CN111662929B - Vector containing Hdac3 mutation and application thereof in gene therapy of tumor - Google Patents

Abstract

The present disclosure provides a vector containing a mutation of Hdac3 and its use in gene therapy of tumors. In particular, the present disclosure relates to an expression cassette, a recombinant vector and a recombinant adeno-associated virus containing the expression cassette. Meanwhile, the disclosure also relates to a pharmaceutical composition containing the components, a carrier and application thereof, and further provides a method for slowly and continuously killing cells. The carrier provided by the disclosure has good tumor inhibition effect.

Description

Vector containing Hdac3 mutation and application thereof in gene therapy of tumor

Technical Field

The present disclosure belongs to the field of biotechnology. In particular, the disclosure relates to vectors containing the mutation of Hdac3 and their use in gene therapy of tumors.

Background

Tumor diseases are one of the important diseases affecting human health, and have plagued people for a long time, and thousands of people die of cancer every year in the world^[1]. Scientists have also been searching for good treatment regimens and for the reasons for the development of cancer. The current tumor treatment schemes include surgical resection, chemo-radiotherapy, immunotherapy and the like, and the tumor treatment methods are mature and effective treatment methods.

In recent years, the development of tumor immunotherapy has been dramatically advanced in tumor therapy, and great results have been achieved in tumor therapy. Discovery of immunodetection point inhibitors such as PD-1, CTLA-4 and the like and clinical application of related antibodies^[2,3]. Cancer patients activate the body's anti-tumor immunity after using blockers of the immunodetection site. However, the antibody drugs have large toxic and side effects, and usually cause autoimmune diseases of patients and inflammatory reactions of organisms.

With the progress of research, researchers have found that epigenetics plays a great role in cancer therapy, and the regulation of a certain gene in tumor cells (e.g., histone deacetylase 3, Hdac3) is carried out by applying the principle of epigenetics^[4]So as to change the signal path in the tumor cell and enhance the immunogenicity of the tumor cell, thereby activating the immune system and achieving the purposes of killing the tumor cell and treating cancer^[5,6]。

In recent years, researchers have found that oncolytic viruses play a great role in tumor therapy, with adeno-associated virus (AAV) being a very good virus, a non-enveloped, free and soluble dsDNA virus.

AAV is uniquely characterized in that it is attractive as a vector for delivering exogenous DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic and natural infections in humans and other animals are silent and asymptomatic. Moreover, AAV infects many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can essentially persist for the life of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in a plasmid, which makes the construction of recombinant genomes possible. In addition, since the signals directing AAV replication, genome encapsidation and integration are contained in the ITRs of the AAV genome, part or all of the internal approximately 4.3kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with exogenous DNA such as a gene cassette containing a promoter, DNA of interest and polyadenylation signals. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and robust virus. It is susceptible to the conditions used to inactivate adenovirus (56 ℃ to 65 ℃ for hours), making cold storage of AAV less important. AAV may even be lyophilized. Finally, AAV-infected cells are intolerant to repeated infections.

Meanwhile, adeno-associated virus has extensive tissue tropism, and belongs to the most deeply studied viral vector for gene therapy and oncolytic. In addition to having strong lytic activity, adeno-associated virus also has many significant advantages in pharmacological applications. The adeno-associated viral genome is amenable to genetic manipulation. In terms of virus production, adeno-associated virus can be produced at high titer, and the particles have excellent physicochemical stability^[7]. With respect to the safety aspect of clinical applicability of adeno-associated virus, its tumor-selective replication has been considered to be effective in preventing excessive damage of non-target healthy tissues by adeno-associated virus^[8]。

However, clinically, the existing tumor treatment schemes include surgical removal of tumor, chemotherapy or radiotherapy treatment, and tumor immunology treatment schemes (immunodetection point blocking, such as the clinical application of PD-1 antibody). However, these treatment regimens have more or less problems in the clinical setting and do not satisfy the patient's desire for good treatment. For example, in surgical resection treatment, the phenomenon of incomplete resection exists; the chemotherapy or radiotherapy treatment scheme is pulled to move the whole body, and has large damage to the body of a patient; the treatment mode of immunodetection point blocking, the antibody medicine of immunodetection point blocking used, its toxic and side effect is bigger, can cause patient's autoimmune disease and organism's inflammatory reaction usually.

Meanwhile, the prior art does not disclose any report that histone deacetylase 3 is integrated into adeno-associated virus for treating tumor diseases.

Non-patent document

1.Torre,L.A.,et al.,Global Cancer Incidence and Mortality Rates and Trends—An Update.2016.25(1):p.16-27.

2.Sharma,P.and J.P.J.S.Allison,The future of immune checkpoint therapy.2015. 348(6230):p.56-61.

3.Hodi,F.S.,et al.,Improved Survival with Ipilimumab in Patients with Metastatic Melanoma.2010.363(8):p.711-723.

4.HuLl,E.E.,M.R.Montgomery,and K.J.J.B.R.I.Leyva,HDAC Inhibitors as Epigenetic Regulators of the Immune System:Impacts on Cancer Therapy and Inflammatory Diseases.2016.2016:p.8797206.

5.Eckschlager,T.,et al.,Histone Deacetylase Inhibitors as Anticancer Drugs.2017. 18(7):p.1414.

6.West,A.C.and R.W.J.J.o.C.I.Johnstone,New and emerging HDAC inhibitors for cancer treatment.2014.124(1):p.30-39.

7.Niemann,J.and F.J.V.G.Kuhnel,Oncolytic viruses:adenoviruses.2017.53(5):p. 700-706.

8.Hill,C.and R.J.E.O.o.D.D.Carlisle,Achieving systemic delivery of oncolytic viruses. 2019.16(6):p.607-620.

Disclosure of Invention

Problems to be solved by the invention

The purpose of the disclosure is to carry the gene sequence of Hdac3 with active site mutation to enter tumor tissues through the vector of adeno-associated virus, thereby playing a role in inhibiting histone deacetylase 3(Hdac3), further activating anti-tumor immunity, inhibiting tumor growth and achieving the purpose of treating cancer.

In one embodiment, the present disclosure provides a method of treating a tumor. Specifically, the target of Hdac3 for anti-tumor therapy is combined with the advantages of adeno-associated virus vectors in tumor therapy to prepare the adeno-associated virus carrying Hdac3 sequence with enzyme active site mutation, and then tumor patients are treated.

Means for solving the problems

The present disclosure provides the following technical solutions.

(1) An expression cassette, wherein the expression cassette comprises a sequence encoding a mutated Hdac3 protein.

(2) The expression cassette of (1), wherein the mutant Hdac3 protein has at least reduced or abolished activity of a wild-type Hdac3 protein as compared to the wild-type Hdac3 protein;

wherein the amino acid sequence of the wild type Hdac3 protein is shown as SEQ ID NO: 1, or a fragment thereof.

(3) The expression cassette of any one of (1) to (2), wherein the sequence of the mutated Hdac3 protein and the amino acid sequence set forth in SEQ ID NO: 1 has more than 90% homology with the sequence shown in the formula (1); alternatively, the amino acid sequence encoding the mutated Hdac3 protein is as set forth in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

(4) A recombinant vector comprising the sequence of the expression cassette of any one of (1) to (3).

(5) The recombinant vector according to (4), wherein the recombinant vector is selected from recombinant viral vectors.

(6) The recombinant vector according to (5), wherein the recombinant viral vector is a recombinant adeno-associated viral vector.

(7) A pharmaceutical composition comprising a therapeutically effective amount of the recombinant vector according to any one of (4) to (6).

(8) The pharmaceutical composition of (7), further comprising a pharmaceutically acceptable carrier; optionally, the pharmaceutical composition further comprises a radiotherapeutic agent, a chemotherapeutic agent or an immunotherapeutic agent.

(9) Use of the recombinant vector of any one of (4) to (6) or the pharmaceutical composition of any one of (7) to (8) in the preparation of a medicament for killing cells.

(10) The use according to (9), wherein the cell is selected from the group consisting of a proliferative, neoplastic, pre-cancerous or metastatic cell; preferably, the cells are selected from metastatic cells; more preferably, the metastatic cells are selected from metastatic tumor cells.

(11) Use of the recombinant vector of any one of (4) to (6) or the pharmaceutical composition of any one of (7) to (8) for the preparation of a medicament for treating a patient having a tumor.

In a specific embodiment, the therapy is gene therapy.

(12) A method of slow and sustained killing of cells, comprising contacting the cells with the recombinant vector of any one of (4) - (6) or the pharmaceutical composition of any one of (7) - (8).

(13) A method of treating a disease, comprising administering to a patient a recombinant vector of any one of (4) to (6) or a pharmaceutical composition of any one of (7) to (8); optionally, the disease is a tumor or cancer.

In one embodiment of the disclosure, the disclosure provides for the treatment of a tumor or cancer-related disease by means of gene therapy.

ADVANTAGEOUS EFFECTS OF INVENTION

In one embodiment, the adeno-associated virus is used as a vector, carries a histone deacetylase 3 sequence with an active site mutation of enzyme, and is used for infecting the tumor tissues of mice. As a result, the growth of the tumor tissue is inhibited, which indicates that the histone deacetylase 3 with the mutated active site interferes with the normal function of Hdac3 in the tumor cell after entering the cells of the tumor tissue, thereby playing the role of inhibiting the tumor.

Drawings

Fig. 1 shows the construction of HDAC3 knock-out MCA205 cell line and the results of animal model experiments with this cell line. Wherein, part A shows that Hdac3 gene in cells is knocked out by using Western blot after Hdac3 is knocked out by using CIRPR/Cas 9 technology. Part B shows the validation of the knockout of the Hdac3 gene using methods of gene sequencing. Section C shows the results of mouse subcutaneous tumor-implanted animal model experiments with tumor cell lines, and statistical plots of tumor growth in C57/BL6 mice injected subcutaneously with wild-type and Hdac3 gene-deleted MCA205 cells.

FIG. 2 shows that deletion of the Hdac3 gene enhances the ability of tumor cells to present antigens. Part A is ELISPOT experiment for testing tumor cell antigen presentation, and we used a mode of mixing MCA205 WT cell over-expressing OVA protein with MCA205 Hdac3 KO cell over-expressing OVA protein and OT-1 mouse peripheral blood to test the influence of Hdac3 gene deletion on tumor cell antigen presentation. Part B is a statistical plot of the results of the experiments of part a.

FIG. 3 shows the results of an immune component analysis experiment of tumor tissues of MCA205 cells knocked out of Hdac 3. Wherein, the tumor tissue of the transplanted tumor model is made under the skin of the mouse, the tumor tissue is taken down when the tumor cells are transplanted under the skin of the mouse for 7 days, part A is a statistical chart of an immunofluorescence experiment of the tumor tissue, and statistics is that under a laser confocal microscope, in a range of 600 × 600um, wild type and Hdac3 gene knockout group CD4⁺T cell, CD8⁺The number of T cells. Part B is the experimental result of flow assay of immune component analysis, specifically showing wild type and Hdac3 gene knockout group CD4⁺T cell, CD8⁺The proportion of T cells in the tumor tissue cells and the proportion of cells that can secrete IFN-gamma.

FIG. 4 shows the effect of tumor necrosis factor (TNF-. alpha.) on Hdac 3-depleted tumor cells. Wherein, part A shows that wild type cells and Hdac 3-deleted tumor cells undergo apoptosis to different degrees under the action of TNF-alpha, and the results are detected by flow assay. Part B is a statistical plot of the results of part a. The results of the protein extraction from the cells after TNF-. alpha.action and the confirmation of the occurrence of apoptosis by WB are shown in section C. Section D shows that TNF- α activates the apoptotic signaling pathway. We treated cells with Z-VAD, a blocker of Caspase, under which conditions TNF-. alpha.had an effect on Hdac 3-depleted tumor cells. Section E is a statistical plot of the results for section D.

FIG. 5 shows that Hdac3 gene having a mutation in the active site of enzyme has an inhibitory effect on the growth of tumor. Wherein, part A shows the result of tumor cell line construction of MCA205 of Hdac3 gene sequence over-expressing mutant enzyme activity (in which the sites 134-135, 143-144, 170, 172, 259, 266, 296, 298, 424 of histone deacetylase 3 are mutated to alanine), and the result of tumor animal transplantation experiment using this cell line and wild type cell line under the skin of mice shows that the tumor cells over-expressing mutant enzyme activity Hdac3 sequence grow obviously slowly in mice. Part B shows that the gene sequence of histone deacetylase 3 with mutated active site is loaded into adeno-associated virus and injected into tumor tissue of mice to treat tumors, and the tumor tissue has obvious treatment effect on the tumors.

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.

The terms "inhibit," "reduce," or "prevent," or any variation of these terms, as used in the claims and/or the specification, include any measurable reduction or complete inhibition to achieve a desired result (e.g., cancer treatment). Desirable results include, but are not limited to, alleviation, reduction, slowing, or eradication of cancer or a proliferative disorder or cancer-related symptoms, as well as improved quality of life or prolongation of life.

The vector vaccination methods of the present disclosure are useful for treating cancer in mammals. The term "cancer" as used in this disclosure includes any cancer, including, but not limited to, melanoma, sarcoma, lymphoma, cancer (e.g., brain, breast, liver, stomach, lung, and colon), and leukemia.

The term "mammal" in the present disclosure refers to humans as well as non-human mammals.

The methods of the present disclosure comprise administering to a mammal a vector expressing a tumor antigen to which the mammal has a pre-existing immunity. The term "pre-existing immunity" as used in this disclosure is meant to include immunity induced by vaccination with an antigen as well as immunity naturally occurring in mammals.

The term "radiotherapeutic agent" in the present disclosure includes the use of drugs that cause DNA damage. Radiotherapy has been widely used in cancer and disease treatment and includes those commonly referred to as gamma rays, X-rays and/or the targeted delivery of radioisotopes to tumor cells.

The term "chemotherapeutic agent" in the present disclosure is a chemical compound useful for the treatment of cancer. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogen and selective estrogen receptor modulators, anti-progestins, estrogen receptor downregulators, estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, anti-androgens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemotherapeutic agents useful in the treatment methods of the present disclosure include cytostatic and/or cytotoxic agents.

The term "immunotherapeutic agent" in the present disclosure includes "immunomodulators" and agents that promote or mediate antigen presentation that promotes a cell-mediated immune response. Among these, "immune modulators" include immune checkpoint modulators, such as immune checkpoint protein receptors and their ligands that mediate the inhibition of T cell-mediated cytotoxicity and are typically expressed by tumors or on anergic T cells in the tumor microenvironment and allow the tumor to evade immune attack. Inhibitors of the activity of immunosuppressive checkpoint protein receptors and their ligands can overcome the immunosuppressive tumor environment to allow cytotoxic T cell attack of the tumor. Examples of immune checkpoint proteins include, but are not limited to, PD-1, PD-L1, PDL2, CTLA4, LAG3, TIM3, TIGIT, and CD 103. Modulation (including inhibition) of the activity of such proteins may be accomplished by immune checkpoint modulators, which may include, for example, antibodies, aptamers, small molecules that target checkpoint proteins, and soluble forms of checkpoint receptor proteins, among others. PD-1 targeted inhibitors include the approved pharmaceutical agents pembrolizumab and nivolumab, while plepima (ipilimumab) is an approved CTLA-4 inhibitor. Antibodies specific for PD-L1, PD-L2, LAG3, TIM3, TIGIT, and CD103 are known and/or commercially available and can also be produced by those skilled in the art.

In the present disclosure, the term "pharmaceutically acceptable formulation", "physiologically acceptable formulation" or "pharmaceutically acceptable carrier" means a biologically acceptable formulation, gas, liquid or solid or mixture thereof, suitable for one or more routes of administration, in vivo delivery, in vitro delivery or contact, and may include formulations or carriers used in therapy for other diseases (e.g., gene therapy or cell therapy for other ocular diseases). A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material can be administered to a subject without causing a substantial undesirable biological effect. Thus, such pharmaceutical compositions can be used, for example, to administer a protein, polynucleotide, plasmid, viral vector, or nanoparticle to a cell or subject. Such compositions include, but are not limited to, solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or retarding agents compatible with pharmaceutical administration or contact or delivery in vivo or in vitro. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents, lubricants and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents, and immunosuppressive agents) can also be incorporated into the compositions. The pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or known to those of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

As used in this disclosure, the term "amino acid mutation" includes "substitution, duplication, deletion or addition of one or more amino acids". In the present disclosure, the term "mutation" refers to an alteration in the amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

As used in this disclosure, the term "sequence identity" or "percent identity" in a comparison of two nucleic acids or polypeptides refers to the identity or a specific percentage number of identical sequences when compared and aligned for maximum correspondence as measured using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection. That is, the identity of nucleotide or amino acid sequences can be defined by the ratio of the number of nucleotides or amino acids that are identical when two or more nucleotide or amino acid sequences are aligned in such a manner that the number of nucleotides or amino acids that are identical is maximized, and gaps are added as necessary, to the total number of nucleotides or amino acids in the aligned portion.

As used in the present disclosure, sequence identity between two or more polynucleotides or polypeptides may be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides or missing nucleotides. Polypeptides may differ at one position, for example, by containing different amino acids or deleting amino acids. Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

Illustratively, in the present disclosure, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residues when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. The determination/calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 10 residues, a region of at least about 15 residues, a region of at least about 18 residues, a region of at least about 20 residues in length. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the biopolymers (i.e., nucleic acids or polypeptides) to be compared.

In the present disclosure, the term "adeno-associated virus (AAV)" is a replication-defective parvovirus whose single-stranded DNA genome is about 4.7kb in length, including an Inverted Terminal Repeat (ITR) of 145 nucleotides. There are a variety of serotypes of AAV in the prior art, and the nucleotide sequences of the genomes of the aforementioned AAV serotypes are known. Exemplary nucleotide sequences for the AAV serotype 2 (AAV-2) genome are set forth, for example, in Ruffing et al, J.Gen.Virol 75:3385-3392 (1994). As another example, the complete genome of AAV-1 is provided in GenBank accession NC-002077; the complete genome of AAV-3 is provided in GenBank accession NC-1829; the complete genome of AAV-4 is provided in GenBank accession NC-001829; the AAV-5 genome is provided in GenBank accession No. AF 085716; the complete genome of AAV-6 is provided in GenBank accession No. NC _ 001862; at least part of the AAV-7 and AAV-8 genomes are provided in GenBank accession Nos. AX753246 and AX753249, respectively (see also U.S. Pat. Nos. 7,282,199 and 7,790,449 relating to AAV-8); AAV-9 genomes are provided in Gao et al, journal of virology, 78:6381, 6388 (2004); AAV-10 genomes are provided in molecular therapy (mol. ther.), 13(1):67-76 (2006); and AAV-11 genomes are provided in Virology (Virology), 330(2), 375-.

In one embodiment of the present disclosure, the adeno-associated virus can be selected from any one or more of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11.

In one embodiment of the disclosure, an expression cassette is provided comprising a nucleic acid molecule as described in the disclosure and one or more regulatory sequences operably linked to the nucleic acid sequence. In another embodiment, there is provided a vector comprising a nucleic acid molecule as described in the present disclosure or an expression cassette as described in the present disclosure.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from the group consisting of: recombinant adenovirus vectors, recombinant lentiviral vectors, recombinant herpes simplex virus vectors, recombinant sendai virus vectors and recombinant retroviral vectors. In some embodiments, the vector is a recombinant adeno-associated viral vector or plasmid.

In other embodiments, the vector is a plasmid or a non-viral vector. In some embodiments, the non-viral vector is selected from the group consisting of a naked nucleic acid (naked nucleic acid), a liposome, a dendrimer, and a nanoparticle.

In a specific embodiment of the disclosure, the sequence of the Hdac3 protein is a sequence encoded by a protein that reduces or loses the activity of the original Hdac3 protein compared to the wild-type Hdac3 protein.

In a specific embodiment of the present disclosure, the sequence of the Hdac3 protein is SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

"methods in general Biology in the art" in the present disclosure can be referred to corresponding methods described in publications such as "Current Protocols in Molecular Biology, Wiley publication", "Molecular Cloning, A Laboratory Manual, Cold spring harbor Laboratory publication", and the like.

In the present disclosure, the numbering of nucleotides or amino acids as indicated by the different sequence numbers has the following meaning:

SEQ ID NO: 1 is the amino acid sequence of wild type Hdac3 protein;

SEQ ID NO: 2 is the amino acid sequence of an exemplary mutant Hdac3 protein;

SEQ ID NO: 3 is the nucleotide sequence of an exemplary mutant Hdac3 protein.

Embodiments of the present disclosure

The present disclosure is based on the discovery that adeno-associated viruses having a mutated active site of the Hdac3 gene sequence have a very good therapeutic effect in the treatment of cancer, and we have found that this effect is demonstrated in a number of previous experiments.

We know that inhibitors of the class I Hdac family have significant inhibitory effects on tumor growth. For example, Entinostat (MS-275) strongly inhibits Hdac1 and Hdac 3; RGFP966 is an Hdac3 inhibitor and so on, and a plurality of reports are made on the effect in the literature, and the class I Hdac family is known to be a very good target for tumor treatment. Here we selected Hdac3 in class I family of Hdacs to knock out tumor cells to study their effect on tumor growth, and found that deletion of Hdac3 had a very significant inhibitory effect on tumors. Therefore, it is desirable to find a safe and effective method for treating tumors by using Hdac3 as a tumor treatment target.

We first knocked out Hdac3 gene in MCA205 cells (mouse fibrosarcoma cells) by applying CRISPR/Cas9 technology, then used cell lines with the Hdac3 gene knocked out to perform animal experiments (subcutaneous tumor implantation) of transplanted tumor models in C57/BL6 mice, and used wild-type tumor cell lines as control, and found that after the Hdac3 gene knockout, the subcutaneous tumor tissues of the mice grow slowly and all regress when the tumors grow to about the tenth day. According to the results, the fact that the interference of the expression of Hdac3 in tumor cells can inhibit the growth of tumors is suggested, and the method plays a good role in the subsequent research.

Subsequently, in order to verify the effect of deletion of the Hdac3 gene on tumor immunogenicity, particularly tumor antigen presenting ability, we performed an enzyme-linked immunospot assay (ELISPOT) of tumor cells. Firstly, OVA protein is over-expressed on MCA205 WT cells and MCA205 Hdac3 KO cells, the OVA protein is expressed on the surface of cell membranes, then the OVA protein is mixed with T cells expressing OVA protein receptors, then the activation condition of the T cells is detected, and the quantity of IFN-gamma secreted by the T cells is detected through ELISPOT experiments to judge the influence of deletion of the Hdac3 gene on the antigen presenting capacity of tumor cells. The experimental result shows that the secretion of IFN-gamma is obviously much higher in the group of experiments of the tumor cells lacking the Hdac3 gene than in the group of wild cells, which indicates that the antigen presenting capability of the tumor cells can be obviously increased by the deletion of the Hdac3 gene in the tumor cells, and this provides a sufficient basis for the following experiments.

We then further investigated how deletion of Hdac3 affected tumor growth and whether gene knockout of Hdac3 activated anti-tumor immunity in mice. In a transplanted tumor model experiment, when subcutaneous tumor tissues of mice grow to the seventh day, the tumor tissues are taken down and frozen sections are made, and an immunofluorescence experiment shows that a great number of CD4 genes are obviously found in the tumor tissues with the Hdac3 gene knockout function⁺T cells and CD8⁺T is thinInfiltration of the cells. The Hdac3 gene is knocked out in the tumor cells, so that the immunogenicity of the tumor cells can be increased, and the anti-tumor immunity can be activated. This suggests that we can clinically interfere with the expression of Hdac3 in tumor tissue of patients, thereby achieving the purpose of inhibiting tumor growth.

Furthermore, we also studied how the Hdac3 gene affects tumor growth, and as a result, we found that MCA205 tumor cells lacking Hdac3 gene were apoptotic under the action of tumor necrosis factor (TNF- α) compared to wild-type tumor cells. TNF-alpha is known to activate apoptotic signaling pathways in cells. Therefore, the fact that the deletion of Hdac3 makes tumor cells more prone to apoptosis is suggested, and theoretical basis is provided for the application of Hdac3 as a target point for treating tumors.

Based on the above experimental basis, we hoped to find a method that can inhibit Hdac3 in tumors and can be applied clinically, and then we thought to find a method to solve the problem from the genetic level. It is well known that mutant protein coding sequences can cause loss of protein function, and we would like to find a clinically applicable approach by this theory.

Firstly, we performed multiple site gene mutation on Hdac3, and transformed the HDAC3 sequence with active site mutation (134-135, 143-144, 170, 172, 259, 266, 296, 298, 424 site mutation of histone deacetylase 3 into alanine) into tumor cells, and observed that the tumor growth would not be affected at this time, and we found that after the Hdac3 sequence with active site mutation was transformed into the wild-type tumor cell MCA205, the tumor cells growth in mice was fully inhibited.

Then, we hope to utilize the advantage of tumor-selective replication of adeno-associated virus, and let the adeno-associated virus carry the gene sequence of Hdac3 with mutated active site to enter the tumor tissue based on adeno-associated virus, so that the adeno-associated virus can exert its infection function to inhibit the action of histone deacetylase 3 in tumor cells, thereby inhibiting the growth of tumor.

Here, weThe animal experiment of subcutaneous transplantation tumor was performed on C57/BL6 mice, which were injected with 2 x 10⁶MCA205 wild-type cells, experiments were performed in two groups of five mice each; one group, mice tumor tissue was injected with adeno-associated virus 10 by the seventh day of tumor tissue growth¹²Number of particles; the other group was given unloaded adeno-associated virus. As a result, it was found that the tumor growth of mice administered with adeno-associated virus treatment was significantly slower. The adeno-associated virus has certain tumor treatment effect and certain clinical popularization value.

In conclusion, in the present disclosure, we first discovered that the Hdac3 gene is a very good target for tumor immune regulation based on the study of the role of the Hdac3 gene in tumor immunogenicity regulation, and that the infiltration of immune cells inside tumor tissues is increased and tumor tissues show regression in a short time after the deletion of the Hdac3 gene in tumor cells. Based on the above findings, we transferred the Hdac3 gene with mutated enzyme active site into wild-type tumor cells, thereby inhibiting normal histone deacetylase 3 from playing a role, and further inhibiting tumor growth. Using this finding, we loaded the Hdac3 gene with mutated enzyme active site into adeno-associated virus to prepare a vector for treating tumor.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

All reagents and starting materials used in this disclosure are commercially available unless otherwise indicated.

Example 1: hdac3 gene knockout MCA205 cell line is obtained by using CRISPR/Cas9 technology, and the cell line is used The cell line is used for animal experiment and the immunity is checkedVariation of the original Properties

1.1 Experimental reagents, materials

(1) Cell: MCA205 cells (mouse fibrosarcoma cells), HEK293T cells (human embryonic kidney cells), all purchased from ACTCC.

(2) Animals: C57/BL6 (Beijing Wittingle laboratory animal technology Co., Ltd.)

(3) Reagent and apparatus

DMEM(Gibco)

Lipo-fectamine 3000(invitrogen，L3000-015)

CO₂Incubator (Thermo fisher)

Inverted microscope (Olympus)

1.2 Experimental procedures

(1) Gene knockout: sgRNA used for synthetic gene knockout:

F：CACCGCCCAATGAAACCTCATCGCC(SEQ ID NO：4)

R：AAACGGCGATGAGGTTTCATTGGGC(SEQ ID NO：5)

used to construct a Lenti V2 plasmid containing this sgRNA.

1) HEK293T cells were cultured, used after the cells were in good condition, plated in 6-well plates at 1 × 10 per well⁶The cell of (1).

2) The following day, lentivirus packaging began when HEK293T cells in six well plates were between 30% and 50% dense.

Construction of the Lenti V2 backbone plasmid: the V2 plasmid was first cleaved with Bsmb I enzyme, electrophoresed on a 1% agarose gel, run, and then cleaved to recover the desired band, resulting in the linearized V2 plasmid. The linearized V2 plasmid and the sgRNA were ligated together with DNA ligase to construct a complete plasmid, which was amplified.

The lentivirus packaging method comprises the following steps: first step, mixing of plasmids: lenti V2 was used as a backbone plasmid, and pVSVg/psPAX2 was used as an envelope plasmid to package lentiviruses. Wherein the amount of each plasmid in each six-well plate: the amount of sgRNA-ligated V2 was 1.33. mu.g, pVSVg was 0.67. mu.g, and psPAX2 was 1. mu.g. In the second step, Lipofectamine 30006. mu.L was mixed with opti-MEM 90. mu.L, and then mixed with the three plasmids of the first step, and allowed to stand at room temperature for 5 minutes. Third, 6. mu.L of P3000 was mixed with 90. mu.L of opti-MEM, and then mixed with the above mixed system, followed by standing at room temperature for 25 minutes. After 25 minutes, the above mixed liposome liquid was added to a six-well plate in which 1.5mL of medium was required to culture HEK293T cells. Put in CO₂The incubator is used for 48 hours. Then collecting cell culture medium, filtering with 0.22um filter membrane to obtain lentivirus virus liquid for infecting cells.

3) Culturing MCA205 WT cells, and culturing MCA205 WT cells in good condition 1 x 10⁶The next day, 1mL of virus solution was added to the cell culture medium per well and infected for 48 hours.

4) puro screening. Cells after 48h of virus infection were cultured in medium containing puro until no cells died.

5) Monoclonality of the knockout cells. A portion of the cells were removed and one cell was driven into each well in a 96-well plate using flow sorting techniques. In CO₂Culturing in an incubator for 7-14 days to amplify the monoclonal cells.

(2) Identification of gene knockout: and (3) identifying whether the Hdac3 gene is knocked out by using a Western blot verification method and a gene sequencing method. And (5) identifying the Western blot. And after the cells in the 96-well plate are full, transferring the cells into a 24-well plate, amplifying the cells, taking a part of the cells, extracting proteins, performing a WB (wideband) experiment, identifying whether the target genes are expressed in the cells, and if the target genes are not expressed, indicating that the cell line after the monoclonal is a gene knockout cell line. When the gene sequencing method is used for identification, DNA of a monoclonal cell is extracted, sequencing is carried out, and whether gene deletion exists or not is compared with that of a wild cell.

(3) Animal experiments: after obtaining the MCA205 cell line with the Hdac3 gene knockout, animal model experiments of subcutaneous tumor implantation were performed in two groups, five mice in each group, C57/BL6, one group of wild-type MCA205 tumor cells, one group of Hdac3 gene knockout MCA205 cells, and 2 x 10 cells were injected subcutaneously into each mouse⁶And (4) cells. And observing the growth condition of the tumor, and drawing a tumor growth curve.

1.3 conclusion of the experiment

The results of the experiment are shown in FIG. 1. From this experiment, it was found that after knock-out of HDAC3 in tumor cells, tumor growth was inhibited in a tumor animal model of tumor cell transplantation. It can be shown that histone deacetylase 3(Hdac3) is a good site for treating tumors, suggesting that inhibition of Hdac3 may have a tumor treatment effect.

Example 2: effect of deletion of Hdac3 Gene on antigen-presenting ability of tumor cells

2.1 materials of the experiment

(1) Animals: OT-1 mice: the mouse T cells specifically express the OVA protein receptor. Purchased from Baiosai Tokyo Gene Biotechnology Ltd

(2) Reagent:

1640 medium (Gibco)

Erythrocyte lysate (solarbio, Cat: R1010)

ELISPOT kit (BD company)

(3) The instrument comprises the following steps:

enzyme-linked immunosorbent spot analyzer (CTL, S6)

2.2 Experimental procedures

(1) Construction of cell lines overexpressing OVA

In this experiment we were required to overexpress OVA proteins on MCA205 WT and MCA205 KO Hdac3 cells. The specific steps for the construction of the over-expressed cell line are as follows:

1) HEK293T cells were cultured, lentivirus was packaged, and the cells were used after they were in good condition, plated in 6-well plates 1 × 10 per well⁶The cell of (1). The following day, lentivirus packaging began when HEK293T cells in six well plates were between 30% and 50% dense.

The lentivirus packaging method comprises the following steps: first step, mixing of plasmids: the slow virus is packaged by taking the plasmid VP64-OVA-GFP as an expression plasmid and taking pVSVG and psPAX2 as envelope plasmids. Wherein the amount of each plasmid in each six-well plate: VP64-OVA-GFP was 1.33. mu.g, pVSVg was 0.67. mu.g, and psPAX2 was 1. mu.g. In the second step, Lipofectamine 30006. mu.L was mixed with opti-MEM 90. mu.L, and then mixed with the three plasmids of the first step, and then allowed to stand at room temperatureStanding for 5 minutes. Third, 6. mu.L of P3000 was mixed with 90. mu.L of opti-MEM, and then mixed with the above mixed system, followed by standing at room temperature for 25 minutes. After 25 minutes, the liposome mixture was added to a six-well plate containing 1.5mL of culture medium in which HEK293T cells were cultured. Put in CO₂The incubator is used for 48 hours. Then collecting cell culture medium, filtering with 0.22um filter membrane to obtain lentivirus virus liquid for infecting cells.

2) Lentiviruses infect cells. Culturing MCA205 WT and MCA205 Hdac3 KO cells, and plating in six-well plate with 1 × 10 wells after the cells are in good condition⁶The cell of (1). The following day, cells were infected with lentivirus starting when the cell density in the six well plates was between 30% and 50%, and 1mL of the above-obtained virus solution was added to the supernatant of the cell culture medium. The cells were then cultured for 48 hours.

3) The over-expressed cell lines were sorted out. And (3) observing whether the cells have green fluorescence or not under a fluorescence microscope after 48 hours of virus infection, if the cells express the green fluorescence, indicating that the cells in the culture dish have cells which are successfully overexpressed, digesting the cells, resuspending the cells by using 500 mu L of PBS, and sorting the cells with the green fluorescence by using a flow sorting method to obtain a cell line which overexpresses OVA protein.

(2) ELISPOT (enzyme-linked immunospot)

1) Plate paving: the antibody was diluted with PBS and added to a 96-well plate of ELISPOT at 100. mu.L per well overnight at 4 ℃. The next day, the antibody was discarded, and 200. mu.L (1640 medium + 10% FBS) of blocking solution was added to each well, washed once with the blocking solution, and blocked at room temperature for 2 h.

2) Preparation of cells and blood of OT-1 mice: MCA205-OVA cells and MCA205 Hdac3 KO-OVA cells were cultured 5 x 10 inside one well of each cell culture well plate as required in ELISPOT experiments⁴The amount of cells (2) to prepare cells. Meanwhile, blood of OT-1 mice is prepared, blood is taken from the micro-veins of the mice, the blood volume of the mice required is calculated firstly, T cells in the blood of the OT-1 mice are required in the total six wells of a 96-well plate in the experiment, so six drops of blood of the OT-1 mice are taken,the blood was then split red with red cell split fluid and split red on ice for 15 minutes, then 3500rpm for 10 minutes. The supernatant was discarded, 600. mu.L of cell culture medium was resuspended, 100. mu.L per well was added to a 96-well plate, and at the same time 100. mu.L of the corresponding cell suspension was added to the corresponding well plate. Culturing at 37 deg.C for 16-18 hr.

3) Discard Medium and use ddH₂O Wash twice, 200. mu.L of ddH per well₂And O. The plate was then washed 3 times with PBST, 200. mu.L/well.

4) Detection antibodies were prepared, diluted in 10% FBS, 100. mu.L/well and incubated at room temperature for 2 h. After incubation, the antibody was discarded and the plate washed 3 times with PBST, 200 μ L/well.

5) Enzyme conjugate was prepared and 100. mu.L/well was added to the well plate and incubated at room temperature for 1h, and then the plate was washed 3 times with PBST, 200. mu.L/well. The plate was then washed 2 times with PBS, 200. mu.L/well. The PBS was discarded.

6) mu.L of Final Substrate Solution was added to the well plate and left in the dark at room temperature for 10-30 minutes. The liquid was discarded and 200. mu.L/well of ddH was added₂O, developing the color of the mixture,

7) after a significant color change, the ddH is discarded₂And O, placing the mixture at room temperature, drying the pore plate, and reading by using an enzyme-linked immunosorbent spot analyzer.

2.3 results of the experiment

The results of the experiment are shown in FIG. 2. According to our experiments, compared with the wild MCA205 cell, the tumor cell which lacks the Hdac3 gene has a much increased antigen presenting capability and correspondingly increased immunogenicity, which provides a sufficient basis for the immunotherapy of tumors.

Example 3: immune cell infiltration of tumor tissue in transplanted tumor model of tumor cells lacking Hdac3 gene Analysis of

3.1 test materials, reagents

(1) Experimental Material

DMEM(Gibco)

PBS(Gibco，C1001050013T)

DNaseⅠ(Roche)

Collagenase liberase TL Research Grade (Roche, 05401020001)

O.C.T.(Sakura，4583)

Mouse-CD8(Abcam，ab217344)，

Mouse-CD4(Abcam，ab133616)，

Alexa Fluor 568rabbit anti-mouse IgG(Life，A11004)。

Mouse-CD8(BioLegend，100414)，

Mouse-CD4(BioLegend，100723)，

IFN-γ(eBioscience，12-7311-82)

PMA(Abcam，ab120297)

Ionomycin(Abcam，ab120116)

Brefeldina(Abcam，ab120299)

(2) Laboratory apparatus

Freezing and slicing the machine: from Leica (CM1950)

Laser confocal microscopy: from Leica (SP8 type)

Flow cytometry: from Life Technologies (Atture NxT)

3.2 Experimental procedures

Detecting infiltration of immune cells in tumor tissues, in a transplanted tumor animal model experiment, the infiltration is carried out in two groups, wherein each group comprises five C57 mice, one group comprises wild type MCA205 tumor cells, the other group comprises Hdac3 knockout MCA205 cells, and each mouse is injected with 2 x 10 subcutaneous injection⁶And (4) cells. When the tumor grows to the seventh day, tumor tissues are taken out from the subcutaneous part, a flow cytometry experiment of immunofluorescence and immune component analysis of a frozen section is carried out, and the infiltration condition of immune cells in the tumor tissues is judged by detecting markers such as CD4, CD8 and the like.

(1) Immunofluorescence assay

1) After the animal was modeled as a transplanted tumor, tumor tissues of the mice were collected on the seventh day, and tumor tissues of the wild type and the Hdac3 knockout group were collected from three mice, respectively.

2) Fixed dehydration of tumor tissue. Soaking the tissue in 4% paraformaldehyde, and fixing for 24 h; then dehydrating, soaking in 30% sucrose for 48h to complete dehydration. Tumor tissues were embedded with OCT and stored at-20 ℃.

3) And (5) freezing and slicing. Tumor tissues stored at-20 ℃ were taken out, and sectioned with a cryomicrotome to a thickness of 5 μm, and about 10 pieces of each tissue were cut. Storing at-20 ℃ for later use.

4) The cut tissue pieces were removed from-20 ℃ and left at room temperature for 5-20 minutes. Then, the PBS immersion was repeated twice for 5 minutes, and the OCT was washed off. The water surrounding the tissue on the slide was wiped dry and a loop was drawn around the tissue with an immunohistochemical pen.

5) The tissue is fixed. 100 μ L of 2% paraformaldehyde was added to each tissue and fixed for 15 minutes at room temperature (note: paraformaldehyde requires pre-cooling). PBS was then washed three times for 5 minutes each.

6) And (4) membrane permeation. 100 μ L of 0.2% Triton membrane-penetrating agent was added to each piece of tissue at room temperature for 10 minutes, and this membrane-penetrating time was suitably prolonged. PBS was then washed three times for 5 minutes each.

7) And (5) sealing. The tissue was blocked with 10% sheep serum, approximately 100 μ L of each tablet was added (sheep serum was diluted with PBS), blocked for 1 hour at room temperature, and then washed once with PBS.

8) Applying and breeding the primary antibody. Prepare primary antibody working solution, dilute primary antibody with PBS, dilute according to the antibody specification, and then apply over night at 4 ℃. The next day, the pieces were removed from 4 ℃ and left at room temperature for a while, the temperature was returned to room temperature, and then washed three times with PBS for 5 minutes each.

9) The secondary antibody was incubated with the working solution of the secondary antibody, diluted with PBS, incubated at room temperature for 1 hour, and then washed three times with PBS for 5 minutes each.

10) Staining of the cell nuclei. Staining of nuclei with hoschost, 1: hoschest was diluted 1000, stained for 5 min in the dark, and then washed three times with PBS.

11) And (6) sealing the sheet. Mounting with anti-quencher, take care not to dry the tumor tissue. The coverslip was then covered.

12) The images were observed by confocal laser microscopy and photographed.

(2) Experimental procedure for flow cytometry for immune component analysis

1) After the mice were subcutaneously transplanted with tumor models, tumor tissues of the mice were collected on the seventh day, and tumor tissues of three mice were collected for the wild type and the Hdac3 knockout groups, respectively. At the same time, the spleen of one mouse was used in the following experiment to make a single-stained tube.

2) Preparing tissue digestive juice and digesting tissues. Consists of DMEM medium, DNase I and collagenase. Each tissue was placed in a 1.5mL EP tube, and 1mL of tissue digest was added and minced with scissors. Placing the cut tumor tissue together with the tissue digestive juice into an EP tube together with CO₂The incubator is used for 30 minutes, and the tube is square and flat when in dressing in the dressing box.

3) The plated tumor cells were filtered through a 70 μm filter, washed several times with pre-cooled PBS, then centrifuged and the cells washed again with PBS.

4) And (5) staining the antibody. Preparing antibody staining, and staining antibodies respectively: CD4, CD8, and the like. The cells were stained on ice for 15 minutes, washed once with PBS, reselected with 500. mu.L of PBS, and then detected on a flow-machine.

5) Cytokine stimulators for lymphocytes are configured. The cells separated in the third step were again selected with cytokine stimulators (DMEM supplemented with PMA: 100 mg/mL; Ionomycin: 1. mu.g/mL; Brefeldina (1: 1000)), and cultured in a cell culture incubator for 4 hours. Gently shake once an hour each to allow repeated mixing of cells and cytokine stimulator. After 4 hours, the cells were washed once with PBS, then the cells were broken and stained for IFN-. gamma.. The cells were stained on ice for 15 minutes, washed once with PBS, reselected with 500. mu.L of PBS, and then detected on a flow-machine.

3.3 results of the experiment

The results of the experiment are shown in FIG. 3. In flow experiments and immunofluorescence experiments of immune component analysis, we can find that tumor cells after the Hdac3 gene is knocked out can recruit a large amount of immune cells into tumor tissues in a mouse body and kill the tumor cells to achieve the effect of inhibiting tumors compared with a control group.

Example 4: tumor cells are more sensitive to TNF-alpha and cause apoptosis after deletion of Hdac3

4.1 test materials, reagents

TNF-α(MACS,130101687)

Z-VAD(selleck，S810202)

Annexin V(BD，51-65874X)

DAPI(BBI life science，E607303-0002)

Caspase 3(CST，9665S)

Cleaved-caspase 3(CST，9664S)

Caspase 8(CST，4790S)

Cleaved-caspase 8(CST，8592S)

TubuLin(CST，5335)

4.2 Experimental procedures

(1) Culturing wild MCA205 cells and Hdac3 gene knockout MCA205 cells, respectively plating the two cells in a 12-well plate when the cell state is good, adding TNF-alpha with the concentration of 20 ng/mu L when the cell density is about 50%, and detecting the apoptosis condition in a flow mode after 12 hours.

(2) Apoptosis detection was performed by flow cytometry. TNF-alpha treated wild type MCA205 cells and Hdac3 gene knockout MCA205 cells are taken, the cells are washed once by PBS and resuspended by 100 microliter of PBS, then 2 microliter of Annexin V and DAPI are respectively added into each sample, the samples are stained for 15 minutes in a dark place on ice, washed once by PBS, resuspended by 500 microliter of PBS and detected by a flow-type machine.

(3) Western blot experiments were used to determine whether TNF- α activated apoptosis of tumor cells. The method comprises the following steps of taking TNF-alpha treated wild type MCA205 cells and Hdac3 gene knockout MCA205 cells, treating the cells by using a cell lysate, carrying out ice lysis for 30 minutes, adding SDS-PAGE loading buffer, carrying out metal bath at 100 ℃ for 10 minutes, taking running protein glue, exposing Caspase family proteins, and observing whether the Caspase family proteins are activated by TNF-alpha.

(4) According to the above experiments, it is known that tumor cells lacking the Hdac3 gene can induce apoptosis under the action of TNF-alpha, and in order to further verify the correctness of the discovery, we firstly use protein blocker Z-VAD of Caspase family to block protein of Caspase family, block apoptosis of cells, and observe whether TNF-alpha can also induce apoptosis of cells lacking the Hdac3 gene.

We first cultured wild-type MCA205 cells and Hdac3 gene-knocked-out MCA205 cells, and when the cell status was good, the two cells were plated separately in 12-well plates, and when the cell density was about 50%, 10 ng/. mu.L of Z-VAD was added to the cell culture medium, then after 30 minutes 20 ng/. mu.L of TNF-. alpha.was added to the cell culture medium, and then 5% of CO was added₂The cells were cultured in an incubator for 24 hours, after which the cells were examined for apoptosis by flow cytometry.

4.3 results of the experiment

The results are shown in FIG. 4. According to our experiments, compared with the wild type MCA205 cell, the tumor cell with the deletion of the Hdac3 gene can trigger the apoptosis under the condition that TNF-alpha acts for 24 hours, but the wild type MCA205 cell has no obvious change, and through the test of protein level, the protein of Caspase family downstream of the TNF-alpha induced apoptosis signal pathway is activated, and after the Caspase is blocked by using an inhibitor Z-VAD of the Caspase, the TNF-alpha can not induce the apoptosis of the Hdac3 deleted cell. Therefore, we know that tumor cells can easily be induced by TNF-alpha to undergo apoptosis in the absence of Hdac 3.

Experiment 5: mouse tumors were treated with adeno-associated virus.

5.1 test materials, reagents

(1) Cell: HEK293T cells

(2) Plasmid:

pAAV2/2(addgene：#104963)，

rAAV2-retro helper(addgene：#81070)，

AAV-CAG-GFP(addgene：#28014)。

(3) animals: c57BL/6 (Beijing Weitonglihua laboratory animal technology Co., Ltd.)

(4) Laboratory apparatus

Microcentrifuge (Thermo fisher, G121)

Magnetic stirrer (IKA, RH digital type)

5.2 Experimental procedures

(1) The Hdac3 sequence mutated at multiple sites was overexpressed in MCA205 cells, and the cells were then observed for growth in mice.

1) A plasmid having the Hdac3 gene sequence with a multi-site gene mutation was constructed.

A PCR method is utilized to synthesize a multi-site gene mutation Hdac3 gene sequence in multiple segments (wherein 134-135, 143-144, 170, 172, 259, 266, 296, 298, 424 sites in the protein sites of histone deacetylase 3 are mutated into alanine), then DNA ligase is used for connecting into a complete gene segment, the enzyme cutting sites at two ends of the gene sequence are ensured to be consistent with the enzyme cutting sites of the insertion sites of plasmids, the plasmids and the gene segments are cut by two restriction endonucleases, then the cut plasmids and the gene segments are connected for half an hour at room temperature by the DNA ligase to be connected into a complete circular plasmid, then the constructed plasmid is transferred into competent escherichia coli, and the plasmid is amplified to obtain a large number of plasmids.

2) Construction of an overexpression cell line:

packaging of the over-expressed virus. In constructing a cell line overexpressing a certain gene, the packaging of the overexpressed virus is the same as that of the lentivirus at the time of gene knockout, and the details are shown in the packaging of the lentivirus in experiment I.

Virus infected cells and over-expression cell lines. Culturing cells in a six-well plate, discarding half of culture medium when the cell density is about 30%, adding a proper amount of virus liquid, infecting the virus for 48 hours, wherein generally over-expressed plasmids carry GFP genes, and at the moment, green fluorescent protein is expressed in the cells and can be observed under a fluorescent microscope. Then separating out the cells with green fluorescent protein by using a flow sorting technology to obtain a cell line of the over-expressed gene.

3) Animal experiments with transplanted tumor models

We used the over-expressed cell lines obtained above for mice subcutaneous transplantation tumor experiments, wild type cells as controls, 5 mice per group, each mice injected subcutaneously 2 x 10⁶And (4) cells. And observing the growth condition of the tumor, and drawing a tumor growth curve.

(2) Production of adeno-associated virusPreparing: we used HEK293T cells to package adeno-associated virus in this disclosure, using AAV 2-type serotypes to package adeno-associated virus. Purifying the adeno-associated virus by iodixanol concentration gradient purification method, and finally concentrating the adeno-associated virus to 10¹³Concentration of particles per ml.

1) An adeno-associated virus gene vector plasmid with the Hdac3 gene sequence with the enzyme active site mutation is constructed. The PCR method is used, and the template is as follows: the plasmid obtained when the overexpression cell line was constructed as above. The Hdac3 gene sequence of the multi-site gene mutation is amplified. Cleavage plasmid (AAV-CAG-GFP) and gene fragment: restriction enzymes EcoR I and Not I are used for enzyme digestion for 2 hours, then DNA ligase is used for connecting the plasmid after enzyme digestion and the gene fragment, the plasmid is transferred into competent escherichia coli to be amplified to obtain a large number of plasmids, the detailed experimental steps are carried out in the experiment, and the detailed description is omitted.

2) Packaging of adeno-associated virus.

The packaging of adeno-associated virus is similar to the packaging of lentiviruses above and will not be described in any greater detail herein. The specific operation steps can be referred to ashttps://www.addgene.org/protocols/aav-production- hek293-cells/)

A, when HEK293T cells were cultured in a T175 cell culture flask and the cell state was good and the cell density was about 30%, adeno-associated virus was ready to be transfected and packaged.

B, plasmid pAAV2/2, rAAV2-retro helper, AAV-CAG-GFP was prepared according to the three-plasmid 1: 1: 1, 727.6. mu.L, 1185.4. mu.L and 584.2. mu.L were each prepared under the condition that the respective plasmids were 1. mu.g/. mu.L. The mixed plasmid was mixed with 176mL of Opti-MEM, and 7.5mL of polyurethane amine was added. Incubate for 15 minutes at room temperature and then transfer to HEK293T cell culture flasks. And incubating for 48-72 hours.

C, after 72 hours, the cells and cell supernatant were collected, centrifuged at 1000g for 10 minutes, the cell supernatant was removed, filtered through a 0.45um filter, 25mL of polyethylene glycol was added to each 100mL of supernatant, and stirred at 4 ℃ for 1 hour. Then centrifuged at 2818g for 15 minutes. The supernatant was discarded and the pellet resuspended in 10mL of PBS.

D, 50 units of benzoate enzyme per ml of the above resuspension was added, followed by incubation at 37 ℃ for 45 minutes and then centrifugation at 2415g for 10 minutes. The supernatant was collected and stored at 4 ℃ for the following purification experiments.

3) Concentration of adeno-associated virus.

Firstly, an iodixanol centrifuge tube with a concentration gradient is prepared, wherein the concentration gradient is 60%, 40%, 25% and 15% from bottom to top in sequence. The virus solution of adeno-associated virus was added to the upper layer of the prepared centrifuge tube, the centrifuge tube was sealed, and the centrifuge tube was centrifuged at 350000g for 90 minutes. The virus can be concentrated, and the concentrated virus liquid can be stored at-80 deg.C.

The procedures for concentrating the adeno-associated virus are conventional in the art, and for exemplary purposes, reference is made to (A), (B)https://www.addgene.org/protocols/aav-purification-iodixanol-gradient- uLtracentrifugation/)

4) The application of the adeno-associated virus to the treatment of tumors: an animal model experiment of transplanted tumors was performed subcutaneously in C57 mice using MCA205 cells in two groups of five C57 mice, one group was treated with adeno-associated virus in tumor tissues and the other group was treated with placebo on the seventh day of the experiment, and the treated growth was observed.

5.3 results of the experiment

The results of the experiment are shown in FIG. 5. Compared with the control group, the tumor growth of the mice treated by the adeno-associated virus is obviously slowed down, which indicates that the adeno-associated virus has the effect of inhibiting the tumor growth and has the effect of clinical application.

The present disclosure is not intended to be limited in scope by the specifically disclosed embodiments, which are provided, for example, to illustrate aspects of the present disclosure. Various modifications to the compositions and methods will be apparent from the description and teachings of the disclosure. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

Sequence listing

<110> Suzhou systematic medical institute

<120> vector containing Hdac3 mutation and its use in gene therapy of tumor

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Gln Tyr Leu Asp Gln Ile Arg Gln Thr Ile Phe Glu Asn Leu Lys Met

355 360 365

Leu Asn His Ala Pro Ser Val Gln Ile His Asp Val Pro Ala Asp Leu

370 375 380

Leu Thr Tyr Asp Arg Thr Asp Glu Ala Asp Ala Glu Glu Arg Gly Pro

385 390 395 400

Glu Glu Asn Tyr Ser Arg Pro Glu Ala Pro Asn Glu Phe Tyr Asp Gly

405 410 415

Asp His Asp Asn Asp Lys Glu Ser Asp Val Glu Ile

420 425

<210> 3

<211> 1284

<212> DNA

<213> Homo sapiens

<400> 3

atggccaaga ccgtggcgta tttctacgac cccgatgtgg gcaacttcca ctatggagct 60

ggacacccaa tgaaacctca tcgcctggca ttgactcata gcctagtcct gcattatggt 120

ctctataaga agatgatcgt cttcaagcct taccaggcct cccagcatga catgtgccgc 180

ttccattctg aggactacat cgacttcctg cagagagtca gccccaccaa tatgcagggt 240

ttcaccaaga gccttaatgc cttcaacgtg ggtgatgact gcccagtgtt tccaggactt 300

ttcgagttct gctcccgtta cacaggcgca tctctgcaag gagcaacaca gctaaacaac 360

aagatctgtg atattgccat caactgggcc ggtggtctag ccgccgccaa gaaatttgag 420

gcctctgccg cctgctatgt caatgacata gtaattggta tcctggagct gcttaagtac 480

caccctcggg tgctctacat tgatatcgcc atcgcccatg gtgacggggt tcaggaagcc 540

ttctacctca ctgaccgggt catgactgtg tccttccaca aatacggaaa ttacttcttt 600

cctggaacag gtgacatgta tgaagttgga gcagagagtg gccgctacta ttgtctcaat 660

gtgcccttac gagatggcat tgatgcccag agttacaagc accttttcca gccagtcatc 720

agccaggtgg tggacttcta ccagccgacg tgcatcgtgc tccagtgtgg cgctgactcc 780

ctgggctgtg atcgagccgg ctgcttcaat ctcagcattc gaggacatgg ggaatgtgtt 840

gaatatgtca agagtttcaa tatccctctc ctggtactgg gaggtgccgg cgccactgtc 900

cgaaatgttg cccggtgttg gacatatgaa acatctctgc tggtagaaga ggccattagt 960

gaggaacttc cctatagtga atacttcgag tactttgccc cagatttcac actccatcca 1020

gatgtcagca cccgcatcga gaatcagaac tcacgccagt atctggacca gatccgccag 1080

acaatctttg aaaacttgaa gatgctgaac catgcaccca gtgtccagat tcatgatgtc 1140

ccggcagacc tcctgacgta tgacaggact gacgaggccg acgctgaaga gagaggtccc 1200

gaggagaact acagcaggcc agaagcaccc aatgagttct atgatggcga ccatgacaac 1260

gacaaggaaa gtgatgtgga gatt 1284

<210> 4

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 4

caccgcccaa tgaaacctca tcgcc 25

<210> 5

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 5

aaacggcgat gaggtttcat tgggc 25

Claims (11)

1. An expression cassette comprising a sequence encoding a mutant Hdac3 protein, said mutant Hdac3 protein having at least reduced or abolished activity of a wild-type Hdac3 protein as compared to the wild-type Hdac3 protein; the amino acid sequence of the protein Hdac3 encoding the mutation is shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

2. A recombinant vector comprising the sequence of the expression cassette of claim 1.

3. The recombinant vector according to claim 2, wherein said recombinant vector is selected from recombinant viral vectors.

4. The recombinant vector according to claim 3, wherein the recombinant viral vector is a recombinant adeno-associated viral vector.

5. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant vector according to any one of claims 2-4.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the pharmaceutical composition further comprises a radiotherapeutic agent, a chemotherapeutic agent, or an immunotherapeutic agent.

8. Use of the recombinant vector of any one of claims 2-4 or the pharmaceutical composition of any one of claims 5-7 in the preparation of a medicament for killing a cell, wherein the cell is a fibrosarcoma cell.

9. Use of the recombinant vector according to any one of claims 2-4 or the pharmaceutical composition according to any one of claims 5-7 for the preparation of a medicament for the treatment of a patient suffering from a tumor, wherein said tumor is fibrosarcoma.

10. Use of the recombinant vector of any one of claims 2-4 or the pharmaceutical composition of any one of claims 5-7 for the preparation of a medicament for slow and sustained killing of fibrosarcoma cells, wherein said use comprises contacting said fibrosarcoma cells with the recombinant vector of any one of claims 2-4 or the pharmaceutical composition of any one of claims 5-7.

11. Use of the recombinant vector according to any one of claims 2-4 or the pharmaceutical composition according to any one of claims 5-7 for the manufacture of a medicament for the treatment of fibrosarcoma, wherein said use comprises administering the recombinant vector according to any one of claims 2-4 or the pharmaceutical composition according to any one of claims 5-7 to a patient.

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