection. Infecting lentivirus into SK-OV-3 cells by a Polybrene reagent, switching to an RPMI1640 culture medium with puromycin for screening after transfection for 48h, and detecting possible off-target region indel mutation by PCR product high-throughput sequencing after 72h, wherein an amplification primer is as follows: f: ATGAACTTTCTGCTGTCTTG, R: TCACCGCCTCGGCTTGTCACATC. The results show that there is no significant off-target effect, wherein the sgRNA1 gene editing efficiency reaches 75.3%, while the sgRNA control gene editing efficiency is only 46.5%, which indicates that the selection for the target has a great influence on the editing efficiency. Cells in which the mutant sequence of the target gene is present as indicated by "TCTACCTCCACATGCCAAG" were selected and expanded.

Example 3 cell identification

The cells selected in example 2 were diluted and inoculated into a 96-well plate, trypsinized, confluent SK-OV-3 cells were harvested, resuspended in PBS, and the supernatant was collected by centrifugation in a boiling water bath for 15min and 12000r/min and subjected to polyacrylamide gel electrophoresis. Transferring the mixture to a PVDF membrane after electrophoresis, sealing the mixture for 1h at room temperature by using 5% skimmed milk powder, and then adding an endogenous VEGF antibody at a ratio of 1: 500 to incubate for 1h at room temperature; washing the membrane 3 times and 5 min/time with 1 XTSST, incubating at room temperature for 1h with goat anti-mouse IgG labeled with HRP (1: 5000), washing the membrane 3 times and 5 min/time with 1 XTSST, and detecting the Western blot band by chemiluminescence method. SK-OV-3 cells in normal culture were used as controls. The results are shown in FIG. 2.

From the results in FIG. 2, it is shown that VEGF protein is not expressed at all in the gene-edited cells, while VEGF is highly expressed in normal SK-OV-3 cells, and thus, a cell line SK-OV-3-VEGF-gRNA1 in which VEGF protein is completely knocked out has been successfully obtained.

Example 4 growth characterization of cells after knockdown

CCK8 is used for detecting cell proliferation ability, and cell line SK-OV-3-VEGF-gRNA1 and normal SK-OV-3 control cells are taken for proliferation experiment. Pancreatin digesting cells, counting and adjusting the cell concentration, respectively inoculating two groups of cells into a 96-well plate according to 5000 cells per well, adding 150 mu L of culture medium into each well, and culturing at 37 ℃ with 5% CO 2; culturing for 24h, 48h, 72h and 96h, adding 100 mu L of culture medium and 20 mu L of CCK8 reagent into each well, and incubating for 2h in a dark place; and (3) measuring the absorbance at 450nm by using a microplate reader, and judging according to the absorbance value, wherein the larger the OD value is, the stronger the cell activity is, and the more the cell proliferation is. Three duplicate wells were set for each experiment. The results are shown in FIG. 3.

As can be seen from FIG. 3, after the VEGF gene is knocked out, the growth rate of SK-OV-3-VEGF-gRNA1 cells is obviously reduced, at 96h, the OD450 is only 0.24, and the growth rate of SK-OV-3 cells is 0.9, which indicates that the knocking-out of VEGF can effectively inhibit the proliferation of cancer cells.

Example 5 clonogenic experiments of cells after knockdown

Taking a cell line SK-OV-3-VEGF-gRNA1 and normal control cells for trypsinization, counting and adjusting the concentration, adding 10mL of culture medium into various 10cm culture dishes according to the number of 10000 of each cell, culturing for 2-3 weeks at 37 ℃ in 5% CO2, fixing with 4% paraformaldehyde and staining with 1% crystal violet, observing the cell clone number, and calculating the plate clone formation rate. Three replicates per experimental setup. The results are shown in FIG. 4.

In order to detect the influence of VEGF on the proliferation capacity of tumor cells, the regulation of VEGF knockout on the proliferation of the breast cancer cells is analyzed by utilizing a clone formation experiment, and the experiment result shows that the VEGF knockout can inhibit the formation of the clone number of the breast cancer cells, the clone formation number is obviously reduced by about 20 compared with a control group, the clone formation number of the control cells is as large as about 290, and a large colony is formed locally.

Example 6PD-1 monoclonal antibody treatment of cell line SK-OV-3-VEGF-gRNA1 tumor-bearing nude mice

SPF-grade female BALB/c nude mice, 4-5 weeks old, were bred for 3 days in an adaptive manner, and were randomly divided into five groups: blank control group (not inoculated with cancer cells), normal control group (inoculated with normal SK-OV-3 cells), experimental group (inoculated with cell line SK-OV-3-VEGF-gRNA1), combined experimental group (inoculated with cell line SK-OV-3-VEGF-gRNA1, intraperitoneal injection of PD-1 monoclonal antibody 10mg/kg/5d), and control combined experimental group (inoculated with cell line SK-OV-3, intraperitoneal injection of PD-1 monoclonal antibody 10mg/kg/5 d).

Wherein SK-OV-3 cells and SK-OV-3-VEGF-gRNA1 cells are trypsinized, counted, centrifuged and supernatant is discarded, PBS is used for preparing single cell suspension, and the cell concentration is adjusted to be 2 multiplied by 10 per milliliter ⁷ Each nude mouse was inoculated with 100. mu.l of cell suspension in the axillary mammary gland.

When the tumor diameter is 5mm, PD-1 is administered to the combined experimental group and the control combined experimental groupAntibody therapy was carried out at a dose of 10mg/kg body weight by intraperitoneal injection, 1 time per 5 days for 5 times, and the other groups were administered with the same amount of physiological saline, while measuring the major axis (a) and the minor axis (b) of the subcutaneous tumor with a vernier caliper, and the tumor volume (V) was calculated as a.times.b ² And/2, drawing a tumor growth curve. Nude mice were sacrificed after 5 times of administration, tumor tissues were dissected off, and tumors were weighed. The results are shown in FIG. 5.

From the results in fig. 5, it can be seen that tumor-bearing nude mice die without death, the blank control group has no tumor growth, the normal control group has a faster tumor growth rate, and on the contrary, the experimental group and the combined experimental group have smaller tumor weight, especially the combined experimental group has a minimum weight of only 0.04g, which indicates that the monoclonal antibody can inhibit the tumor growth more effectively in cooperation with the gRNA knockout.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

<110> Beijing Yunshenda Biotechnology development Co., Ltd

<120> use of gene editing technology in the treatment of cancer

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115

Claims (3)

1. Use of a VEGF inhibitor consisting of a VEGF-knockout CRISPR/cas9 system element, wherein the gRNA sequence is TCTACCTCCACCATGCCAAG, and a PD-1 monoclonal antibody in the manufacture of a kit for inhibiting ovarian cancer; the variable region of the light chain of the PD-1 monoclonal antibody is shown as SEQ ID NO: 2, the sequence of the heavy chain variable region is shown as SEQ ID NO: 3, respectively.
2. 2. Use according to claim 1, characterized in that the PD-1 monoclonal antibody comprises Fab, Fab ', F (ab)2, Fv fragments, F (ab') 2, scFv, di-scFv or dAb.
3. 3. Use according to claim 2, characterized in that the monoclonal antibody is a fully human antibody.

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