Vector containing Hdac3 mutation and application thereof in gene therapy of tumor
阅读说明：本技术 含有Hdac3突变的载体及其在基因***中的应用 (Vector containing Hdac3 mutation and application thereof in gene therapy of tumor ) 是由 杨衡 李利利 刘俊骁 马瑜婷 于 2020-04-22 设计创作，主要内容包括：本公开提供一种含有Hdac3突变的载体及其在基因治疗肿瘤中的应用。具体来说,本公开涉及一种表达盒,含有所述表达盒的重组载体和重组腺相关病毒。与此同时,本公开还涉及含有上述成分的药物组合物、载体及其应用,并且进一步提供了缓慢持续杀伤细胞的方法。本公开提供的载体具有良好地抑制肿瘤的效果。(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.)
1. An expression cassette, wherein the expression cassette comprises a sequence encoding a mutated Hdac3 protein.
2. The expression cassette of claim 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 claims 1-2, wherein the sequence of the mutated Hdac3 protein and the amino acid sequence as 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 claims 1-3.
5. The recombinant vector according to claim 4, wherein said recombinant vector is selected from recombinant viral vectors.
6. The recombinant vector according to claim 5, wherein the recombinant viral vector is a recombinant adeno-associated viral vector.
7. A pharmaceutical composition comprising a therapeutically effective amount of a recombinant vector according to any one of claims 4-6.
8. The pharmaceutical composition of claim 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 claims 4-6 or the pharmaceutical composition of any one of claims 7-8 in the manufacture of a medicament for killing cells.
10. The use according to claim 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 claims 4-6 or the pharmaceutical composition of any one of claims 7-8 in the manufacture of a medicament for treating a patient having a tumor.
12. A method of slow and sustained killing of cells comprising contacting the cells with the recombinant vector of any one of claims 4-6 or the pharmaceutical composition of any one of claims 7-8.
13. A method of treating a disease comprising administering to a patient the recombinant vector of any one of claims 4-6 or the pharmaceutical composition of any one of claims 7-8.
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, histonecetolase 3, Hdac3) is achieved 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 andTrends—An Update.2016.25(1):p.16-27.
2.Sharma,P.and J.P.J.S.Allison,The future of immune checkpointtherapy.2015. 348(6230):p.56-61.
3.Hodi,F.S.,et al.,Improved Survival with Ipilimumab in Patients withMetastatic Melanoma.2010.363(8):p.711-723.
4.HuLl,E.E.,M.R.Montgomery,and K.J.J.B.R.I.Leyva,HDAC Inhibitors asEpigenetic Regulators of the Immune System:Impacts on Cancer Therapy andInflammatory Diseases.2016.2016:p.8797206.
5.Eckschlager,T.,et al.,Histone Deacetylase Inhibitors as AnticancerDrugs.2017. 18(7):p.1414.
6.West,A.C.and R.W.J.J.o.C.I.Johnstone,New and emerging HDACinhibitors 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 ofoncolytic 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 the experimental study of transplanted tumor model, when the subcutaneous tumor tissue of the mouse grows to the seventh day, the tumor tissue is taken out, frozen sections are made, and the result of immunofluorescence experiment shows that the Hdac3 gene isThere were clearly many CD4 in the knocked-out tumor tissue+T cells and CD8+Infiltration of T 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, we performed animal experiments with subcutaneous transplants of tumors in C57/BL6 mice injected subcutaneously 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
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.