Gene and radiation combination therapy for treatment of cancer

Abstract

The present invention relates to an expression system comprising a first and / or a second nucleic acid construct, in particular for use in gene and radiation combination therapy for the treatment of diseases, in particular cancer. A first nucleic acid construct encodes a target STING that is responsive to an STING agonist that recognizes the target STING rather than human STING and natural variants thereof, where the sequence encoding the target STING is operatively linked to an element that allows the expression and / or translation of the target STING. The second nucleic acid construct comprises a therapeutic gene sequence operatively linked to a STING response promoter. The expression system is administered to a target tissue or organ that was previously, concurrently, or subsequently subjected to low dose radiation, with or without a further round of radiation therapy and / or treatment with a STING agonist.

Description

Field of the Invention

[0001] The present invention relates to an expression system comprising a first and / or second nucleic acid construct, particularly for gene and radiation therapy for treating a disease, especially cancer. The first nucleic acid construct encodes a target STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants, wherein the sequence encoding the target STING is operably linked to an element that allows the target STING to be expressed and / or translated. The second nucleic acid construct comprises a therapeutic gene sequence operably linked to a STING responsive promoter. The expression system is administered to a target tissue or organ that has previously received low-dose radiation, followed by another round of radiotherapy and / or treatment with a STING agonist. Background of the Invention

[0003] Cancer is a group of diseases involving abnormal cell growth and proliferation, with approximately 19 million new cases each year and is the cause of approximately 10 million deaths each year (Global Cancer Observatory, 2020). Although significant advances have been made in cancer treatment over the past few decades, the annual mortality rate shows that a large number of people still die from cancer.

[0004] A serious impediment to cancer treatment is that there are several different types of cancer, each with its own challenges to treat. For example, some cancers are easily accessible and therefore can be treated locally (i.e., surgical resection, localized radiation therapy), while other cancers can only be treated with systemic approaches (i.e., chemotherapy, hormone therapy, immunotherapy).

[0005] There are many different treatment methods, such as radiotherapy, chemotherapy, surgical resection, hormone therapy and immunotherapy. Some of these treatments target tumors by systemically interfering with cell proliferation, thereby stopping tumor growth. However, these treatments cause serious side effects to patients, such as fatigue, nausea and vomiting, anemia and infection, because these systemic treatments also do not expect to target physiological dividing cells such as hematopoietic cells, cells in the oral cavity, digestive tract and reproductive system. In addition, depending on the location of the tumor, local therapies such as local radiotherapy can cause serious side effects. In contrast, the concept of immunotherapy is to stimulate the body's immune system to fight cancer cells, thereby reducing unwanted side effects. Immunotherapy can be divided into active or passive immunotherapy. Active immunotherapy is characterized in that the patient is immunized with an agent that enhances the immune response to tumor antigens. Examples include therapeutic cancer vaccines, cell-based immunotherapy and targeted antibody therapy. In contrast, passive immunotherapy does not directly target tumor cells, but enhances the ability of the immune system to attack cancer cells. Examples include checkpoint inhibitors and cytokines. However, to date, immunotherapy has shown several disadvantages, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, and v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821).

[0006] STING (also known as endoplasmic reticulum interferon stimulator (ERIS), mediator of IRF3 activation (MITA), or transmembrane protein 173 (TMEM173) is a four-transmembrane ER protein present in the endoplasmic reticulum (ER) that acts as a sensor for the presence of double-stranded DNA (dsDNA) in the cytosol. As such, STING is activated by DNA viruses, DNA damaging insults such as radiation therapy, and micronuclei that are often present in the cytosol of malignant cells (Li et al. Journal of Hematology & Oncology 2019, 12:35). STING activates TANK-binding kinase 1 (TBK1), leading to phosphorylation and activation of interferon-regulated transcription factor 3 (IRF3), and upregulation of type I interferon (IFN) expression. STING also recruits IκB kinase (IKK) and activates the NF-κB pathway, which promotes the production of proinflammatory molecules, including IL-6 and TNFα, and cooperates with TBK1-IRF3 to produce type I IFN (Li et al. Journal of Hematology & Oncology 2019,12:35). Type I IFN stimulates immune effector cells including dendritic cells (DC) and natural killer (NK) cells, and promotes the production of chemokines such as CXCL9 and CXCL10, which attract T lymphocytes to tumors, which is crucial for anti-tumor immunity (Mowat, C. et al. J. Exp. Med. 2021, Vol. 218, No. 9, e20210108). In fact, STING agonists, like cyclic dinucleotides (CDNs), exert powerful anti-tumor effects in different tumor models (Sivick Ke et al. Cell Reports 2018; 25, 3074-3085). The importance of STING in cancer pathology can also be derived from the fact that STING expression is often suppressed or lost in many cancers.

[0007] Unfortunately, there are significant STING polymorphisms in the human population, which influence the molecular responses induced by activation of the protein and the degree of sensitivity to stimulatory ligands. Therefore, this can greatly affect the effectiveness and safety of molecular entities used for clinical purposes.

[0008] Based on these numerous findings, STING is now considered a master regulator in the cancer immunity circuit.

[0009] A well-described STING activator disclosed in WO 2004 / 039363 A1 is 5,6-dimethylxanthone-4-acetic acid (DMXAA), but other STING agonists are also known in the art. Clinical trials of DMXAA have shown that it is well tolerated and exhibits only mild side effects. However, despite the anti-tumor effects of STING described in animal models, clinical trials have shown that DMXAA failed in trials due to lack of efficacy. Further studies have demonstrated the reason for this treatment failure, showing that DMXAA strongly activates mouse STING, but cannot activate human STING due to amino acid differences in the cyclic dinucleotide (CDN) binding site of STING.

[0010] Documents US 5,126,132 A and US2005 / 214274 A1 disclose cell-based immunotherapy, in which immune active cells such as T cells from a patient are isolated, cultured and modified ex vivo so that these cells recognize a specific type of cancer and then reinjected into the patient. However, in addition to the complex process of generating these ex vivo modified cells, they only target a specific type of cancer.

[0011] Immunotherapy, which uses the cytokine interleukin-2 (IL-2) to stimulate the immune system to target cancer cells, was approved for the treatment of melanoma and kidney cancer as early as 1992. However, studies have shown that only about 7% to 16% of patients respond to the treatment, and serious side effects are common (Boni et al. Expert Opin Drug Saf. 2017 Dec; 16(12): 1347-1357).

[0012] The use of autologous cancer cells as vaccines to enhance anti-tumor immunity has been explored for some time. However, due to the weak immunogenicity of many cancers, the downregulation of MHC molecules, the lack of sufficient co-stimulatory molecule expression and the secretion of immunosuppressive cytokines by cancer cells, the response to such vaccines does not produce long-term efficacy (see, e.g., Armstrong TD and Jaffee EM, Surg Oncol Clin N Am. 1 1 (3): 681-96, 2002). Many cytokines have been shown to play a role in regulating the immune response to tumors. For example, U.S. Patent No. 5,098,702 describes the use of a combination of TNF, IL-2 and IFN-β in a synergistically effective amount to combat existing tumors. U.S. Patent Nos. 5,078,996, 5,637,483 and 5,904,920 describe the use of GM-CSF for treating tumors. However, direct administration of cytokines for cancer treatment may not be practical because they are often systemically toxic (Asher et al., J. Immunol. 146:3227-3234, 1991).

[0013] Document US2021 / 038684 A1 provides compositions and methods for treating cancer using IL-2 immunotherapy. The method of the invention includes systemic administration of interleukin-2 / interleukin-2-receptor alpha fusion protein to the patient, wherein administration results in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient, while there is no dose-dependent increase in circulating immunosuppressive T regulatory (Treg) cells. However, despite the intention to overcome tumor immune escape, the invention includes known disadvantages of cancer immunotherapy, such as weak tumor cell killing ability, and only some patients respond to treatment (Boni et al., Journal of Clinical Oncology 2021, 39: 15_suppl, 2513-2513). In addition, cytokine-based immunotherapy usually needs to be repeated (avoiding excessive stimulation that brings additional medical expenses and burdens to patients) to ensure sufficient immune system stimulation.

[0014] Documents WO 2020 / 172553 and WO 2021 / 127556A1 disclose anticancer therapies that combine different types of therapies (such as combining cell-based immunotherapy or checkpoint inhibitor immunotherapy with radiotherapy). However, not every one of these therapies to be combined targets cancer, so a synergistic effect cannot be achieved by combining these therapies.

[0015] Therefore, there remains a need for effective and safe treatments for proliferative diseases such as cancer, in which many different types of cancer are efficiently targeted, which treatments show no or mild undesirable side effects, and to which a wide variety of patients respond.

[0016] Therefore, an object of the present invention is to provide an improvement or alternative to the prior art.

[0017] This problem is solved by providing a recombinant expression system for combined gene and radiation therapy according to claim 1 or 18. Specific embodiments are subject matter of further dependent claims. Summary of the invention:

[0019] In a first aspect, the present invention provides a combined expression system for gene and radiation therapy of a disease in a recipient mammal, wherein

[0020] a. The recombinant expression system comprises a first nucleic acid construct encoding a target STING, wherein the target STING responds to a STING agonist that recognizes the target STING but not human STING and its natural variants (human non-responsive STING agonist), wherein the sequence encoding the target STING is operably linked to an element that allows expression and / or translation of the target STING; and / or a second nucleic acid construct comprising a therapeutic gene sequence, wherein the therapeutic gene sequence is operably linked to a STING-responsive promoter or a radiation-inducible promoter; and

[0021] b. administering the recombinant expression system to the recipient mammal before, simultaneously with, or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal with low-dose radiation to be transfected or transduced with the recombinant expression system; and / or

[0022] c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation and / or administering a STING agonist that recognizes the target STING but not human STING and its natural variants (human non-responsive STING agonist) to the recipient mammal.

[0023] In a similar aspect, the present invention relates to a method for treating a disease in a recipient mammal using a vector comprising a recombinant expression system, the method comprising:

[0024] a. providing a recombinant expression system, wherein the recombinant expression system comprises a first nucleic acid construct encoding a target STING, wherein the target STING responds to a STING agonist that recognizes the target STING but not human STING and its natural variants, wherein the sequence encoding the target STING is operably linked to an element that allows expression and / or translation of the target STING; and / or a second nucleic acid construct comprising a therapeutic gene sequence, wherein the therapeutic gene sequence is operably linked to a STING-responsive promoter or a radiation-inducible promoter; and

[0025] b. administering the recombinant expression system to the recipient mammal before, simultaneously with, or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system; and / or

[0026] c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation and / or administering a STING agonist that recognizes the target STING but not human STING and its natural variants to the recipient mammal.

[0027] It should be noted that in the case where the recombinant expression system is administered to the recipient mammal before the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system is irradiated with low-dose radiation in step b, the subsequent low-dose radiation is provided by the low-dose radiation of step c. Therefore, the administration of the recombinant expression system in step b is explicitly followed by the low-dose radiation of step c, whereby no additional low-dose radiation is applied in step b. Therefore, in the case, only one low-dose radiation (i.e., the low-dose radiation of step c)) needs to be applied.

[0028] It is noteworthy that the embodiments described below are applicable to the recombinant expression system of the present invention for combined gene and radiation therapy and a method of treating a disease in a recipient mammal using the recombinant expression system.

[0029] It is contemplated that the use of the gene and radiation combination therapy or the method for treating the disease of a recipient mammal according to the present invention may be combined with other treatments as anticancer therapies. Several anticancer therapies are known in the art, such as chemotherapy, hormone therapy, radiotherapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia or other immunotherapies.

[0030] The use of a combined gene and radiation therapy or method of treating a disease in a recipient mammal according to the present invention provides several advantages over prior art cancer therapies.

[0031] In the second nucleic acid construct, therapeutically relevant expression of the therapeutic gene sequence is ensured by placing the transgene under the control of a STING-responsive promoter or a radiation-inducible promoter.

[0032] The STING-responsive promoter is characterized in that it promotes transcription of therapeutic gene sequences only when STING is active in the cell. STING can be activated by a variety of treatment-related means, thereby allowing spatiotemporal control of STING activity in a given tissue or organ. Several STING agonists that allow specific STING activation are known in the art. In addition, STING is known to be activated by encountering DNA damaging attacks such as radiotherapy and by micronuclei that are frequently present in the cytoplasm of malignant cells. Therefore, the expression of therapeutic gene sequences controlled by STING-responsive promoters is associated with different cancer-related stimuli, which can be further and selectively stimulated by STING agonist drug therapy.

[0033] Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces expression of a therapeutic gene sequence when a cell is irradiated. These promoters may be natural, synthetic or artificially produced, selected from recombinant products of natural promoters. For example, these promoters may contain at least one of the following sequences, either individually or in a repetitive manner:

[0034] a. a eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), also known as a CArG element or [CC(A+T-rich)6GG], such as a radiation-inducible sequence of the early growth response-1 (Egr-1) promoter or the pE9 promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:86 or is identical to SEQ ID NO:86 (Egr1 promoter), or alternatively the sequence comprises at least two sequences according to SEQ ID NO:87, wherein the at least two sequences may be separated by additional nucleotides;

[0035] b. a low frequency CCAAT box sequence, such as the CCAAT box sequence of the survivin promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 88 or is identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter);

[0036] c. cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter;

[0037] d. Late herpes simplex virus 1 promoters, such as glycoprotein C promoter or U S 11 promoter;

[0038] e. Combinations of the above or artificial mutations thereof.

[0039] The inventors found that the first nucleic acid of the expression system according to the present invention induces upregulation of STING-related genes or proteins when the target STING is specifically activated by administering a STING agonist, and the STING agonist recognizes the target STING rather than human STING and its natural variants. It was further found that low-dose radiotherapy also induces upregulation of STING-related genes or proteins. It can be demonstrated that enrichment of STING (for example, by transducing cells with target STING) makes the cells more sensitive to STING agonists such as DMXAA (classical pathway) or low-dose radiotherapy (non-canonical pathway), respectively, and when the two stimuli (STING agonist and low-dose radiotherapy) are applied together, the synergistic effect between them can be fully demonstrated (see Fig. 9A -C). Thus, in one aspect of the invention, it is sufficient that the expression system only comprises a cassette encoding a target STING that is responsive to a STING agonist that does not activate human STING.

[0040] The expression system may also include a first and a second nucleic acid construct representing two expression cassettes: a) a first nucleic acid construct encoding a target STING that is responsive to and does not activate human STING and its natural variants (hereinafter also referred to as a "human non-responsive STING agonist"), and b) a second nucleic acid construct encoding a therapeutic gene, preferably an immunomodulatory gene or protein, which is under the control of a promoter that is activated when STING is activated.

[0041] According to the present invention, the expression system comprises two boxes or only one of the boxes. The combined expression of two nucleic acid constructs and therefore two expression boxes has the following advantages, namely, at the same time or after the systemic or intratumoral injection of the expression system (preferably a vector, more preferably a viral vector) containing the expression box, the systemic administration of the agonist of the target STING enables the selective activation of STING in the tumor without affecting the endogenous human STING in the rest of the body, thus avoiding toxicity. By inducing endogenous genes or by activating the promoter of the second expression box encoding immunomodulatory genes or proteins (such as IL-12), the intratumoral activation of the target STING induces the production of type I IFN, chemokines and other cytokines in the tumor. In this way, the systemic administration of the agonist of the target STING triggers the intratumoral production of type I IFN and other immunostimulatory peptides produced by STING activation, as well as the production of immunomodulatory genes or proteins expressed by the second expression box. The local intratumoral production of this combination of immunomodulatory genes or proteins will promote anti-tumor immunity without systemic toxicity.

[0042] The use of a system consisting of two expression cassettes injected into freshly irradiated tumors enables local activation of the transgenic target STING by ionizing radiation (atypical pathway), and this activation can be further increased by initiating the typical pathway of STING activation using a human non-responsive STING agonist such as DMXAA. By combining irradiation with DMXAA, the inventors found that a synergistic effect was obtained, resulting in a strong local STING response, causing strong activation of the STING responsive promoter that directs the expression of the immunomodulatory peptide encoded by the second expression cassette, resulting in high intratumoral levels of the combination of immunomodulatory molecules.

[0043] By injecting the tumor with one or more long-term expression vectors (such as adeno-associated viral vectors) containing both expression cassettes in the same or separate vectors, the production of the combination of immunomodulatory genes or proteins in the tumor can be induced by repeated systemic administration of an agonist of the target STING.

[0044] As mentioned above, cancer immunotherapy has several limitations, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821). The immunotherapy according to the present invention is a more efficient and specific immunotherapy because it is also combined with gene therapy. Therefore, the immunotherapy according to the present invention is not just immunotherapy itself, but a combination of immunotherapy and gene therapy, hereinafter referred to as "immunogene therapy", to effectively treat proliferative diseases such as cancer.

[0045] The inventors were surprised to find that the combination of the present invention comprising a combination of immunogene therapy of a viral vector and radiotherapy does not simply add the effects obtained by each individual treatment method, but works in a synergistic manner because both treatment effects are enhanced when the two therapies are combined. Therefore, the combined therapy according to the present invention significantly reduces the progression of tumors in experimental models. In addition, the therapy results in a reduction in tumor size, even complete remission. The therapy also shows a distant effect because the tumor size of tumors not directly treated is also reduced by the therapy.

[0046] The synergistic effect of the present invention is manifested in three aspects: 1) radiotherapy before treatment with viral vectors increases the efficiency of gene delivery to target cells, thereby increasing the number of cells producing therapeutic genes, and 2) radiotherapy / multiple radiotherapy given immediately before or after administration of the vector induces expression of the therapeutic gene sequence by activating the STING response promoter, and 3) the therapeutic gene expressed by the viral vector enhances the immunostimulatory effect caused by radiotherapy. Therefore, as shown in the examples of the present application, the effect of the treatment is multiplied, rather than simply added.

[0047] As a potential mechanism for this synergistic effect, it is hypothesized that radiation therapy given immediately before vector administration leads to activation of the DNA damage response (which lasts for several days, (Zhao Z et al., Genomics 2020; 112: 1309-1317]), as well as the presence of cytoplasmic DNA in irradiated cells. All these changes maintain the activation of STING for a period of time. For this reason, activation of STING-responsive promoters also occurs when radiation therapy is given immediately before vector administration.

[0048] The system of the present invention allows for the expression of therapeutic gene sequences on demand and in a highly localized manner, thereby avoiding the adverse aspects of systemic immunotherapy known in the art, such as toxicity.

[0049] In addition, in the case of recombinant expression of a first nucleic acid construct encoding a target STING, it was found that when the target STING was specifically activated by administering a STING agonist that recognizes the target STING but not human STING and its natural variants, the expression of the construct induced upregulation of STING-related genes or proteins.

[0050] Thus, enrichment of STING (e.g., by transducing cells with targeted STING) renders cells more sensitive to STING agonists such as DMXAA (canonical pathway) or low-dose radiation therapy (non-canonical pathway), respectively, and enables full display of the synergistic effect between the two stimuli (STING agonist and low-dose radiation therapy) when they are applied together (see Fig. 9A -C and Fig. 10A -D), thereby providing a third synergistic effect according to the present invention.

[0051] Thus, in one aspect of the invention, it is sufficient for the recombinant expression system to comprise only a first nucleic acid construct encoding a target STING that is responsive to a STING agonist that does not activate endogenous human STING.

[0052] In addition, the inventors have found that the administration of a recombinant expression system (preferably a viral vector) and the expression in combination with radiotherapy according to the present invention is non-toxic and shows less side effects in vivo. It is noteworthy that the inventors have found that when administered in vivo, the administration of a recombinant expression system (preferably a viral vector) and the expression in combination with radiotherapy lead to tumor reduction, ultimately leading to complete remission. Even, by locally administering the recombinant expression system of the present invention (preferably a viral vector) in a tumor, not only the size of the targeted tumor is reduced, but also non-targeted tumors show a significant size reduction, which is also referred to as the abscopal effect. DETAILED DESCRIPTION OF THE INVENTION

[0054] The recombinant expression system generates specific and basal immune pathways in transfected cancer cells that utilize otherwise endogenous pathway components.

[0055] It is noteworthy that the term "recombinant expression system" includes any nucleotide, its analog and its polymer. Therefore, any recombinant expression system disclosed herein refers to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. A more comprehensive definition is included in the definition section of this application.

[0056] The first component of this pathway is the so-called "target STING", which is defined by its responsiveness to STING agonists that cannot activate human STING, thereby allowing selective activation of target STING. The first component is given by the first nucleic acid construct of the present invention and can be regarded as the "co-effector" and "activator" part of the pathway. It is worth noting that this first component can also be administered alone, so that cells expressing this first component respond to STING agonists and low-dose radiotherapy, so that when these two stimuli (STING agonists and low-dose radiotherapy) are applied together, the synergistic effect between them is fully demonstrated.

[0057] In the nucleic acid construct according to the present invention, the target STING is operably linked to an element that allows expression and / or translation of the target STING. In the case where the recombinant expression system is a DNA-based expression system, the element is typically a promoter that allows transcription of mRNA encoding the target STING. In the case where the recombinant expression system is an RNA-based expression system, the element is typically an RNA element that allows translation of mRNA encoding the target STING, such as a CAP, a 5′UTR, a 3′UTR or a poly A site.

[0058] The second component of the pathway is an artificial gene construct, which includes an immunomodulatory gene or protein, the sequence of which is operably linked to a STING-responsive promoter. The immunomodulatory gene or protein activates innate and adaptive immune responses to produce a specific cancer immune response. The second component is given by the second nucleic acid construct of the present invention and can be regarded as the "synergistic effector" part of the pathway.

[0059] The recombinant expression system according to the present invention comprises a recombinant expression system and allows for the spatiotemporal expression of a therapeutic gene sequence according to STING activity and / or the spatiotemporal expression of a target STING according to a nucleic acid construct contained in the recombinant expression system. In the case of a second nucleic acid construct, this is achieved by operably linking the therapeutic gene sequence to a STING responsive promoter. One of the several ways to activate STING is to expose cells to ionizing radiation that produces DNA damage that is recognized by the cGAS / STING pathway, inducing the expression of STING downstream targets such as type I interferons or inflammatory cytokines.

[0060] Ionizing radiation is widely used to treat diseases. The most prominent use is to treat cancer, which is a proliferative disease characterized by rapid division of cancer cells. The DNA of cells receiving ionizing radiation is damaged, thereby inhibiting cell division, or killing cells in the case of excessive DNA damage. Therefore, the initial low-dose radiation of tissues or organs as described in b) of the present invention plays a dual role: i) low-dose radiation damages the DNA of target cells, thereby acting in its original manner, and ii) low-dose radiation enhances the transduction of AAV vectors to cells. This is so because radiotherapy triggers a DNA damage response (DDR), which, among other effects, promotes the conversion of AAV single-stranded DNA to transcriptionally active double-stranded DNA (Peng D. et al. J. Hepatol. 2000; 32: 975-85). As a result, the number of target cells expressing transgenes delivered by viral vectors increases.

[0061] On the other hand, DNA damaging agents have been shown to increase cytoplasmic DNA in tumor cells (Li, C et al. Molecular Immunology 2021; 131, 180-190) subsequently leading to STING activation (Deng, L et al., Immunity 2014; 41, 843-852; Dunphy et al. Molecular Cell, 2018; 71, 745-760). Therefore, radiotherapy before intratumoral injection of AAV vectors will lead to enhanced tumor transduction by AAV vectors and STING activation. The latter effect will promote the production of immunostimulatory cytokines, which are expressed by vectors under the control of STING-responsive promoters.

[0062] The low-dose radiation described in c) of the present invention also plays a dual role. First, low-dose radiation damages the DNA of the target cells, thereby exerting its effect in its original way. Secondly, DNA damage causes activation of the cGAS / STING pathway, thereby inducing the expression of therapeutic gene sequences delivered by a recombinant expression system (preferably a viral vector). Therefore, the spatiotemporal expression of therapeutic gene sequences and target STING can be controlled by local low-dose radiation, although other measures can also be taken to induce STING activity.

[0063] In general, low-dose radiation and immunogene therapy work synergistically, because each therapy has an effect in itself, however, as described above, when the therapies interact, the effect is multiplied, rather than simply added. This is also shown in vitro (see Example 9) and in vivo (see Example 10). Therefore, the combination of immunogene therapy according to the present invention and radiotherapy is an effective therapy for treating diseases in recipient mammals.

[0064] In another preferred embodiment, the recombinant expression system for use according to the present invention is used in combination therapy with at least one other therapy selected from the group consisting of: a therapy comprising at least one check point inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine and / or a therapy comprising at least one exogenous cytokine.

[0065] The inhibitor of the therapy including at least one checkpoint inhibitor can be selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR inhibitors, more preferably selected from the group consisting of: PD-1, PD-L1, PD-L2 or CTLA-4 inhibitors. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells, which help regulate immune responses and prevent excessive or prolonged immune activation. However, cancer cells can use these checkpoint proteins to escape detection by the immune system. By blocking these checkpoint proteins, immune checkpoint inhibitors can release the brakes on the immune response and allow the immune system to attack and destroy cancer cells. In the context of this aspect of the invention, it is particularly advantageous to use immune checkpoint inhibitors as additional therapy because the expression system containing therapeutic gene sequences causes T cells to infiltrate the tumor environment. Since the function of these T cells may be impaired due to immune checkpoints, methods to overcome this obstacle can greatly benefit treatment outcomes.

[0066] The therapy including at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using tumor infiltrating leukocytes (TIL). In some cases, tumor silences the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of escaping T cell immunity. These problems can be avoided using adoptive cell therapy because these adoptive cells are generally able to identify and target tumor cells. Therefore, a synergistic effect can be expected in a combined approach.

[0067] Furthermore, combining the expression system of the present invention with the cancer vaccine or exogenous cytokine for use according to the present invention can produce a synergistic effect because the recruited T cells are directed toward cancer cells by the cancer vaccine or exogenous cytokine.

[0068] Advantageously, in the specification of the present invention, it can be specified that the disease is selected from the group consisting of: cancer, autoimmune diseases, inflammatory diseases or infectious diseases. For example, it may be feasible to express an immunomodulator by an expression system. As used herein, the term "immunomodulator" refers to an agent that regulates (e.g., stimulates) the immune system (which can then be used to treat diseases, preferably proliferative diseases such as cancer). Immunomodulators can cause stimulation of the immune system, which is commonly used in immunotherapy for targeted cancer. However, other diseases such as autoimmune diseases, diabetes, cardiovascular diseases and inflammation can also be treated with immunomodulators. For example, IFN-β is used to treat multiple sclerosis. In addition, it has been shown that IL-2 regulates the balance of regulatory T cells and effector T cells, thereby maintaining the function of β cells. Immunotherapy can be used to treat non-cancer diseases, such as lupus, rheumatoid arthritis, diabetes.

[0069] The cancer may also be a solid cancer selected from the group consisting of: cancers of the bladder (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); hematopoietic tumors of the lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; brain cancer, central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; thymic cancer, including fibrosarcoma. Tumors of mesenchymal origin including sarcoma, rhabdomyosarcoma, and osteosarcoma; Other tumors including melanoma, peridermia pigmentosa, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratoma; Melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, neuroblastoma , pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colon cancer, esophageal cancer, lung cancer, mesothelioma, multiple myeloma, Merkel cell carcinoma, skin cancer, testicular cancer, uterine cancer, plasma cell tumors and head and neck cancer, gastric cancer, germ cell tumors, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, sinonasal natural killer cells (sinonasal natural killer), tumors, plasma cells; neuroendocrine tumors, bile duct cancer or carcinoid tumors.

[0070] Preferably, the solid cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, neuroendocrine tumors, bile duct cancer or carcinoid tumor.

[0071] Basically, every disease that can be treated by radiotherapy is a disease suitable for targeting by the combination therapy according to the present invention, because the synergistic effect of enhanced gene delivery and induction of expression of the therapeutic gene sequence is universally applicable and further combined with the effect of the radiotherapy alone. Therefore, non-cancer diseases such as arthrosis, Dupuytren's disease, Ledderhose disease, calcaneal spurs, tennis elbow, vertebral hemangioma, vertebral hemangioma, chronic synovitis, pigmented villonodular synovitis (PVNS), chronic joint effusions, rheumatoid arthritis and active joint diseases (each of which can be treated by radiotherapy) can also be treated particularly easily by the combination therapy according to the present invention.

[0072] In another aspect, the present invention relates to a recombinant expression system comprising a nucleic acid construct encoding a therapeutic gene sequence, the therapeutic gene sequence being operably linked to a STING-responsive promoter. Therefore, the present invention relates to a recombinant expression system comprising a second nucleic acid construct as described in the present application.

[0073] It is noteworthy that according to this aspect, a recombinant expression system comprising a second nucleic acid construct is also claimed and not only for use in a combination therapy according to the invention, or for a method of treating a disease in a recipient mammal using said expression system according to the invention.

[0074] In a preferred embodiment, the expression system is provided as an expression vector, which is more preferably a viral vector; even more preferably as a viral particle comprising a viral vector.

[0075] It is noteworthy that the embodiments described below for the expression system comprising the second nucleic acid are applicable to the expression system of the present invention itself, and also to the expression system used for gene and radiation combination therapy or the method of treating a disease in a recipient mammal using the expression system.

[0076] It is also conceivable within the scope of the present invention that the therapeutic gene sequence of the second nucleic acid construct encodes a protein or gene selected from the group consisting of: cytokines such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones; shRNAs against cytokines, nuclear factors or hormones.

[0077] In a preferred embodiment, the therapeutic gene sequence encodes an immunomodulatory protein.Similar to the term "immunomodulator", the term "immunomodulatory protein" refers to a protein that modulates (eg, stimulates) the immune system.

[0078] A non-limiting list of therapeutic proteins representing immunomodulatory proteins that can be used in the second nucleic acid construct of the recombinant expression system of the present invention (preferably in the form of a viral vector) includes the following proteins: interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, single-chain interleukin-12, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, interleukin-24, interleukin-25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, interleukin-30, interleukin-31, interleukin-32, interleukin-33, interleukin-34, interleukin-35 Interleukin-14, Interleukin-15, Interleukin-15-sushi, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14 , CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXC L1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CX CL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte-macrophage colony stimulating factor, type I interferon, IFN-γ, TNF-α, FLT3-ligand, blocking peptides targeting TGF-β, blocking peptides targeting IL-10, blocking peptides targeting FoxP3, monoclonal antibodies or single chain variable fragments (scFv) or nanobodies neutralizing PD1, PDL1, CTLA4, CD137, TIM3, LAG3 and fragments or variants thereof. A non-limiting list of therapeutic genes useful in the present invention is: shRNA targeting TGF-β, shRNA targeting IL-10 or shRNA targeting FoxP3.

[0079] Those skilled in the art will recognize that any gene or protein that slows, stops or reverses disease progression or cures disease is a suitable molecule that may be encoded by the therapeutic gene sequence of the second nucleic acid construct of the present invention.

[0080] It can be provided that the gene or protein encoded by the therapeutic gene sequence of the second nucleic acid construct of the recombinant expression system of the present invention is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10, single-chain interleukin-12 (scIL-12), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-s ushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony-stimulating factor (GM-CSF), type I interferon (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT3-ligand; immunostimulatory monoclonal antibodies or single-chain variable fragments (scFV) or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides or shRNAs against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

[0081] In one embodiment of the present invention, the gene encoded by the therapeutic gene sequence of the second nucleic acid construct of the recombinant expression system of the present invention is an shRNA directed against the mRNA of an immunosuppressive factor such as a cytokine, a nuclear factor or a hormone. Preferably, the shRNA is directed against TGF-β-mRNA, IL-10-mRNA or FoxP3 mRNA.

[0082] It is known in the art that several immunomodulatory proteins are used for immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used for immunotherapy in cancer treatment for more than 20 years. However, only a very small number of patients respond to IL-2-related immunotherapy, and these therapies are known to cause serious side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSC) into efficient antigen presenting cells (APC). In addition, IL-12 strongly stimulates T cells and NK cells and increases the production of IFN-γ, thereby inducing a Th1-type response, which can control the growth of the treated tumor and distant metastasis (Lasek et al. Cancer Immunol. Immunother. 2014; 63: 419). In addition, IL-12 has an anti-angiogenic effect, which can impair tumor vascularization, thereby limiting tumor growth. IL-12 is a heterodimeric cytokine encoded by two independent genes. In order to promote its expression in a recombinant expression system, it is feasible to use a recombinant single-chain IL-12 (scIL-12) transgene encoding two subunits of IL-12. Thus, in a preferred embodiment, the therapeutic gene sequence encodes IL-12.

[0083] In one embodiment, the at least one immunomodulatory protein is a complex or fusion protein of IL-15 and the Sushi domain of the IL-15 receptor α chain to enhance the agonist activity of IL-15 through trans expression such as provided by compound P22339 (Hu, Q., Ye, X., Qu, X. et al., SciRep 2018, 7675).

[0084] Optionally, IL-12 may be selected from the group consisting of: an α subunit of IL-12, a β subunit of IL-12, a single-chain IL-12 comprising an α and β subunit of IL-12. In the specification of the present invention, single-chain IL-12 (scIL-12) is defined as a fusion protein comprising an α and β subunit, wherein the peptide sequence is either directly connected to a linker peptide or separated by a linker peptide, wherein the linker peptide may have a length ranging from 1 to 100 amino acids. Preferably, the length of the linker ranges from 4 to 60 amino acids, more preferably from 8 to 40 amino acids, and particularly preferably from 12 to 25 amino acids. The length of the linker can be 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids. In addition, IL-12 can be a single-chain IL-12 (human or mouse single-chain IL-12) comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The sequence identity of the polypeptide sequence to SEQ ID NO: 9 or 10 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have interleukin-12 biological activity, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-12 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10.

[0085] The biological activity of IL-12 can be assessed in vitro by the ability of the peptide to increase IFN-γ production, which is produced by cells expressing the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, a reporter cell line can be used as, for example, a Promega bioluminescent IL-12 bioassay (Catalog No. JA2601, JA2605).

[0086] In vivo IL-12 bioactivity can be assessed by its ability to elevate IFN-γ serum levels.

[0087] In addition, the at least one immunomodulatory protein may be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β. The sequence identity of the polypeptide sequence to SEQ ID NO: 79 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have IFN-β biological activity, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of IFN-β assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 79.

[0088] The biological activity of IFN-β can be estimated by the ability of inducing type I IFN response genes. For this purpose, human cell lines (such as HEK 293) can be incubated with polypeptides, and the expression of IFN response genes can be measured by RT-qPCR. Alternatively, IFN reporter cells encoding luciferase under the control of a promoter containing ISRE can be used to assess the IFN-β biological activity of the polypeptide, which is carried out by measuring the expression of luciferase in a luminometer 3h after the polypeptide is added to the cell culture.

[0089] In vivo IFN-β bioactivity can be assessed by induction of IFN response genes in peripheral blood mononuclear cells (RT-qPCR).

[0090] In addition, the at least one immunomodulatory protein may be IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL- 15. The sequence identity of the polypeptide sequence to SEQ ID NO: 80 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have the biological activity of interleukin-15, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-15 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 80.

[0091] The biological activity of IL-15 can be assessed in vitro by a CTLL-2 cell proliferation assay.

[0092] In vivo IL-15 bioactivity can be assessed by its ability to induce NK cell and T cell proliferation using FACS analysis.

[0093] In addition, the at least one immunomodulatory protein may be CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10. The sequence identity of the polypeptide sequence to SEQ ID NO: 81 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have the biological activity of CXCL10, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of CXCL10 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 81. CXCL10 is also known as IP-10.

[0094] The biological activity of CXCL10 can be assessed by its ability to attract T lymphocytes and natural killer cells in vivo or in vitro migration assays.

[0095] In a preferred embodiment, the second nucleic acid construct of the present invention comprises two or more sequences, each sequence encoding a different immunomodulatory protein, wherein the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β; or the second immunomodulatory protein may be an IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL-15-sushi; or the second immunomodulatory protein may be a chemokine (preferably CXCL10) comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: NO:81. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity, wherein the polypeptide has the biological activity of IL-15-sushi.

[0096] In addition, other combinations of immunomodulatory proteins are also feasible. Single-chain IL-12 can be the first immunomodulatory protein, and the second immunomodulatory protein can be selected from the group consisting of: a peptide that blocks TGF-β, a peptide that blocks FoxP3, or a monoclonal antibody against the immune checkpoint inhibitor PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitor PD1 or PD-L1.

[0097] It should be noted that in the context of a second nucleic acid comprising two immunomodulatory proteins, the terms first and second immunomodulatory proteins do not describe the order of the coding sequences on the second nucleic acid, but are only used to illustrate two different immunomodulatory proteins. Therefore, the coding sequence of the second immunomodulatory protein can be located upstream of the coding sequence of the first immunomodulatory protein on the nucleic acid. Here, upstream means that the coding sequence of the second immunomodulatory protein is located before the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid.

[0098] TGF-β blocking peptides are peptides that interfere with TGF-β itself or interfere with the receptors that bind TGF-β, so that at least some TGF-β signaling is reduced or even completely prevented. Examples of TGF-β blocking peptides are P17 and P144.

[0099] A FoxP3 blocking peptide is a peptide that interferes with FoxP3 itself or with a receptor that binds FoxP3, such that at least some FoxP3 signaling is reduced or even completely prevented. An example of a FoxP3 blocking peptide is P60.

[0100] In another preferred embodiment, the second nucleic acid construct is a bicistronic or multicistronic expression cassette comprising two or more therapeutic gene sequences, each of which encodes a different immunomodulatory gene or protein, or a protein or RNA with anti-cancer properties, wherein the sequences are separated by a sequence encoding an IRES; and / or the sequences are separated by a sequence encoding a 2A peptide (preferably a sequence encoding a P2A peptide). The IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TREs).

[0101] In the present specification, a protein or RNA having anti-cancer properties is any such molecule that is capable of delaying, stopping or reversing the progression of cancer.

[0102] A bicistronic or polycistronic expression cassette is a gene construct that contains two or more independent coding regions or genes in a single transcript. Generally, it contains two or more different sequences in a single transcript. In order to allow independent translation of two or more immunomodulatory proteins and / or transcription of immunomodulatory genes, a TRE is needed.

[0103] One possible solution is to use an internal ribosome entry site (IRES). An IRES is a specific RNA sequence in a transcript that allows translation to be initiated at non-AUG codons, bypassing the normal cap-dependent initiation process. This mechanism allows regulation of gene expression and co-expression of multiple proteins from a single mRNA molecule.

[0104] Another possible solution is provided by 2A peptides (also referred to as 2A self-cleaving peptides). 2A peptides are a class of 18-22aa long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DxExNPGP (SEQ ID NO:32) and are present in a wide range of viral families. They help produce polyproteins by causing ribosomes to be unable to form peptide bonds. Due to its short length, 2A peptides are very suitable for viral vector systems with limited capacity, such as AAV. Another advantage of these peptides is that they allow equimolar expression of the coding sequences separated by these peptides. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The flanks of these peptides can have linker peptides of different lengths, which can lead to improved functionality. For example, the F2A element with a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO:82) in front shows significant expression of the protein separated by the element.

[0105] Table 1: Examples of combinations of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector

[0106] <![CDATA[1 st Immunomodulatory proteins]]> TRE <![CDATA[2 nd Immunomodulatory proteins]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[IFN-B B ]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > IRES FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > IRES TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > IRES Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > IRES Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > IRES Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > IRES Nanobodies targeting PD-L1 <![CDATA[IFN-B B ]]> IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IFN-B B ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > P2A FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > P2A TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > P2A Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > P2A Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > P2A Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > P2A Nanobodies targeting PD-L1 <![CDATA[IFN-β B ]]> P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IFN-B B <!-- 13 -->]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IL,15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > F2A FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > F2A TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > F2A Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > F2A Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > F2A Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > F2A Nanobodies targeting PD-L1 <![CDATA[IFN-B b ]]> F2A <![CDATA[IL-15-sushi C ]]>

[0107] A - preferably human single chain IL-12 as described in SEQ ID NO: 9

[0108] B - preferably human IFN-β as set forth in SEQ ID NO: 79

[0109] C-preferably human L-15-sushi as described in SEQ ID NO: 80

[0110] D-preferably human CXCL10 as set forth in SEQ ID NO: 81

[0111] Immunotherapy involving systemic availability of immunomodulatory proteins usually causes unwanted side effects. However, the advantageous aspect of the present invention is that the recombinant expression system used herein induces local expression of therapeutic gene sequences, which can be immunomodulatory proteins, thereby inducing highly local and targeted immune responses. For example, intratumoral administration of the recombinant expression system (which is a preferred embodiment of the present invention) induces an immune response by producing immunostimulatory cytokines such as interleukins or chemokines in the tumor mass. Without wishing to be bound by theory, it is believed that the release of immunomodulatory proteins in or near the tumor causes T cells to infiltrate near the tumor, and can result in the recognition of tumor cells that usually escape the recognition of the immune system, thereby enabling the immune system to target and kill these cells. Therefore, the progression of the tumor is at least delayed, preferably stopped, and more preferably reversed.

[0112] In a preferred embodiment, the therapeutic gene sequence of the second nucleic acid construct of the recombinant expression system of the present invention encodes an immunomodulatory gene or protein, more preferably an immunostimulatory gene or protein, i.e., a gene or protein that stimulates the immune system, such as by directly stimulating the immune system (e.g., IL-12) or inhibiting immunosuppressive molecules (e.g., shRNA targeting TGF-β).

[0113] According to a further possibility, the second nucleic acid construct may be provided with a STING responsive promoter operably linked to a therapeutic gene sequence, the promoter being selected from the group consisting of an IFN-β responsive promoter (preferably according to SEQ ID NO: 25), an IFNβ-minimal promoter (preferably according to SEQ ID NO: 31), a CXCL9 responsive promoter (preferably according to SEQ ID NO: 23), a CXCL10 responsive promoter (preferably according to SEQ ID NO: 24), a STAT motif promoter (preferably according to SEQ ID NO: 26), an IRSF3 motif promoter (preferably according to SEQ ID NO: 27 or SEQ ID NO: 28), an ISRE motif promoter (preferably according to SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13), a NFκB motif promoter (preferably according to SEQ ID NO: 29 or SEQ ID NO: 30) and a STAT6 motif promoter. In a preferred embodiment, the STING responsive promoter comprises at least one interferon stimulated response element (ISRE).

[0114] By operatively linking the expression of a therapeutic gene sequence to the activity of STING using a STING-responsive promoter according to the present invention, targeted and localized expression of the therapeutic gene sequence can be achieved, thereby avoiding potential side effects caused by systemic expression of the transgene encoded by the second nucleic acid construct.

[0115] The STING-responsive promoter of the second nucleic acid construct of the recombinant expression system of the present invention may optionally be selected from the list consisting of:

[0116] a. a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11;

[0117] b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart;

[0118] c. A nucleotide sequence according to SEQ ID NO: 12, comprising four copies of ISRE separated by 20 nucleotides;

[0119] d. The nucleotide sequence according to SEQ ID NO: 13, comprising four copies of ISREs separated by 6 nucleotides.

[0120] The use of STING-responsive promoters in the present specification ensures controlled and targeted expression of therapeutic gene sequences. In addition, the aforementioned STING-responsive promoters have been shown to have additional advantages. As described in detail in the examples of the present application, STING-responsive promoters have been shown to be inducible by administering human non-responsive STING agonists in the presence of target STING. In addition, the induction of these promoters has been shown to be specific, as only limited expression occurs in the absence of a selective STING agonist. In particular, a promoter carrying four copies of an ISRE as described in SEQ ID NO: 12 is highly inducible while also exhibiting high specificity.

[0121] Preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. does not contain an AP-1 site or an NFκB site. Even more preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. does not contain any other motifs that contribute to the inducibility, leakiness or expression efficiency of the promoter. In other words, the nucleotide sequence of the promoter described in a. or b. may comprise several nucleotides before, between and / or after the ISRE of SEQ ID NO: 11, wherein the nucleotides are selected in such a way that these additional nucleotides do not form any transcriptional regulatory elements (e.g. randomly). Particularly preferably, the nucleotide sequence of the STING-responsive promoter as described in a. and b. consists of 1-10 copies, preferably 2-7 copies, more preferably 3-5 copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 11 or identical to SEQ ID NO: 11.

[0122] Furthermore, the nucleotide sequences of the STING-responsive promoters described in a. to d. may already represent functional promoters, and thus these promoters do not require additional elements to induce expression of an operably linked transgene.

[0123] It is known that some viral particles, especially AAV, show tropism for hepatocytes. This means that when the viral particles enter the blood circulation system, they preferentially transduce hepatocytes, ultimately resulting in the expression of the encoded transgene in the liver. In addition, in the case of intratumoral injection of the viral particles, some of these particles will enter the blood circulation system, because tumors are highly vascularized. In order to prevent the unwanted expression of the transgene encoded by the viral particles in the liver, it is therefore appropriate to control its expression. Several methods are known to those skilled in the art to achieve this goal.

[0124] In general, a suitable method for restricting transgene expression to a specific tissue or a specific cell type is to use a viral particle with a distinct tropism for the tissue or cell type. A method for achieving this is by changing the serotype of the AAV or by changing the capsid of the AAV. Several AAV serotypes are known in the art, each with a different tropism. However, since the present invention is intended to target several different types of cancer, it is necessary to optimize the serotype or capsid for each cancer or tumor type. Nevertheless, this may be a suitable way to achieve tissue or cell type specific expression of a transgene.

[0125] The skilled person may also use DNA or RNA binding repressors to avoid unwanted expression of the transgene encoded by the viral particle in tissues or cells that are not intended to express the protein, preferably the liver. Thus, the second nucleic acid construct may comprise an operator that binds a repressor. The repressor may be operably linked to a promoter that is active in hepatocytes, thereby inducing expression of the repressor in these cells, thereby inhibiting expression of the transgene.

[0126] Another possibility is to use RNA interference (RNAi). RNAi is characterized in that RNA molecules reduce or suppress the expression of nucleic acid sequences with substantial or complete homology to the RNA molecules. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA or microRNA (miR). In order to use RNAi to reduce or suppress transgenic expression in specific tissues, it is conceivable that, for example, shRNA targeting the transgenic nucleotide sequence can be effectively connected to a tissue-specific promoter. When the shRNA is used, it therefore reduces or suppresses transgenic expression in tissues. Although this method is feasible in the specification of the present invention, it may be disadvantageous because it is necessary to additionally use the shRNA to limit the expression of transgenic in target tissues such as the liver.

[0127] A good way to avoid this shortcoming is to effectively link the miRNA target sequence to the nucleotide sequence of the transgene. Therefore, in the tissue where there is a miRNA associated with the miRNA target sequence, the expression of the transgene will be reduced or inhibited. Therefore, a preferred embodiment of the present invention includes a target sequence of a microRNA whose sequence motif is highly abundant in the liver. Therefore, a single administration of the nucleic acid construct is sufficient to avoid transgene expression in the liver, while transcription in other tissues or organs is not weakened.

[0128] According to a further possibility, it can be provided that the second nucleic acid construct of the recombinant expression system comprising the therapeutic gene sequence also comprises at least one sequence motif, which inhibits the transgenic expression of the therapeutic gene sequence in tissues or cells that are not intended to express the protein, wherein the tissue is preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

[0129] Optionally, it is envisioned that the microRNA highly abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c or let7f. In addition, the microRNA miR-122 abundant in the liver can be advantageous. The advantage of miR-122 is that it is highly expressed in the liver, and more importantly, specifically expressed in the liver. Therefore, by selecting the miR-122 target sequence, a significant reduction of the transgene in the liver is ensured, while the expression in other tissues is not weakened. In addition, it has been shown that miR-122 is downregulated in most liver tumors, so the recombinant expression system according to the present invention is also suitable for treating liver cancer.

[0130] According to a preferred embodiment of the present invention, the corresponding miR-122 target sequence is a miR-122 target having at least 90% or at least 95% sequence identity relative to SEQ ID NO: 14 or being identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence as described in the previous sentence. Increasing the number of copies of the miR-122 target sequence increases the miR-mediated reduction, while no significant improvement is observed for numbers higher than the disclosed 5 copies. In a preferred embodiment, the copies are separated by 1 to 40 nucleotides, preferably 1 to 30 nucleotides, more preferably 2 to 20 nucleotides, and particularly preferably 2 to 8 nucleotides.

[0131] Preferably, the nucleotide sequence comprises 5 copies of the miR-122 target sequence as set forth in SEQ ID NO: 15. As described in the Examples of this application, the nucleotide sequence set forth in SEQ ID NO: 15 has been shown to substantially inhibit transgene expression in the liver, resulting in undetectable plasma levels of the transgene by the method described in Example 4.

[0132] It can be provided that the low-dose radiation is based on photon radiation such as X-rays or gamma rays, or particle radiation, wherein the particles of the particle radiation are preferably selected from the group consisting of beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions. Each type of radiation has its own characteristics and characteristic spectrum of therapeutic applications. A person skilled in the art selects the corresponding radiation based on the disease itself, the location and the progression of the disease.

[0133] Optionally, it is contemplated that low dose radiation may be administered with a total radiation dose of 100 Gy or less, preferably 40 Gy or less, and more preferably 25 Gy or less. The total radiation dose may be less than 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 Gy. In some embodiments, the dosage of 40Gy is 100Gy, 150Gy, 100Gy, 150Gy, 100Gy, 150Gy, 150Gy, 100Gy, 150Gy, 150Gy, 150Gy, 16 ...50Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy, 150Gy,

[0134] There are several advantages to using low-dose radiation. First, radiotherapy is always accompanied by side effects. By using low-dose radiation, these side effects are significantly reduced. Secondly, there is a limit to the amount of radiation that a part of your body can safely receive throughout your life, which is the so-called lifetime limiting dose. Using low-dose radiation is not only more likely to stay below this limit, but also allows for a higher number of radiation treatments. In the context of the present invention, this is particularly useful because it allows therapeutic gene sequences to be repeatedly expressed under the control of the STING response promoter. Third, low-dose radiation enhances the susceptibility of cells to gene delivery. AAV is a single-stranded DNA vector that needs to be converted into double-stranded DNA to form a transcription template. Not wishing to be constrained by therapy, it is believed that the DNA damage response caused by radiotherapy helps convert single-stranded DNA into double-stranded DNA. Using radiotherapy to enhance tumor transduction through AAV vectors is an attractive option because radiotherapy is often used to treat solid tumors. Published data on AAV transduction and radiation involve in vitro studies using different radiation doses and in vivo studies using high radiation doses. However, high radiation doses can cause vascular damage and mark changes in tissue structure. Thus, relatively low radiation doses preserve tumor vasculature, thereby maintaining lymphocyte trafficking to the tumor.

[0135] According to another possibility, the low-dose radiation may be administered in the form of stereotactic radiosurgery (SRS), intraoperative radiotherapy (IORT), stereotactic body radiotherapy (SBRT), single-dose radiotherapy (SDRT), intensity-modulated radiotherapy (IMRT), image-guided radiotherapy (IGRT), stereotactic ablative radiotherapy (SABR) or hypofractionated radiotherapy preferably delivered by a linear accelerator (LINAC) machine, a gamma knife machine or proton beam therapy.

[0136] The advantage of using the above-mentioned administration method is that they allow high-precision targeting of target tissues or organs. In addition to limiting the unwanted side effects from radiation, transgenic expression is also induced at specific sites. In the specification of the present invention, this limits the expression of the target gene sequence to a clearly defined site, thereby avoiding the systemic availability of the transgene, and thus avoiding unwanted side effects. As described in the following examples, although the target tumor receives radiotherapy combined with immunogene therapy, other non-target tumors also show a reduction in size (remote effect). Therefore, by using high-precision radiotherapy, not only side effects are reduced, but also distant tumors are still affected by treatment.

[0137] Advantageously, in the description of the present invention, it can be specified that the time interval between the low-dose radiation of b. and the subsequent administration of the viral vector is between 1 minute and 20 days, preferably between 10 minutes and 2 days, and more preferably between 30 minutes and 24 hours. The time interval can be any time between 1 minute and 20 days. The subsequent administration of the viral vector begins at the earliest 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days or 19 days after receiving the low-dose radiation. This ensures enhanced gene delivery and increased expression of the transgene in the target tissue or organ.

[0138] Advantageously, in the specification of the present invention, it can be specified that the time interval between the administration of the viral vector and the subsequent c. low-dose irradiation is between 2 hours and 30 days, preferably between 1 day and 5 days, more preferably between 1 day and 3 days. The time interval can be any time between 2 hours and 30 days. Subsequent low-dose radiation begins at the earliest 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 25 days, 26 days, 27 days, 28 days, 29 days after the administration of the viral vector. Subsequently, low-dose radiation can be repeated at intervals between 1 day and 30 days. Subsequent low-dose radiation may begin as early as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, and 29 days after the previous dose of radiation.

[0139] According to the present invention, the recombinant expression system further comprises a first nucleic acid construct comprising a sequence encoding a target STING, wherein the sequence is responsive to a STING agonist that recognizes the target STING but not human STING and its natural variants, wherein the sequence encoding the target STING is operably linked to a promoter.

[0140] It is worth noting that the target STING differs from the native human STING in that it is activated by human non-responsive agonists such as DMXAA. However, after activation, the target STING shows the biological activity of human STING because the target STING is still able to bind and activate downstream effector proteins, which are also activated by human STING such as iRhom2 and TANK binding kinase 1 (TBK1). After activation, STING undergoes conformational changes, leading to TBK1 recruitment. In turn, TBK1 phosphorylates transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB), which translocate to the nucleus and stimulate the production of proinflammatory molecules such as type I interferon, IL6, TNF-α or CXCL10. (Ahn, J. et al. Exp Mol Med 51, 1-10, 2019).

[0141] In the present specification, the use of exogenous target STING has several advances. First, by using target STING, STING activity of exogenous target STING rather than endogenous human STING can be selectively induced. This means that STING activity is induced only in target cells that have been transfected or transduced with viral vectors, and therefore STING activity is confined locally. Therefore, systemic STING activation is avoided, and unwanted side effects caused by systemic STING activation are avoided. Therefore, the expression of therapeutic gene sequences is locally confined to target cells. Therefore, STING activity can be controlled not only by local administration of radiotherapy, but also by using target STING and corresponding agonists. Secondly, in many human tumors, the cGAS / STING pathway is epigenetically silent, and tumors with low cGAS / STING expression are associated with poor prognosis, which may be due to tumor evasion of immunity (Chabanon RM et al., Nature Reviews Cancer 202121:701). Thus, by providing exogenous targeted STING to tumor cells, the availability and functionality of cGAS / STING is ensured. Furthermore, the activity of exogenous targeted STING can be controlled in two different ways as described above.

[0142] It may be provided that after administration of the recombinant expression system (preferably a viral vector) comprising the first nucleic acid construct, a human non-responsive STING agonist that does not react with human STING is administered to the recipient mammal. The non-human responsive STING agonist may be selected from the group consisting of: 5,6-dimethylxanthone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tiloron) and 10-carboxymethyl-9-acridone (CMA), more preferably, wherein the STING agonist is DMXAA. It is recognized by those skilled in the art that analogs of the molecules disclosed herein and any stereoisomers thereof may also be used. In addition, one or more atoms of the above-mentioned molecules, their analogs and / or their stereoisomers may be replaced by their respective isotopes.

[0143] When used for human treatment, the use of human non-responsive STING agonists in the description of the present invention has some advantages. By definition, human non-responsive STING agonists cannot bind to human wild-type STING or its natural variants in a way that causes STING activation. Therefore, it can be administered systemically without causing a systemic STING response, thereby avoiding unwanted side effects. Since the present invention is intended to express a target STING that responds to a human non-responsive STING agonist, it is possible to specifically activate this transgenic STING, thereby causing local STING activation. Therefore, in the description of the present invention, the human non-responsive STING agonist has two effects: it is used to define STING used as a target STING in gene therapy, and in addition, it is also used in subsequent drug therapy to activate the target STING, thereby achieving a highly selective treatment method.

[0144] Preferably, the target STING is responsive to DMXAA. The use of DMXAA-responsive STING has the advantage that the STING agonist DMXAA has been demonstrated in clinical studies to be a molecule with a favorable safety profile and only mild side effects.

[0145] Furthermore, it could be shown that activation of the target STING by DMXAA showed a dose-dependent induction of downstream STING targets such as IFN-β (see Example 1). This allows the immune response to be controlled by simply adjusting the DMXAA dose during treatment.

[0146] Intratumoral injection of DMXAA has been shown to effectively elicit CD8+ T cell responses and promote the elimination of established tumors in a STING-dependent manner in mice (Corrales et al. Cell Reports, 2015; 11, 1018-1030). Clinical trials demonstrating the anti-tumor properties of DMXAA have shown that it exhibits only mild side effects. However, these trials showed that DMXAA cannot activate human wild-type STING or its natural variants. Nevertheless, murine STING and rat STING respond to DMXAA as they display the biological activity of STING.

[0147] Furthermore, it is contemplated within the scope of the present invention that the target STING is a mutant form of a natural variant of human STING that further comprises at least one, at least two or at least three amino acid substitutions that render the human STING variant sensitive to a human non-responsive STING agonist as determined in an in vitro agonist assay.

[0148] In vitro agonist assay tests can be used to identify human non-responsive STING agonists. Several possible methods are known to those skilled in the art to perform such assays. In a non-limiting example, a vector encoding a reporter gene (e.g., luciferase) under the control of a STING responsive promoter can be constructed. Cells can be transformed, transfected, or transduced with the vector and additionally with a vector that induces expression of human STING or its natural variant, mutated human STING variant, or non-human STING. These cells should then be contacted with a STING agonist (preferably DMXAA), and luciferase activity should be measured. If luciferase activity in cells transformed, transfected, or transduced with a vector that induces expression of a mutated human STING variant or non-human STING is detectable, but the luciferase activity in cells transformed, transfected, or transduced with a vector that induces expression of human STING or its natural variant is not significantly different from that of a mock-treated control group, the agonist is considered to be "human non-responsive". In this case, no significant difference from the mock-treated control group means that the luciferase activity is less than 30%, preferably less than 10%, more preferably less than 5%, and particularly preferably less than 1% of the luciferase activity obtained by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6). Another example of an in vitro agonist assay test is to directly measure IFN-β levels when cells transformed, transfected or transduced with a vector that induces a mutation in a human STING variant, a non-human STING, or human STING or a natural variant thereof are contacted with a STING agonist. In this case, a human non-responsive STING agonist will provide at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the IFN-β levels obtained by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO: 6).

[0149] It is also envisaged within the scope of the present invention that the target STING is a non-human STING that is sensitive to a non-human responsive STING agonist as determined in an in vitro agonist assay test, whereby the human non-responsive STING agonist is preferably DMXAA. Non-limiting examples of non-human STING that are sensitive to a human non-responsive STING agonist as determined in an in vitro agonist assay test are murine STING or a natural variant thereof, rat STING or a natural variant thereof, or a hybrid STING. In a preferred embodiment, the non-human STING is murine STING according to SEQ ID NO: 7, which encodes a protein of SEQ ID NO: 8.

[0150] In a preferred embodiment of the invention, the target STING is a DMXAA-sensitive human STING as determined in an in vitro DMXAA assay test. As described above, the advantages of using DMXAA for human therapy are that DMXAA has been shown to have a favorable safety profile, exhibits only minor side effects, and because it cannot activate human STING, it can be administered systemically without causing systemic STING activation.

[0151] In vitro DMXAA assay tests can be used to identify STING that responds to DMXAA. Cells can be transduced with vectors that induce expression of any STING (e.g., human wild-type STING, mouse STING). If, after the addition of DMXAA, the cells respond by showing a statistically significant increase in the mRNA levels of IFN-β and other cytokines measured by quantitative PCR (qPCR) compared to mock-treated controls in triplicate assays, the expressed STING is considered to be DMXAA-sensitive (see, e.g., FIG. 1 ). In this case, a statistically significant increase means that the mRNA level is at least 10%, preferably at least 20%, more preferably at least 30%, and in particular at least 50% higher than the mock-treated control. An example of a DMXAA-non-responsive STING is human wild-type STING or a natural variant thereof. Another example of an in vitro DMXAA assay is to directly measure the IFN-β level of cells transformed, transfected, or transduced with a vector encoding STING (e.g., human wild-type STING, mouse STING) after contact with DMXAA. In this case, DMXAA-sensitive human STING will provide at least 25%, preferably at least 50%, more preferably at least 75%, and even more preferably 90% of the IFN-β levels as obtained by using hSTING[R]-S162A / G230I / Q266I (SEQ ID NO:6) treated with DMXAA.

[0152] Advantageously, in the description of the present invention, it may be provided that the target STING is a DMXAA-sensitive human STING variant selected from the group consisting of:

[0153] a. a human STING[R] variant, further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I;

[0154] b. a human STING[AQ] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, A230I, E260I, and E260V;

[0155] c. a human STING[H] variant further comprising at least 1, at least 2, or at least 3 amino acid substitutions selected from the group consisting of S162A, G230I, and Q266I; and

[0156] d. A truncated human STING[H] variant further comprising at least one or at least two amino acid substitutions selected from the group consisting of S162A and G230I.

[0157] The skilled person noted that all of the above mutations are located in the cyclic dinucleotide (CDN) binding site (binding pocket) and / or lid region of the binding pocket of STING. Therefore, in another embodiment, endogenous human STING may contain a mutation in one of the amino acids located in the binding pocket region and / or lid region to make it DMXAA-sensitive human STING.

[0158] For example, several natural variants of human STING have been identified as a result of several single polymorphisms (SNPs) and SNP haplotypes that produce several allelic polymorphic forms of the protein. The amino acid sequence of the most common natural variant or allele of human STING is SEQ ID NO: 1, which is referred to as wild-type human STING. The most common natural allelic variants of human STING include: hSTING [R] as described in SEQ ID NO: 1, usually referred to as hSTING R232, i.e., the amino acid at position 232 is arginine, which STING is also considered to be a wild-type isoform; hSTING [H] as described in SEQ ID NO: 2, usually referred to as hSTING H232, i.e., the amino acid at position 232 is histidine; hSTING [AQ] as described in SEQ ID NO: 3, whose sequence differs from hSTING [R] by the incorporation of amino acid substitutions G230A and R293Q. In addition, a truncated form of hSTING [H] as described in SEQ ID NO: 4 is characterized. Another natural variant is STING[HAQ], which has amino acid substitutions R71H, G230A, and R293Q. Thus, the target STING is different from one of the known haplotypes of human STING (i.e., hSTING[R] as described in SEQ ID NO: 1, hSTING[H] as described in SEQ ID NO: 2, hSTING[AQ] as described in SEQ ID NO: 3, hSTING[H] as described in SEQ ID NO: 4, STING[Q] with amino acid substitution R293Q, or STING[HAQ] with amino acid substitutions R71H, G230A, and R293Q). In addition, the target STING can be responsive to any one of the human non-responsive STING agonists selected from the group consisting of 5,6-dimethylxanthone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone) and 10-carboxymethyl-9-acridone (CMA). Thus, administration of the agonist results in activation of exogenous STING provided by the first nucleic acid of the recombinant expression system, wherein endogenous STING encoded in the genome of the human subject is not activated.

[0159] In another possibility, a DMXAA-sensitive human STING variant encoded by the sequence of the first nucleic acid construct may be provided selected from the group consisting of: hSTING[R]-S162A / G230I / Q266I, hSTING[R]-S162A / Q266I, hSTING[R]-G230I, hSTING[R]-S162A, hSTING[R]-Q266I, hSTING[R]-S162A / G230I, hSTING[AQ]-S162A, hSTING[AQ]-Q266I, hSTING[R]-S162A / Q230I, hSTING[AQ]-S162A, hSTING[ hSTING[AQ]-S162A / R169A, hSTING[AQ]-S162A / T267A, hSTING[AQ]-E260I, hSTING[AQ]-E260V, hSTING[H]-S162A / G230I / Q266I, hSTING[H]-S162A / Q266I, hSTING[H]-G230I, hSTING[H]-S162A, hSTING[H]-S162A / G230I and hSTING[H]-Q266I, and preferably hSTING[R]-S162A / G230I / Q266I.

[0160] Experiments have been performed showing that mutation S162A is critical in allowing DMXAA to bind and activate human STING, and that mutations Q266I and G230I or A230I increase sensitivity to DMXAA (Gao et al., 2014, Cell Reports 8, 1668-1676).

[0161] According to another advantageous aspect, a DMXAA-sensitive human STING variant can be provided selected from the group consisting of: SEQ ID NO: 5 (which is a polynucleotide sequence of hSTING[R]-S162A / G230I / Q266I) or a polynucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to SEQ ID NO: 5. The sequence identity of the polynucleotide sequence to SEQ ID NO: 5 can be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Of note, the polypeptide encoded by any of the aforementioned polynucleotides has a biological activity in response to the agonist DMXAA as determined in an in vitro DMXAA assay test.

[0162] According to another advantageous aspect, it can be provided that the DMXAA-sensitive target STING is selected from the group consisting of the following polynucleotide sequences: a polynucleotide sequence encoding a polypeptide comprising SEQ ID NO: 6 (which is the sequence of hSTING[R]-S162A / G230I / Q266I) or a polynucleotide sequence encoding a polypeptide having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 6. The sequence identity of the polypeptide encoded by the polynucleotide sequence relative to SEQ ID NO: 6 may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Notably, a polypeptide encoded by any of the foregoing polynucleotides has biological activity that is responsive to a human non-responsive STING agonist as determined in an in vitro agonist assay.

[0163] Both hSTING[R]-S162A / G230I / Q266I and mouse STING have been extensively studied and are known to be highly activated by DMXAA and CMA. In addition, mouse STING can also be activated by FAA and tilorone. However, in cancer treatment, the use of variants of human STING may be preferred over the use of non-human STING because the former may show better interactions with signaling molecules in human cells. In addition, the use of non-human STING may induce unwanted immune responses.

[0164] It is noteworthy that the polynucleotide sequence from the first nucleic acid construct comprising a sequence encoding the target STING described above may comprise at least one modification of the polypeptide, which is an amino acid substitution, an amino acid insertion, an amino acid deletion, a C-terminal truncation or an N-terminal truncation. In addition, according to the present invention, a polynucleotide sequence that hybridizes with the complementary sequence of the above-mentioned polynucleotide sequence under stringent conditions is also included. In addition, according to the present invention, a fragment of at least 50 amino acid residues of a DMXAA-sensitive human STING variant is included. It is noteworthy that, as determined in an in vitro agonist assay test, the polypeptide encoded by any of the aforementioned polynucleotides has a biological activity that responds to a human non-responsive STING agonist.

[0165] Preferably, the promoter that can provide the first nucleic acid construct comprising a sequence encoding the target STING is a constitutive promoter, such as SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh, CBA, human beta actin, Ac5, polyhedrin, TEF1, GDS, CaMV35S, Ubi, H1 or U6. Preferably, the promoter is selected from the list consisting of: SV40, CMV0, UBC, EF1a, PGK, CAG, EFS, CBh and CBA. The use of a constitutive promoter in the specification of the present invention has the advantage of ensuring the expression of the target STING. Generally, constitutive promoters show higher expression levels compared to inducible or tissue-specific promoters. Alternatively, the promoter may be an inducible promoter, preferably selected from the list consisting of: telomerase response promoter, IL-6 response promoter, TTF-β inducible promoter, VEGFR inducible promoter, epidermal growth factor receptor inducible promoter, HER2 / Neu inducible promoter, MUC1 promoter, BRCA1 promoter, BRCA2 promoter, LALBA promoter, PSA promoter, ERBB2 promoter, CEA promoter, uPAR promoter, thyroid transcription factor 1 (TTF1). However, those skilled in the art recognize that in the present specification, cancer-specific promoters are also feasible promoters for nucleotide sequences.

[0166] According to a further possibility, it can be provided that the second nucleic acid construct comprising the therapeutic gene sequence also comprises a transgene encoding thymidine kinase (preferably thymidine kinase according to SEQ ID NO: 16). The nucleotide sequence encoding thymidine kinase is connected to the nucleotide sequence of the therapeutic gene sequence and can be separated by a 2A self-cleaving peptide (2A peptide). 2A peptides constitute a class of 18-22 amino acid long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DXEXNPGP and are present in a wide range of viral families. They help produce polyproteins by causing the ribosome to be unable to form peptide bonds. Those skilled in the art recognize that the use of 2A peptides allows the co-expression of therapeutic gene sequences and thymidine kinase. The co-expression of thymidine kinase is advantageous because it allows tumor transduction to be assessed by PET imaging, and in the case of potential IL-12-mediated toxicity, cells expressing cytokines are eliminated by administering thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir).

[0167] The first and / or second nucleic acid construct (preferably a viral vector) of the recombinant expression system of the present invention may also contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). WPRE is known to increase the expression of a transgene, and therefore is most effective when placed downstream of the transgene (i.e., the target STING or therapeutic gene sequence), close to the polyadenylation signal.

[0168] Conveniently, the first and / or second nucleic acid construct (preferably a viral vector) of the recombinant expression system of the present invention further comprises a polyadenylation signal located at the 3′ position relative to the target STING or therapeutic gene sequence. The length of the Poly (A) tail can be optimized to stabilize the mRNA and subsequently increase protein translation.

[0169] The skilled person is also aware of other methods for increasing protein translation, such as

[0170] a. by sequence modification to optimize G / C content and / or overstabilizing secondary structure codons, or

[0171] b. alteration of the 5′ cap by addition of synthetic or natural 5′ end cap analogs such as m7GpppG or initiator trimers such as “CleanCap”, or stabilization by capping enzymes, or

[0172] c. Adding regulatory elements

[0173] c.1 in the 5′-untranslated region (UTR), such as by optimizing or modifying the length and sequence to avoid overly stable structures through appropriate cis-regulatory sequences, or

[0174] c.2 In the 3′-UTR, stable elements or repetitive motifs or synthetic or natural sequences are introduced by sequence modification, which have been shown to be effective in other mRNAs.

[0175] In a specific embodiment, the recombinant expression system of the present invention consists of a second nucleic acid construct given in the table below, wherein the different elements are shown in the 5′ to 3′ direction as a promoter, a first open reading frame (ORF1), a first translational regulatory element (TRE1), a second open reading frame (ORF2), a second translational regulatory element (TRE2), and a target sequence for a microRNA (microRNA element).

[0176] Table 2: Embodiments of the second nucleic acid construct of the expression system

[0177] Promoter ORF1 TRE1 ORF2 TRE2 microRNA elements <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> IFN-β Minimal <![CDATA[scIL12 B ]]> IFN-β Minimal <![CDATA[scIL12 C ]]> <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> <![CDATA[miR122 target site E <!-- 22 -->]]> IFN-β Minimal <![CDATA[scIL12 B ]]> <![CDATA[miR122 target site E > IFN-β Minimal <![CDATA[scIL12 C ]]> <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > IFN-β Minimal <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > IFN-β Minimal <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> IHRES <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> IHRES <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> p2A <![CDATA[scIL12 B ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[HSV-TK D ]]> p2A <![CDATA[scIL12 C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IHRES <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> IHRES <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> p2A <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 C ]]> p2A <![CDATA[HSV-TK D ]]> WPRE <![CDATA[miR122 target site E >

[0178] A - preferably 4 copies of ISRE, preferably given by SEQ ID NO: 12

[0179] B - preferably human single chain IL-12 as described in SEQ ID NO: 9

[0180] C - preferably murine single chain IL-12 as described in SEQ ID NO: 10

[0181] D—preferably HSV thymidine kinase as described in SEQ ID NO: 16

[0182] E - preferably 5 copies of the miR-122 target sequence as set forth in SEQ ID NO: 15

[0183] Another aspect of the invention is an expression vector encoding the first and second nucleic acid construct according to the invention.

[0184] The use of an expression vector comprising the first and / or second nucleic acid construct has certain advantages. First, the expression vector is specifically designed to allow the nucleotide sequence of the encoding to be introduced into a host cell and to control their expression. Second, the expression vector is a very stable plasmid, so it is protected and not degraded. Third, the expression vector can be used for gene delivery using different methods. Non-limiting examples of the expression vector of the present invention and preferred embodiments are viral vectors, more preferably viral vectors selected from the list consisting of: adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. In particular, viral vectors combine several advantages of expression vectors, such as general safety, low toxicity and stability.

[0185] It is conceivable that the first and second nucleic acid constructs of the expression system according to the invention are contained in a single expression vector. This has the advantage that a single administration of the expression vector is sufficient to express the expression system according to the invention in the patient.

[0186] An alternative aspect of the present invention is a combination of a first and a second expression vector, the first vector encoding a first nucleic acid construct and the second vector encoding a second nucleic acid construct according to the present invention. This has the advantage that the size of each nucleotide sequence is not as limited as combining two nucleic acid constructs in one vector. Size limitation is a common problem for viral vectors. For example, adeno-associated virus (AAV) is limited to a size of about 5 kb, however, it must be taken into account that ITRs, promoters and other elements are not included in the 5 kb.

[0187] Thus, in an alternative aspect, the recombinant expression system of the present invention provides a combination of a first expression vector encoding a first nucleic acid construct and a second expression vector encoding a second nucleic acid construct.

[0188] It is conceivable that the first and second nucleic acid constructs according to the invention are contained in a single viral vector. This has the advantage that a single administration of the viral vector is sufficient to express the expression system according to the invention in the patient.

[0189] It is also advantageous that the first and / or second nucleic acid construct of the one or more viral vectors further comprises 5′ITR and 3′ITR sequences, preferably the AAV ITRs described in SEQ ID NO: 17. It may be advantageous that the one or more viral vectors are AAV vectors, and preferably have serotypes AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10, with AAV2 being the most preferred. AAV is commonly used in biotechnology and is therefore well characterized. An advantage of using AAV vectors is that they are generally considered safe, exhibit low toxicity and are stable. In addition, AAV has been approved for use in humans by several governmental organizations around the world.

[0190] It is also advantageous that the first and / or second nucleic acid construct of the one or more viral vectors further comprises a 5'ITR, a ψ packaging signal and a 3'ITR sequence, preferably a 5'ITR, a ψ packaging signal and a 3'ITR of an adenovirus (Ad). It may be advantageous that the one or more viral vectors are adenoviral vectors, preferably high-capacity adenoviral vectors. Adenoviruses are commonly used in biotechnology and are therefore well characterized. An advantage of adenoviral vectors is that they have a higher size limit than AAV vectors. In addition, adenoviruses have been approved for use in humans by several governmental organizations around the world.

[0191] It is also advantageous that the one or more viral vectors are lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. These vectors are commonly used in biotechnology and are therefore well characterized.

[0192] It is also contemplated that nanoparticles can be used to deliver the first and / or second nucleic acid constructs of the present invention.Nanoparticles are known in the art as gene delivery systems and have advantages such as adjustable size, shape and / or surface, allowing for the regulation of their biological behavior. For effective delivery and cellular uptake, the nucleic acid construct can be packaged into nanoparticles (preferably liposomes) made of a pharmaceutically acceptable carrier, which is composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers (such as standard lipids compatible with biological membranes) in different proportions, wherein the standard lipids are selected from the following list: phospholipids, such as distearoylphosphatidylglycerol, distearoylphosphatidylcholine, (DLPC) 1,2-dilauroyl-sn-glycero-3-phosphocholine, (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DPPC) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, (DSPC) 1,2-distearoyl-sn-glycero-3-phosphocholine, (DOPC) 1,2-dioleoyl- sn-glycero-3-phosphocholine, (DMPE) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, (DPPE) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, (DOPS) 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DPPS) 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, (DMPS) 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, (DOPG) 2,3-dimyristoyl-sn-glycero-1-phosphocholine and their corresponding enantiomers, among which distearoylphosphatidylglycerol and distearoylphosphatidylcholine are most preferred. Another suitable example is that cardiolipin is preferably used in combination with cholesterol.Alternative nanoparticles are dendrimer nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid particles, cationic lipid cholesterol nanoparticles, cationic lipid cholesterol PEG nanoparticles, ionizable lipids, preferably lipids that are positively charged under physiological conditions, including amino lipids such as MC3 (Dlin-MC3-DMA), C12-200, cKK-E12, ZA3-Ep10, 200-Oi10; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (ALC 0159) N,N-dimyristamide of 2-hydroxyacetic acid (O-PEGylated to a PEG chain of about 2 kilodaltons), (ALC 0315) (4-hydroxybutyl) azadiyl] di (hexane-6,1-diyl) bis (2-hexyldecanoate), Lipofectamine, Trans-IT, InstantFECT, (DOTAP) 1,2-dioleoyl-3-trimethylammonium. Other components may include peptides and polypeptides, such as DP7-C (cholesterol-modified cationic peptide DP7) (VQWRIRVAVIRK, SEQ ID NO: 85), polymers such as polyethylene glycol preparations composed of polymer molecules of different molecular sizes, which can be non-covalently bound or covalently linked to the lipids in the preparation.

[0193] It is also possible to use one or more viral particles for delivery of one or more viral vectors containing the first and / or second nucleic acid construct of the expression system of the present invention.

[0194] Another aspect of the invention is a viral particle comprising the aforementioned viral vector, said viral vector comprising the first and second nucleic acid constructs of the invention. This has the advantage that a single administration of said viral particle is sufficient to express the expression system according to the invention in a patient.

[0195] Another aspect of the present invention is the combination of a first and a second viral particle, wherein the first viral particle comprises a viral vector encoding a first nucleic acid construct, and the second viral particle comprises a viral vector encoding a second nucleic acid construct according to the present invention. This has the advantage that the size of each nucleic acid construct is not limited as when two nucleic acid constructs are combined in one viral vector of a single viral particle. Size limitation is a common problem for viral vectors. For example, adeno-associated virus (AAV) is limited to a size of about 5 kb, but it must be taken into account that ITR, promoters and other elements are not included in the 5 kb.

[0196] The one or more viral particles are preferably AAV or one or more adenoviral particles; more preferably one or more AAV viral particles. As described above, Ad and AAV exhibit several advantages over other viral particles, such as they are well characterized, frequently used, and approved for human treatment. These viruses exhibit other advantages that make them particularly useful in gene therapy, such as they are able to transduce dividing as well as non-dividing cells, and they do not integrate into the host genome.

[0197] In alternative embodiments, the combination of the first expression vector and the second expression vector, or the combination of the first viral particle and the second viral particle is provided as a kit-of-parts.

[0198] Another aspect of the invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more viral vectors or one or more viral particles according to the invention. An advantage of pharmaceutical compositions is that they are "ready-to-use" because they can be directly administered to the patient to be treated. In addition, pharmaceutical compositions are tailored to the needs of treatment to optimize the stability and availability of the ingredients while avoiding unwanted side effects to the patient.

[0199] It is noteworthy that radiotherapy can be applied externally or internally. Those skilled in the art know several methods for providing external radiotherapy. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators sufficient for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. For therapeutic purposes, internal radiotherapy can be provided by administering radioisotopes in pharmacologically acceptable forms and dosages. The embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically limits radioactive deposits so that they can not be moved from the position where they have been deposited. Radioactive deposits can be placed in the gap (interstitial radiotherapy), which is used for example for a considerable percentage of prostate tumors and some head and neck tumors, or radioactive deposits can be placed in the cavity (intracavitary radiotherapy), which is used for example for some uterine, bronchial or esophageal tumors. Nonlocalized radiation therapy can be a localized administration of partial diffusion, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the goal of keeping it close to the injection site rather than being widely distributed in the body, or it can be a completely systemic approach, such as the use of synthetic soluble chemicals with radioisotopes that find their site of action by affinity or biologics such as radiolabeled antibodies to better deliver radiation to the target tumor. A typical example of the latter is iodine-131. While different isotopes may be used for different therapeutic paradigms, isotopes useful in the present invention may preferably be selected from the list consisting of yttrium-90, iodine-131, lutetium-177, iridium-192, astatine-211, bismuth-213, actinium-225, samarium-153, radium-223, phosphorus-32, strontium-89, erbium-169, holmium-166, terbium-161, gallium-68, rhenium-188 or rhenium-186.

[0200] As the inventors have found, both in vitro (see Example 9) and in vivo (see Example

[0201] Example 10) There is a synergistic effect between the canonical STING pathway activated by DMXAA and the atypical STING pathway activated by radiotherapy, which supports the beneficial combination of STING agonist treatment and radiotherapy in step c.

[0202] In addition, the second nucleic acid construct has the advantage that it can be applied systemically and that the expression of at least one immunomodulatory gene or protein can be induced by radiation. Thus, radiation has a dual effect, since it not only promotes the transduction of the virus into the irradiated cells, but also induces the expression of at least one immunomodulatory gene or protein.

[0203] The first effect described above relates to an important concept of the present invention, namely that radiotherapy marks the tumor for transgene expression from a gene vector such as an AAV vector. This fact has very important clinical applications because the data of the present application show that when the AAV vector is administered parenterally (e.g., intravenously), tumor irradiation converts the tumor into a target for transgene (e.g., IL-12) expression. Using this method, the inventors were even able to cure subcutaneous tumors when the vector was administered intravenously. After intravenous administration of AAV.ISREx4.IL12miR122T to animals with subcutaneous tumors, the tumor was eliminated if the tumor had been previously irradiated, but not if it had not received any irradiation. In these experiments, tumor irradiation alone (by intravenous administration of a control virus encoding luciferase) could not induce tumor regression.

[0204] In another aspect, the present invention provides a recombinant expression system for combined gene and radiation therapy of a disease in a recipient mammal, wherein

[0205] a. A recombinant expression system consisting of a nucleic acid construct encoding at least two therapeutic gene sequences, the gene sequences being operably linked to a STING-responsive promoter or a radiation-inducible promoter; and

[0206] b. administering the recombinant expression system to the recipient mammal simultaneously or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression vector with low-dose radiation; and / or

[0207] c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation.

[0208] In a similar aspect, the present invention relates to a method for treating a disease in a recipient mammal using a vector comprising a recombinant expression system, the method comprising:

[0209] a. Providing a recombinant expression system, wherein the recombinant expression system is composed of a gene encoding at least two therapeutic genes

[0210] The therapeutic gene sequence is operably linked to a STING-responsive promoter or a radiation-inducible promoter by virtue of the nucleic acid construct composition of the sequence; and

[0211] b. administering the recombinant expression system to the recipient mammal simultaneously or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system with a low dose of radiation; and / or

[0212] c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation.

[0213] It should be noted that in the case where the recombinant expression system is administered to the recipient mammal before the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system is irradiated with low-dose radiation in step b, the subsequent low-dose radiation is provided by the low-dose radiation of step c. Therefore, the administration of the recombinant expression system in step b is explicitly followed by the low-dose radiation of step c, whereby no additional low-dose radiation is applied in step b. Therefore, in the case, only one low-dose radiation (i.e., the low-dose radiation of step c)) needs to be applied.

[0214] It is noteworthy that the embodiments described below are applicable to the recombinant expression system of this aspect of the invention for combined gene and radiation therapy and methods of treating diseases in recipient mammals using the recombinant expression system.

[0215] It is contemplated that, according to this aspect of the invention, the method of using a combination of gene and radiation therapy or treating a disease in a recipient mammal may be combined with other treatments as anticancer therapies. Several anticancer therapies are known in the art, such as chemotherapy, hormone therapy, radiation therapy, photodynamic therapy, stem cell transplantation, surgery, targeted therapy, hyperthermia or other immunotherapies.

[0216] According to this aspect of the invention, the method of using a combination of gene and radiation therapy or treatment of disease in a recipient mammal provides several advantages over prior art cancer therapies.

[0217] In the nucleic acid construct, therapeutically relevant expression of the therapeutic gene sequence is ensured by placing the transgene under the control of a STING-responsive promoter or a radiation-inducible promoter.

[0218] The STING-responsive promoter is characterized in that it promotes transcription of therapeutic gene sequences only when STING is active in the cell. STING can be activated by a variety of treatment-related means, allowing spatiotemporal control of STING activity in a given tissue or organ. Several STING agonists that allow specific STING activation are known in the art. In addition, STING is known to be activated by encountering DNA damaging attacks such as radiotherapy and by micronuclei that are frequently present in the cytoplasm of malignant cells. Therefore, the expression of therapeutic gene sequences controlled by STING-responsive promoters is associated with different cancer-related stimuli.

[0219] Alternatively, a radiation-inducible promoter may be provided. As used herein, a radiation-inducible promoter is a promoter that induces expression of a therapeutic gene sequence when a cell is irradiated. These promoters may be natural, synthetic or artificially produced, selected from recombinant products of natural promoters. For example, these promoters may contain at least one of the following sequences, either individually or in a repetitive manner:

[0220] a. the eukaryotic radiation-inducible sequence CC(A / T)(6)GG (SEQ ID NO:87), also known as CarG element or [CC(A+T-rich)6GG], such as the sequence of the early growth response-1 (Egr-1) promoter or the pE9 promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:86 or is identical to SEQ ID NO:86 (Egr1 promoter), or alternatively the sequence comprises at least two sequences according to SEQ ID NO:87, wherein the at least two sequences may be separated by additional nucleotides;

[0221] b. a low frequency CCAAT box sequence, such as the CCAAT box sequence of the survivin promoter, preferably the sequence has 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 88 or is identical to SEQ ID NO: 88 (CCAAT-rich domain of the survivin promoter);

[0222] c. cyclin-dependent kinase inhibitor 1 (also known as CDKN1A or p21) promoter;

[0223] d. Late herpes simplex virus 1 promoters, such as glycoprotein C promoter or U S 11 promoter;

[0224] e. Combinations of the above or artificial mutations thereof.

[0225] The expression system consists of a nucleic acid construct encoding at least two therapeutic genes (preferably different immunomodulatory proteins) under the control of a promoter that is activated upon STING activation.

[0226] According to this aspect of the invention, the expression system has the advantage that, at the same time or after systemic or intratumoral injection of the expression system (preferably a vector, more preferably a viral vector), local radiation therapy is applied to the tumor so that the STING-responsive promoter in the tumor can be activated, thereby expressing the at least two therapeutic gene sequences. In addition, local irradiation of the tumor induces endogenous intratumoral STING activity, which then induces the production of type I IFN, chemokines and other cytokines in the tumor by inducing endogenous genes. The local intratumoral production of this combination of immunomodulatory proteins will promote anti-tumor immunity without systemic toxicity.

[0227] It is possible to induce the production of the above combinations of immunomodulatory proteins in tumors by repeated irradiation therapy, either systemically or by injecting the tumor with a long-term expression vector containing a recombinant expression system (such as an adeno-associated virus vector).

[0228] As described above, cancer immunotherapy has several limitations, such as i) poor tumor targeting, ii) weak tumor cell killing ability, iii) laborious and therefore costly, iv) tumor immune escape, v) overstimulation of the immune system that can lead to serious side effects (Tan et al. 2020, Biomed Pharmacother. 124: 109821). The immunotherapy according to this aspect of the invention is a more effective and specific immunotherapy because it is also combined with gene therapy. Therefore, the immunotherapy according to this aspect of the invention is not only immunotherapy itself, but also a combination of immunotherapy and gene therapy, hereinafter referred to as "immunogene therapy" for effectively treating proliferative disorders such as cancer.

[0229] The inventors were surprised to find that the combination of the immunogene therapy comprising viral vectors of the present invention and radiotherapy does not simply add the effects obtained by each individual treatment method, but acts in a synergistic manner because both treatment effects are enhanced when the two therapies are combined. Therefore, the combined therapy according to this aspect of the present invention significantly reduces tumor progression in experimental models. In addition, the therapy results in a reduction in tumor size and even complete remission. The therapy also shows a distant effect because the tumor size of tumors not directly treated is also reduced by the therapy.

[0230] The synergistic effect of this aspect of the invention is manifested in three aspects: 1) radiation therapy performed before treatment with the viral vector increases the efficiency of gene delivery to target cells, thereby increasing the number of cells producing the therapeutic gene, and 2) radiation therapy / multiple radiation therapies given immediately before or after administration of the vector induce expression of the therapeutic gene sequence by activating the STING responsive promoter, and 3) the therapeutic gene expressed by the viral vector enhances the immunostimulatory effect initiated by the radiation therapy. Therefore, as also shown in the examples of the present application, the effect of the treatment is multiplied, rather than simply additive.

[0231] As a potential mechanism for this synergistic effect, it is hypothesized that radiation therapy given just before vector administration leads to activation of the DNA damage response (which lasts for several days, (Zhao Z et al., Genomics 2020; 112: 1309-1317]) and the presence of cytoplasmic DNA in irradiated cells. All these changes maintain STING activation for a period of time. For this reason, activation of STING-responsive promoters also occurs when radiation therapy is given immediately before vector administration.

[0232] The system of the present invention allows for the expression of therapeutic gene sequences in a highly localized manner as desired, thereby avoiding the adverse aspects of systemic immunotherapy known in the art, such as toxicity.

[0233] In addition, the inventors have found that the administration of a recombinant expression system (preferably a viral vector) and the expression in combination with radiotherapy according to this aspect of the invention is non-toxic and shows less side effects in vivo. It is noteworthy that the inventors have found that when administered in vivo, the administration of a recombinant expression system (preferably a viral vector) and the expression in combination with radiotherapy lead to tumor reduction, ultimately leading to complete remission. Even, by locally administering the recombinant expression system (preferably a viral vector) of this aspect of the invention in a tumor, not only the size of the target tumor is reduced, but also non-target tumors show a significant size reduction, also known as the abscopal effect.

[0234] The recombinant expression system of this aspect of the invention generates specific and basal immune pathways in transfected cancer cells that utilize otherwise endogenous pathway components.

[0235] It is noteworthy that the term "recombinant expression system" includes any nucleotide, its analog and its polymer. Therefore, any recombinant expression system disclosed herein refers to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule, and therefore include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. A more comprehensive definition is included in the definition section of this application.

[0236] One component of the approach is an artificial gene construct that includes at least two therapeutic gene sequences operably linked to a STING-responsive promoter. The therapeutic gene sequences can encode immunomodulatory proteins that activate innate and adaptive immune responses to generate a specific cancer immune response.

[0237] The recombinant expression system according to this aspect of the invention allows for spatiotemporal expression of therapeutic gene sequences in accordance with STING activity. This is achieved by operatively linking the therapeutic gene sequence to a STING responsive promoter. One of the several ways to activate STING is to expose cells to ionizing radiation that produces DNA damage that is recognized by the cGAS / STING pathway, inducing expression of STING downstream targets such as type I interferons or inflammatory cytokines.

[0238] Ionizing radiation is widely used to treat diseases. The most prominent use is to treat cancer, which is a proliferative disease characterized by rapid division of cancer cells. The DNA of cells receiving ionizing radiation is damaged, thereby inhibiting cell division, or killing cells in the case of excessive DNA damage. Therefore, the initial low-dose radiation of tissues or organs as described in b) of this aspect of the invention has a dual effect: i) low-dose radiation damages the DNA of target cells, thereby acting in its original manner, and ii) low-dose radiation enhances cell transduction through AAV vectors. This is so because radiotherapy triggers the DNA damage response (DDR), which, among other effects, promotes the conversion of AAV single-stranded DNA to transcriptionally active double-stranded DNA (Peng D. et al. J. Hepatol. 2000; 32: 975-85). As a result, the number of target cells expressing the transgene delivered by the viral vector increases.

[0239] On the other hand, DNA damaging agents have been shown to increase cytoplasmic DNA in tumor cells (Li, C et al. Molecular Immunology 2021; 131, 180-190) subsequently leading to STING activation (Deng, L et al., Immunity 2014; 41, 843-852; Dunphy et al. Molecular Cell, 2018; 71, 745-760). Therefore, radiotherapy before intratumoral injection of AAV vectors will lead to enhanced tumor transduction by AAV vectors and STING activation. The latter effect will promote the production of immunomodulatory proteins expressed by the vector under the control of the STING response promoter.

[0240] The low-dose radiation described in c) of this aspect of the invention again plays a dual role. First, the low-dose radiation damages the DNA of the target cells, thereby acting in its original manner. Secondly, the DNA damage causes activation of the cGAS / STING pathway, thereby inducing the expression of the therapeutic gene sequence delivered by the recombinant expression system (preferably a viral vector). Therefore, the spatiotemporal expression of the therapeutic gene sequence can be controlled by local low-dose radiation, although other measures can also be taken to induce STING activity.

[0241] In general, low-dose radiation and immunogene therapy work synergistically because each therapy has an effect on its own, however, as described above, when the therapies interact, the effect is multiplied, rather than simply added. This is also demonstrated in vitro (see Example 9) and in vivo (see Examples 10-15, 18). Therefore, the combination of immunogene therapy and radiation therapy according to this aspect of the invention is an effective therapy for treating diseases in recipient mammals.

[0242] In another preferred embodiment, the recombinant expression system for use according to this aspect of the invention is used in combination therapy with at least one other therapy selected from the group consisting of: a therapy comprising at least one check point inhibitor, a therapy comprising at least one adoptive cell therapy, a therapy comprising at least one cancer vaccine and / or a therapy comprising at least one exogenous cytokine.

[0243] The inhibitor of the therapy including at least one checkpoint inhibitor can be selected from the group consisting of: inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR, more preferably selected from the group consisting of: inhibitors of PD-1, PD-L1, PD-L2 or CTLA-4. Immune checkpoints are molecules expressed on T cells or cells that interact with T cells, which help regulate immune responses and prevent excessive or prolonged immune activation. However, cancer cells can use these checkpoint proteins to escape detection by the immune system. By blocking these checkpoint proteins, immune checkpoint inhibitors can release the brakes on the immune response and allow the immune system to attack and destroy cancer cells. In the context of this aspect of the invention, it is particularly advantageous to use immune checkpoint inhibitors as additional therapy because the expression system comprising at least two therapeutic gene sequences causes T cells to infiltrate the tumor environment. Since the function of these T cells may be impaired due to immune checkpoints, methods to overcome this obstacle can greatly benefit treatment outcomes.

[0244] The therapy including at least one adoptive cell therapy can be adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using tumor infiltrating leukocytes (TIL). In some cases, tumor silences the expression of MHC class I molecules and / or IFN-γ signaling molecules as a means of escaping T cell immunity. These problems can be avoided using adoptive cell therapy because these adoptive cells are generally able to identify and target tumor cells. Therefore, a synergistic effect can be expected in a combined approach.

[0245] Furthermore, combining the expression system of this aspect of the invention with a cancer vaccine or exogenous cytokine used according to this aspect of the invention can produce a synergistic effect because the recruited T cells are directed toward cancer cells by the cancer vaccine or exogenous cytokine.

[0246] Advantageously, in the context of this aspect of the present invention, it can be specified that the disease is selected from the group consisting of: cancer, autoimmune diseases, inflammatory diseases or infectious diseases. For example, it may be feasible to express an immunomodulator by an expression system. As used herein, the term "immunomodulator" refers to an agent that regulates (e.g., stimulates) the immune system (which can then be used to treat a disease, preferably a proliferative disease such as cancer). Immunomodulators can cause stimulation of the immune system, which is typically used for immunotherapy targeting cancer. However, other diseases such as autoimmune diseases, diabetes, cardiovascular diseases and inflammation can also be treated with immunomodulators. For example, IFN-β is used to treat multiple sclerosis. In addition, it has been shown that IL-2 regulates the balance of regulatory T cells and effector T cells, thereby maintaining the function of β cells. Immunotherapy can be used to treat non-cancer diseases, such as lupus, rheumatoid arthritis, and diabetes.

[0247] The cancer may also be a solid cancer selected from the group consisting of bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); hematopoietic tumors of the lymphoid lineage including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; brain cancer, central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; thymic cancer, including fibrosarcoma, rhabdomyosarcoma and Tumors of mesenchymal origin including osteosarcoma; other tumors including melanoma, pigmented dysderma, keratoacanthoma, seminoma, thyroid follicular carcinoma and teratoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, multiforme glioma Blastoma, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colon cancer, esophageal cancer, lung cancer, mesothelioma, multiple myeloma, Merkel cell carcinoma, skin cancer, testicular cancer, uterine cancer, plasma cell tumors and head and neck cancer, gastric cancer, germ cell tumors, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, nasal sinus natural killer cells, tumors, plasma cells; neuroendocrine tumors, bile duct cancer or carcinoid tumors.

[0248] Preferably, the solid cancer is selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, neuroendocrine tumors, bile duct cancer or carcinoid tumor.

[0249] Essentially, every disease that can be treated by radiotherapy is a disease suitable for targeting by combination therapy according to this aspect of the invention, since the synergistic effects of enhanced gene delivery and induction of expression of the therapeutic gene sequence are universally applicable and also combined with the effects of radiotherapy alone. Thus, non-cancer diseases such as arthrosis, Dupuytren's disease, Ledderhose disease, calcaneal spurs, tennis elbow, vertebral hemangioma, vertebral hemangioma, chronic synovitis, pigmented villonodular synovitis (PVNS), chronic joint effusions, rheumatoid arthritis and active joint diseases (each of which can be treated by radiotherapy) can also be particularly easily treated by combination therapy according to this aspect of the invention.

[0250] Within the scope of this aspect of the invention, it is also envisioned that the therapeutic gene sequence of the nucleic acid construct encodes a protein selected from the group consisting of: cytokines such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones.

[0251] In a preferred embodiment, the therapeutic gene sequence encodes an immunomodulatory protein.Similar to the term "immunomodulator", the term "immunomodulatory protein" refers to a protein that modulates (eg, stimulates) the immune system.

[0252] A non-limiting list of therapeutic proteins representing immunomodulatory proteins that can be used in the nucleic acid construct of the recombinant expression system of this aspect of the invention, preferably in the form of a viral vector, includes the following proteins: interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, single-chain interleukin-12, interleukin-12, interleukin-13, Interleukin-14, interleukin-15, interleukin-15-sushi, interleukin-16, interleukin-17, interleukin-18, interleukin-19, interleukin-20, interleukin-21, interleukin-22, interleukin-23, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL 14. CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, C XCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, C XCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, granulocyte-macrophage colony stimulating factor, type I interferon, IFN-γ, TNF-α, FLT3-ligand, blocking peptides targeting TGF-β, blocking peptides targeting IL-10, blocking peptides targeting FoxP3, monoclonal antibodies or single-chain variable fragments (scFv) or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3, and fragments or variants thereof.

[0253] Those skilled in the art will recognize that any protein that slows, stops or reverses disease progression or cures disease is a suitable molecule that can be encoded by one of the therapeutic gene sequences of the nucleic acid construct of this aspect of the invention.

[0254] The protein encoded by the therapeutic gene sequence of the nucleic acid construct of the recombinant expression system of this aspect of the invention is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10, single-chain interleukin-12 (scIL-12), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-s ushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony stimulating factor (GM-CSF), type I interferon (IFN-α and IFN-β), IFN-γ, TNF-α, CXCL10, FLT3 ligand; immunostimulatory monoclonal antibodies or single-chain variable fragments (scFv) or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

[0255] It is known in the art that several immunomodulatory proteins are used for immunotherapy for cancer treatment. For example, IL-2 is a protein that has been used for immunotherapy in cancer treatment for more than 20 years. However, only a very small number of patients respond to IL-2-related immunotherapy, and these therapies are known to cause serious side effects. IL-12 reprograms myeloid-derived suppressor cells (MDSC) into efficient antigen presenting cells (APC). In addition, IL-12 strongly stimulates T cells and NK cells and increases the production of IFN-γ, thereby inducing a Th1-type response, which can control the growth of the treated tumor and distant metastasis (Lasek et al. Cancer Immunol. Immunother. 2014; 63: 419). In addition, IL-12 has an anti-angiogenic effect, which can impair tumor vascularization, thereby limiting tumor growth. IL-12 is a heterodimeric cytokine encoded by two independent genes. In order to promote its expression in a recombinant expression system, it is feasible to use a recombinant single-chain IL-12 (scIL-12) transgene encoding two subunits of IL-12. Thus, in a preferred embodiment, one therapeutic gene sequence of the nucleic acid construct encodes IL-12.

[0256] In one embodiment, one of the immunomodulatory proteins is a complex or fusion protein of IL-15 with the Sushi domain of the IL-15 receptor α chain to enhance the agonist activity of IL-15 through trans expression such as provided by compound P22339 (Hu, Q., Ye, X., Qu, X. et al., Sci Rep 2018, 7675).

[0257] Optionally, IL-12 may be selected from the group consisting of: an α subunit of IL-12, a β subunit of IL-12, a single-chain IL-12 comprising an α and β subunit of IL-12. In the specification of the present invention, single-chain IL-12 (scIL-12) is defined as a fusion protein comprising α- and β, wherein the peptide sequence is either directly connected to a linker peptide or separated by a linker peptide, wherein the linker peptide may have a length ranging from 1 to 100 amino acids. Preferably, the length of the linker ranges from 4 to 60 amino acids, more preferably from 8 to 40 amino acids, and particularly preferably from 12 to 25 amino acids. The length of the linker can be 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids. In addition, IL-12 can be a single-chain IL-12 (human or mouse single-chain IL-12) comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The sequence identity of the polypeptide sequence to SEQ ID NO: 9 or 10 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have the biological activity of interleukin-12, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-12 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 9 or 10.

[0258] The biological activity of IL-12 can be assessed in vitro by the ability of the peptide to increase IFN-γ production, which is produced by cells expressing the IL-12 receptor and the corresponding signaling pathway, such as peripheral blood mononuclear cells or human macrophage cell lines. Alternatively, a reporter cell line can be used as, for example, a Promega bioluminescent IL-12 bioassay (Catalog No. JA2601, JA2605).

[0259] In vivo IL-12 bioactivity can be assessed by its ability to elevate IFN-γ serum levels.

[0260] In addition, one of the immunomodulatory proteins may be IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β. The sequence identity of the polypeptide sequence to SEQ ID NO: 79 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have IFN-β biological activity, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of IFN-β assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 79.

[0261] The biological activity of IFN-β can be estimated by the ability of inducing type I IFN response genes. For this purpose, human cell lines (such as HEK 293) can be incubated with polypeptides, and the expression of IFN response genes can be measured by RT-qPCR. Alternatively, IFN reporter cells encoding luciferase under the control of a promoter containing ISRE can be used to assess the IFN-β biological activity of the polypeptide, which is carried out by measuring the expression of luciferase in a luminometer 3h after the polypeptide is added to the cell culture.

[0262] In vivo IFN-β bioactivity can be assessed by induction of IFN response genes in peripheral blood mononuclear cells (RT-qPCR).

[0263] In addition, one of the immunomodulatory proteins may be IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL- 15. The sequence identity of the polypeptide sequence to SEQ ID NO: 80 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have the biological activity of interleukin-15, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of interleukin-15 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 80.

[0264] The biological activity of IL-15 can be assessed in vitro by a CTLL-2 cell proliferation assay.

[0265] In vivo IL-15 bioactivity can be assessed by its ability to induce NK cell and T cell proliferation using FACS analysis.

[0266] In addition, one of the immunomodulatory proteins may be CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10. The sequence identity of the polypeptide sequence to SEQ ID NO: 81 may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In the context of the present invention, for a polypeptide to be considered to have the biological activity of CXCL10, it must have at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50% of the biological activity of CXCL10 assessed using one of the biological activity tests described below, compared to the biological activity of the polypeptide according to SEQ ID NO: 81. CXCL10 is also known as IP-10.

[0267] The biological activity of CXCL10 can be assessed by its ability to attract T lymphocytes and natural killer cells in vivo or in vitro migration assays.

[0268] In a preferred embodiment, the nucleic acid construct of this aspect of the invention comprises two or more sequences, each sequence encoding a different immunomodulatory protein, wherein the first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity relative to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12. The second immunomodulatory protein may be IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO:79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:79, wherein the polypeptide has the biological activity of IFN-β; or the second immunomodulatory protein may be IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO:80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:80, wherein the polypeptide has the biological activity of IL-15-sushi; or the second immunomodulatory protein may be CXCL10 comprising or consisting of the amino acid sequence of SEQ ID NO:81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:81. NO:81. A polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity, wherein the polypeptide has the biological activity of IL-15-sushi.

[0269] In addition, other combinations of immunomodulatory proteins are also feasible. Single-chain IL-12 can be the first immunomodulatory protein, and the second immunomodulatory protein can be selected from the group consisting of: a peptide that blocks TGF-β, a peptide that blocks FoxP3, or a monoclonal antibody against the immune checkpoint inhibitor PD1 or PD-L1, or a nanobody against the immune checkpoint inhibitor PD1 or PD-L1.

[0270] It should be noted that in the context of a nucleic acid comprising two immunomodulatory proteins, the terms first and second immunomodulatory proteins do not describe the order of the coding sequences on the nucleic acid, but are only used to illustrate two different immunomodulatory proteins. Therefore, the coding sequence of the second immunomodulatory protein can be located upstream of the coding sequence of the first immunomodulatory protein on the nucleic acid. Here, upstream means that the coding sequence of the second immunomodulatory protein is located before the coding sequence of the first immunomodulatory protein in the 5' to 3' direction on the nucleic acid.

[0271] TGF-β blocking peptides are peptides that interfere with TGF-β itself or interfere with the receptors that bind TGF-β, so that at least some TGF-β signaling is reduced or even completely prevented. Examples of TGF-β blocking peptides are P17 and P144.

[0272] A FoxP3 blocking peptide is a peptide that interferes with FoxP3 itself or with a receptor that binds FoxP3, such that at least some FoxP3 signaling is reduced or even completely prevented. An example of a FoxP3 blocking peptide is P60.

[0273] In another preferred embodiment, the second nucleic acid construct is a bicistronic or multicistronic expression cassette comprising two or more therapeutic gene sequences, each of which encodes a different immunomodulatory gene or protein, or a protein or RNA with anti-cancer properties, wherein the sequences are separated by a sequence encoding an IRES; and / or the sequences are separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide. The IRES sequence and the 2A peptide coding sequence are translational regulatory elements (TRE).

[0274] In the present specification, a protein or RNA having anti-cancer properties is any such molecule that is capable of delaying, stopping or reversing the progression of cancer.

[0275] A bicistronic or polycistronic expression cassette is a genetic construct that contains two or more independent coding regions or genes in a single transcript. Generally, it contains two or more different sequences in a single transcript. In order to allow independent translation of two or more immunomodulatory proteins, a TRE is required.

[0276] One possible solution is to use an internal ribosome entry site (IRES). An IRES is a specific RNA sequence in a transcript that allows translation to be initiated at non-AUG codons, bypassing the normal cap-dependent initiation process. This mechanism allows regulation of gene expression and co-expression of multiple proteins from a single mRNA molecule.

[0277] Another possible solution is provided by 2A peptides (also referred to as 2A self-cleaving peptides). 2A peptides are a class of 18-22aa long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DxExNPGP (SEQ ID NO:32) and are present in a wide range of viral families. They help produce polyproteins by causing ribosomes to be unable to form peptide bonds. Due to their short length, 2A peptides are very suitable for viral vector systems with limited capacity, such as AAV. Another advantage of these peptides is that they allow equimolar expression of the coding sequences separated by these peptides. Members of the 2A peptide family are T2A, P2A, E2A and F2A. The flanks of these peptides can have linker peptides of different lengths, which can lead to improved functionality. For example, the F2A element with a linker domain composed of the amino acid sequence GGSG (SEQ ID NO:82) in front shows significant expression of proteins separated by the element.

[0278] Table 3: Examples of combinations of two immunomodulatory proteins separated by a translational regulatory element (TRE) in a bicistronic vector

[0279] <![CDATA[1 st Immunomodulatory proteins]]> TRE <![CDATA[2 nd Immunomodulatory proteins]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[IFN-B B ]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > IRES <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > IRES FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > IRES TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > IRES Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > IRES Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > IRES Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > IRES Nanobodies targeting PD-L1 <![CDATA[IFN-B B ]]> IRES <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IFN-B B ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > P2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > P2A FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > P2A TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > P2A Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > P2A Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > P2A Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > P2A Nanobodies targeting PD-L1 <![CDATA[IFN-β B ]]> P2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IFN-B B ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[IL-15-sushi C ]]> <![CDATA[Single-chain IL-12 A > F2A <![CDATA[CXCL10 D ]]> <![CDATA[Single-chain IL-12 A > F2A FoxP3 blocking peptide <![CDATA[Single-chain IL-12 A > F2A TGF-β blocking peptide <![CDATA[Single-chain IL-12 A > F2A Monoclonal antibodies against PD1 <![CDATA[Single-chain IL-12 A > F2A Monoclonal antibodies targeting PD-L1 <![CDATA[Single-chain IL-12 A > F2A Nanobodies targeting PD1 <![CDATA[Single-chain IL-12 A > F2A Nanobodies targeting PD-L1 <![CDATA[IFN-B b ]]> F2A <![CDATA[IL-15-sushi C ]]>

[0280] A - preferably human single chain IL-12 as described in SEQ ID NO: 9

[0281] B - preferably human IFN-β as set forth in SEQ ID NO: 79

[0282] C-preferably human IL-15-sushi as described in SEQ ID NO: 80

[0283] D-preferably human CXCL10 as set forth in SEQ ID NO: 81

[0284] Immunotherapy involving systemic availability of immunomodulatory proteins usually causes unwanted side effects. However, the advantageous aspect of this aspect of the present invention is that the recombinant expression system used herein induces local expression of therapeutic gene sequences, which can be immunomodulatory proteins, thereby inducing highly local and targeted immune responses. For example, intratumoral administration of the recombinant expression system (which is a preferred embodiment of this aspect of the present invention) induces an immune response by producing immunostimulatory cytokines such as interleukins or chemokines in the tumor mass. Without wishing to be bound by theory, it is believed that the release of immunomodulatory proteins in or near the tumor causes T cells to infiltrate near the tumor, and can result in the recognition of tumor cells that usually escape the recognition of the immune system, thereby enabling the immune system to target and kill these cells. Therefore, the progression of the tumor is at least delayed, preferably stopped, and more preferably reversed.

[0285] In a preferred embodiment, the therapeutic gene sequence of the nucleic acid construct of the recombinant expression system of this aspect of the invention encodes an immunomodulatory protein, more preferably an immunostimulatory protein. These are proteins that stimulate the immune system by directly stimulating the immune system (e.g., IL-12) or by inhibiting immunosuppressive molecules (e.g., blocking peptides targeting TGF-β).

[0286] According to a further possibility, the nucleic acid construct may be provided with a STING responsive promoter operably linked to a therapeutic gene sequence, the promoter being selected from the group consisting of an IFN-β responsive promoter (preferably according to SEQ ID NO: 25), an IFN-β minimal promoter (preferably according to SEQ ID NO: 31), a CXCL9 responsive promoter (preferably according to SEQ ID NO: 23), a CXCL10 responsive promoter (preferably according to SEQ ID NO: 24), a STAT motif promoter (preferably according to SEQ ID NO: 26), an IRSF3 motif promoter (preferably according to SEQ ID NO: 27 or SEQ ID NO: 28), an ISRE motif promoter (preferably according to SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13), a NFκB motif promoter (preferably according to SEQ ID NO: 29 or SEQ ID NO: 30) and a STAT6 motif promoter. In a preferred embodiment, the STING responsive promoter comprises at least one interferon stimulated response element (ISRE).

[0287] According to this aspect of the invention, by effectively linking the expression of the therapeutic gene sequence to the activity of STING using a STING-responsive promoter, targeted and localized expression of the therapeutic gene sequence can be achieved, thereby avoiding potential side effects caused by systemic expression of the transgene encoded by the nucleic acid construct.

[0288] Optionally, the STING responsive promoter of the nucleic acid construct of the recombinant expression system of this aspect of the invention may be selected from the list consisting of:

[0289] a. a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11;

[0290] b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart;

[0291] c. A nucleotide sequence according to SEQ ID NO: 12, comprising four copies of ISRE separated by 20 nucleotides;

[0292] d. The nucleotide sequence according to SEQ ID NO: 13, comprising four copies of ISREs separated by 6 nucleotides.

[0293] The use of a STING responsive promoter in the context of this aspect of the invention ensures controlled and targeted expression of therapeutic gene sequences. In addition, the aforementioned STING responsive promoter has been shown to have additional advantageous aspects. In particular, a promoter carrying four copies of an ISRE as described in SEQ ID NO: 12 is highly inducible while also exhibiting high specificity.

[0294] Preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. does not contain an AP-1 site or an NFκB site. Even more preferably, the nucleotide sequence of the STING-responsive promoter described in a. and b. does not contain any other motifs that contribute to the inducibility, leakiness or expression efficiency of the promoter. In other words, the nucleotide sequence of the promoter described in a. or b. may comprise several nucleotides before, between and / or after the ISRE of SEQ ID NO: 11, wherein the nucleotides are selected in such a way that these additional nucleotides do not form any transcriptional regulatory elements (e.g. randomly). Particularly preferably, the nucleotide sequence of the STING-responsive promoter as described in a. and b. consists of 1-10 copies, preferably 2-7 copies, more preferably 3-5 copies, and particularly preferably 4 copies of a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity relative to SEQ ID NO: 11 or identical to SEQ ID NO: 11.

[0295] Furthermore, the nucleotide sequences of the STING-responsive promoters described in a. to d. may already represent functional promoters, and thus these promoters do not require additional elements to induce expression of an operably linked transgene.

[0296] It is known that some viral particles, especially AAV, show tropism for hepatocytes. This means that when the viral particles enter the blood circulation system, they preferentially transduce hepatocytes, ultimately resulting in the expression of the encoded transgene in the liver. Likewise, in the case of intratumoral injection of the viral particles, some of these particles will enter the blood circulation system, since tumors are highly vascularized. In order to prevent the unwanted expression of the transgene encoded by the viral particles in the liver, it is therefore appropriate to control its expression. Several methods are known to those skilled in the art to achieve this goal.

[0297] In general, a suitable method for restricting transgene expression to a specific tissue or a specific cell type is to use a viral particle with a distinct tropism for the tissue or cell type. A method for achieving this is by changing the serotype of the AAV or by changing the capsid of the AAV. Several AAV serotypes are known in the art, each with a different tropism. However, since the present invention is intended to target several different types of cancer, it is necessary to optimize the serotype or capsid for each cancer or tumor type. Nevertheless, this may be a suitable way to achieve tissue or cell type specific expression of a transgene.

[0298] The skilled person may also use DNA or RNA binding repressors to avoid unwanted expression of the transgene encoded by the viral particle in tissues or cells that are not intended to express the protein, preferably the liver. Thus, the second nucleic acid construct may comprise an operator that binds a repressor. The repressor may be operably linked to a promoter that is active in hepatocytes, thereby inducing expression of the repressor in these cells, thereby inhibiting expression of the transgene.

[0299] Another possibility is to use RNA interference (RNAi). RNAi is characterized in that an RNA molecule reduces or suppresses the expression of a nucleic acid sequence that has a substantial or complete homology with the RNA molecule. Non-limiting examples of RNAi are small interfering RNA, small hairpin RNA or microRNA (miR). In order to use RNAi to reduce or suppress transgenic expression in a particular tissue, it is conceivable that, for example, an shRNA targeting the transgenic nucleotide sequence can be effectively connected to a tissue-specific promoter. When the shRNA is applied, it therefore reduces or suppresses transgenic expression in the tissue. Although this method is feasible in the context of this aspect of the invention, it may be disadvantageous because the shRNA must be additionally applied to limit transgenic expression in target tissues such as the liver.

[0300] A good way to avoid this shortcoming is to effectively link the miRNA target sequence to the nucleotide sequence of the transgene. Therefore, in the tissue where there is a miRNA associated with the miRNA target sequence, the expression of the transgene will be reduced or inhibited. Therefore, a preferred embodiment of this aspect of the invention includes a target sequence whose sequence motif is a microRNA that is highly abundant in the liver. Therefore, a single administration of the nucleic acid construct is sufficient to avoid transgene expression in the liver, while transcription in other tissues or organs is not weakened.

[0301] According to another possibility, it can be provided that the nucleic acid construct of the recombinant expression system comprising at least two therapeutic gene sequences also comprises at least one sequence motif, which inhibits the transgenic expression of the therapeutic gene sequences in tissues or cells that are not intended to express the protein, wherein the tissue is preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

[0302] Optionally, it is envisioned that the microRNA highly abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c or let7f. In addition, the microRNA miR-122 abundant in the liver can be advantageous. The advantage of miR-122 is that it is highly expressed in the liver, and more importantly, specifically expressed in the liver. Therefore, by selecting the miR-122 target sequence, a significant reduction of the transgene in the liver is ensured, while the expression in other tissues is not weakened. In addition, it has been shown that miR-122 is downregulated in most liver tumors, so the recombinant expression system according to this aspect of the invention is also suitable for treating liver cancer.

[0303] According to a preferred embodiment of this aspect of the invention, the corresponding miR-122 target sequence is a miR-122 target having at least 90% or at least 95% sequence identity relative to SEQ ID NO: 14 or being identical to SEQ ID NO: 14. Preferably, the target sequence comprises 1 to 12, preferably 3 to 10, more preferably 4 to 8, and particularly preferably 5 copies of the miR-122 target sequence as described in the previous sentence. Increasing the number of copies of the miR-122 target sequence increases miR-mediated reduction, while no significant improvement is observed for numbers higher than the disclosed 5 copies. In a preferred embodiment, the copies are separated by 1 to 40 nucleotides, preferably 1 to 30 nucleotides, more preferably 2 to 20 nucleotides, and particularly preferably 2 to 8 nucleotides.

[0304] Preferably, the nucleotide sequence comprises 5 copies of the miR-122 target sequence as set forth in SEQ ID NO: 15. As described in the Examples of this application, the nucleotide sequence set forth in SEQ ID NO: 15 has been shown to substantially inhibit transgene expression in the liver, resulting in undetectable plasma levels of the transgene by the method described in Example 4.

[0305] It can be provided that the low-dose radiation is based on photon radiation such as X-rays or gamma rays, or particle radiation, wherein the particles of the particle radiation are preferably selected from the group consisting of beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions. Each type of radiation has its own characteristics and characteristic spectrum of therapeutic applications. A person skilled in the art selects the corresponding radiation based on the disease itself, the location and the progression of the disease.

[0306] Optionally, it is contemplated that low dose radiation may be administered with a total radiation dose of 100 Gy or less, preferably 40 Gy or less, and more preferably 25 Gy or less. The total radiation dose may be less than 100 Gy, 96 Gy, 92 Gy, 88 Gy, 84 Gy, 80 Gy, 76 Gy, 72 Gy, 68 Gy, 64 Gy, 60 Gy, 56 Gy, 52 Gy, 48 Gy, 44 Gy, 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 In some embodiments, the dosage of 40Gy is 100Gy, ...

[0307] There are several advantages to using low-dose radiation. First, radiation therapy is always accompanied by side effects. By using low-dose radiation, these side effects are significantly reduced. Second, there is a limit to the amount of radiation you can safely receive in a part of your body throughout your lifetime, the so-called lifetime limit dose. Using low-dose radiation is not only more likely to stay below this limit, but also allows for a higher number of radiation treatments. In the context of this aspect of the invention, this is particularly useful because it allows for repeated expression of therapeutic gene sequences under the control of the STING-responsive promoter. Third, low-dose radiation enhances the susceptibility of cells to gene delivery. AAV is a single-stranded DNA vector that needs to be converted to double-stranded DNA to form a transcription template. Not wanting to be constrained by therapy, it is believed that the DNA damage response caused by radiation therapy helps convert single-stranded DNA into double-stranded DNA. Using radiation therapy to enhance tumor transduction through AAV vectors is an attractive option because radiation therapy is often used to treat solid tumors. Published data on AAV transduction and radiation involve in vitro studies using different radiation doses and in vivo studies using high radiation doses. However, high radiation doses can cause vascular damage and mark changes in tissue structure. Thus, relatively low radiation doses preserve tumor vasculature, thereby maintaining lymphocyte trafficking to the tumor.

[0308] According to another possibility, the low-dose radiation may be administered in the form of stereotactic radiosurgery (SRS), intraoperative radiotherapy (IORT), stereotactic body radiotherapy (SBRT), single-dose radiotherapy (SDRT), intensity-modulated radiotherapy (IMRT), image-guided radiotherapy (IGRT), stereotactic ablative radiotherapy (SABR) or hypofractionated radiotherapy preferably delivered by a linear accelerator (LINAC) machine, a gamma knife machine or proton beam therapy.

[0309] The advantage of using the above-mentioned administration methods is that they allow high-precision targeting of target tissues or organs. In addition to limiting unwanted side effects from radiation, transgenic expression is also induced at specific sites. In the context of this aspect of the invention, this limits the expression of the target gene sequence to a clearly defined region, thereby avoiding the systemic availability of the transgene and thus avoiding unwanted side effects. As described in the following examples, although the target tumor receives radiotherapy combined with immunogene therapy, other non-target tumors also show a reduction in size (remote effect). Therefore, by using high-precision radiotherapy, not only side effects are reduced, but also distant tumors are still affected by treatment.

[0310] Advantageously, in the context of this aspect of the invention, the time interval between the low-dose irradiation of b. and the subsequent administration of the viral vector may be between 1 minute and 20 days, preferably between 10 minutes and 2 days, more preferably between 30 minutes and 24 hours. The time interval may be any time between 1 minute and 20 days. The subsequent administration of the viral vector may start at the earliest 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days or 19 days after receiving the low-dose radiation. This ensures enhanced gene delivery and increased expression of the transgene in the target tissue or organ.

[0311] Advantageously, in the context of this aspect of the invention, it can be specified that the time interval between the administration of the viral vector and the subsequent low-dose irradiation of c. is between 2 hours and 30 days, preferably between 1 day and 5 days, more preferably between 1 day and 3 days. The time interval can be any time between 2 hours and 30 days. The subsequent low-dose radiation begins at the earliest 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days after the administration of the viral vector. Subsequently, the low-dose radiation can be repeated at intervals between 1 day and 30 days. Subsequent low-dose radiation may begin as early as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, and 29 days after the previous dose of radiation.

[0312] According to a further possibility, it can be provided that the nucleic acid construct comprising the therapeutic gene sequence also comprises a transgene encoding thymidine kinase (preferably thymidine kinase according to SEQ ID NO: 16). The nucleotide sequence encoding thymidine kinase is connected to the nucleotide sequence of the therapeutic gene sequence and can be separated by a 2A self-cleaving peptide (2A peptide). 2A peptides constitute a class of 18-22 amino acid long peptides that can induce ribosome skipping during intracellular protein translation. These peptides share the core sequence motif of DXEXNPGP and are present in a wide range of viral families. They help produce polyproteins by causing the ribosome to be unable to form peptide bonds. Those skilled in the art recognize that the use of 2A peptides allows the co-expression of therapeutic gene sequences and thymidine kinase. The co-expression of thymidine kinase is advantageous because it allows tumor transduction to be assessed by PET imaging, and in the case of potential IL-12-mediated toxicity, cells expressing cytokines are eliminated by administering thymidine kinase substrates (e.g., acyclovir, penciclovir, famciclovir or ganciclovir).

[0313] The nucleic acid construct (preferably a viral vector) of the recombinant expression system of this aspect of the invention may also contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). WPRE is known to increase the expression of a transgene and is therefore most effective when placed downstream of the transgene (i.e., the therapeutic gene sequence) and close to the polyadenylation signal.

[0314] Conveniently, the nucleic acid construct (preferably a viral vector) of the recombinant expression system of this aspect of the invention further comprises a polyadenylation signal located 3' relative to the therapeutic gene sequence. The length of the poly(A) tail can be optimized to stabilize mRNA and subsequently increase protein translation.

[0315] The skilled person is also aware of other methods for increasing protein translation, such as

[0316] a. by sequence modification to optimize G / C content and / or overstabilizing secondary structure codons, or

[0317] b. alteration of the 5′ cap by addition of synthetic or natural 5′ end cap analogs such as m7GpppG or initiator trimers such as “CleanCap”, or stabilization by capping enzymes, or

[0318] c. Adding regulatory elements

[0319] c.1 in the 5′-untranslated region (UTR), such as by optimizing or modifying the length and sequence to avoid overly stable structures through appropriate cis-regulatory sequences, or

[0320] c.2 In the 3′-UTR, stable elements or repetitive motifs or synthetic or natural sequences are introduced by sequence modification, which have been shown to be effective in other mRNAs.

[0321] In a specific embodiment, the recombinant expression system of this aspect of the invention consists of the nucleic acid construct given in the table below, wherein the different elements are shown in the 5′ to 3′ direction as a promoter, a first open reading frame (ORF1), a first translation regulatory element (TRE1), a second open reading frame (ORF2), a second translation regulatory element (TRE2) and a target sequence for a microRNA (microRNA element).

[0322] Table 4: Embodiments of nucleic acid constructs of expression systems

[0323] Promoter ORF1 TRE1 ORF2 TRE2 microRNA elements <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A <![CDATA[IFN-β C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A <![CDATA[IL-15-sushi D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A FoxP3 blocking peptide WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A TGF-β blocking peptide WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A Monoclonal antibodies against PD1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A Monoclonal antibodies targeting PD-L1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A Nanobodies targeting PD1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> P2A Nanobodies targeting PD-L1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[IFN-B C ]]> P2A <![CDATA[II-15-sushi D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES <![CDATA[IFN-B C ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES <![CDATA[IL-15-sushi D ]]> WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES FoxP3 blocking peptide WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES TGF-β blocking peptide WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES Monoclonal antibodies against PD1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES Monoclonal antibodies targeting PD-L1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES Nanobodies targeting PD1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[scIL12 B ]]> IRES Nanobodies targeting PD-L1 WPRE <![CDATA[miR122 target site E > <![CDATA[ISREx4 A ]]> <![CDATA[IFN-β C ]]> IRES <![CDATA[IL-15-sushi D ]]> WPRE <![CDATA[miR122 target site E >

[0324] A - preferably 4 copies of an ISRE preferably given by SEQ ID NO: 12

[0325] B - preferably human single chain IL-12 as described in SEQ ID NO: 9

[0326] C—preferably human single-chain IFN-β as described in SEQ ID NO: 79

[0327] D-preferably human IL-15-sushi as described in SEQ ID NO: 80

[0328] E - preferably 5 copies of the miR-122 target sequence as described in SEQ ID NO: 15

[0329] Another aspect of the present invention is an expression vector encoding a nucleic acid construct comprising at least two therapeutic gene sequences according to the above embodiments.

[0330] The use of expression vectors comprising nucleic acid constructs has certain advantages. First, expression vectors are specifically designed to allow the nucleotide sequence of the encoding to be introduced into host cells and to control their expression. Secondly, expression vectors are very stable plasmids, so they are protected and not degraded. Thirdly, expression vectors can be used for gene delivery using different methods. Non-limiting examples of expression vectors and preferred embodiments of this aspect of the present invention are viral vectors, more preferably viral vectors selected from adeno-associated virus (AAV) vectors, adenovirus vectors, lentiviral vectors, vaccinia virus vectors or herpes simplex virus vectors. In particular, viral vectors combine several advantages of expression vectors, such as general safety, low toxicity and stability.

[0331] It is also advantageous that the nucleic acid construct of the viral vector further comprises 5'ITR and 3'ITR sequences, preferably AAV ITR as described in SEQ ID NO: 17. It may be advantageous that the viral vector is an AAV vector, and preferably has serotype AAV8, AAV1, AAV3, AAV6, AAV9, AAV2, AAV5, AAVrh.10 or any gain-of-function mutant of AAVrh.10, with AAV2 being most preferred. AAV is commonly used in biotechnology and is therefore well characterized. The advantages of using AAV vectors are that they are generally considered safe, exhibit low toxicity and are stable. In addition, AAV has been approved for use in humans by several governmental organizations around the world.

[0332] It is also advantageous that the second nucleic acid construct of the viral vector further comprises a 5'ITR, a ψ packaging signal and a 3'ITR sequence, preferably a 5'ITR, a ψ packaging signal and a 3'ITR of an adenovirus (Ad). It may be advantageous that the viral vector is an adenoviral vector, preferably a high-capacity adenoviral vector. Adenoviruses are commonly used in biotechnology and are therefore well characterized. An advantage of adenoviral vectors is that they have a higher size limit than AAV vectors. In addition, adenoviruses have been approved for use in humans by several government organizations around the world.

[0333] It is also advantageous if the viral vector is a lentiviral vector, a vaccinia virus vector or a herpes simplex virus vector. These vectors are commonly used in biotechnology and are therefore well characterized.

[0334] It is also contemplated that nanoparticles may be used to deliver the nucleic acid constructs of this aspect of the invention.Nanoparticles as gene delivery systems are known in the art and have advantages such as adjustable size, shape and / or surface, allowing for modulation of their biological behavior. For effective delivery and cellular uptake, the nucleic acid construct can be packaged into nanoparticles (preferably liposomes) made of a pharmaceutically acceptable carrier, which is composed of physiologically compatible lipids, proteins, polysaccharides or synthetic polymers (such as standard lipids compatible with biological membranes) in different proportions, wherein the standard lipids are selected from the following list: phospholipids, such as distearoylphosphatidylglycerol, distearoylphosphatidylcholine, (DLPC) 1,2-dilauroyl-sn-glycero-3-phosphocholine, (DMPC) 1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DPPC) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, (DSPC) 1,2-distearoyl-sn-glycero-3-phosphocholine, (DOPC) 1,2-dioleoyl- sn-glycero-3-phosphocholine, (DMPE) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, (DPPE) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, (DOPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, (DOPS) 1,2-dioleoyl-sn-glycero-3-phospho-L-serine, (DPPS) 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, (DMPS) 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine, (DOPG) 2,3-dimyristoyl-sn-glycero-1-phosphocholine and their corresponding enantiomers, among which distearoylphosphatidylglycerol and distearoylphosphatidylcholine are most preferred. Another suitable example is that cardiolipin is preferably used in combination with cholesterol.Alternative nanoparticles are dendrimer nanoparticles, protamine liposomes, cationic polymers, cationic polymer liposomes, polysaccharide particles, cationic lipid particles, cationic lipid cholesterol nanoparticles, cationic lipid cholesterol PEG nanoparticles, ionizable lipids (preferably lipids that are positively charged under physiological conditions), including amino lipids such as MC3 (Dlin-MC3-DMA), C12-200, cKK-E12, ZA3-Ep10, 200-Oi10; (SM-102) heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, (ALC 0159) N,N-dimyristamide of 2-hydroxyacetic acid (O-PEGylated to a PEG chain of about 2 kilodaltons), (ALC 0315) (4-hydroxybutyl) azadiyl] di (hexane-6,1-diyl) bis (2-hexyldecanoate), Lipofectamine, Trans-IT, InstantFECT, (DOTAP) 1,2-dioleoyl-3-trimethylammonium. Other components may include peptides and polypeptides, such as DP7-C (cholesterol-modified cationic peptide DP7) (VQWRIRVAVIRK, SEQ ID NO: 85), polymers such as polyethylene glycol preparations composed of polymer molecules of different molecular sizes, which can be non-covalently bound or covalently linked to the lipids in the preparation.

[0335] It is also possible to use viral particles to deliver viral vectors containing nucleic acid constructs comprising at least two therapeutic gene sequences of the expression system of this aspect of the invention.

[0336] The aforementioned viral particles are preferably AAV or adenoviral particles; more preferably AAV viral particles. As described above, Ad and AAV exhibit several advantages over other viral particles, such as they are well characterized, frequently used, and approved for human treatment. These viruses exhibit other advantages that make them particularly useful in gene therapy, such as they are able to transduce dividing as well as non-dividing cells, and they do not integrate into the host genome.

[0337] Another aspect of the present invention is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a viral vector or viral particle containing a nucleic acid construct comprising at least two therapeutic gene sequences of the above-mentioned expression system. The advantage of pharmaceutical compositions is that they are "ready-to-use" because they can be directly administered to the patient to be treated. In addition, pharmaceutical compositions are customized according to treatment needs to optimize the stability and availability of the ingredients while avoiding unwanted side effects on the patient.

[0338] It is noteworthy that radiotherapy can be applied externally or internally. Those skilled in the art know several methods for providing external radiotherapy. For example, external radiotherapy can be provided by gamma rays, electron beams or proton accelerators sufficient for therapeutic purposes. However, for some tumors, it may be advantageous to use internal radiotherapy. For therapeutic purposes, internal radiotherapy can be provided by administering radioisotopes in pharmacologically acceptable forms and dosages. The embodiment of internal radiotherapy can be brachytherapy or non-localized radiotherapy. Brachytherapy physically limits radioactive deposits so that they can not be moved from the position where they have been deposited. Radioactive deposits can be placed in the gap (interstitial radiotherapy), which is used for example for a considerable percentage of prostate tumors and some head and neck tumors, or radioactive deposits can be placed in the cavity (intracavitary radiotherapy), which is used for example for some uterine, bronchial or esophageal tumors. Nonlocalized radiation therapy can be a localized administration of partial diffusion, such as selective internal radiation therapy (SIRT), in which a suspension of material is injected into a specific artery or vein with the goal of keeping it close to the injection site rather than being widely distributed in the body, or it can be a completely systemic approach, such as the use of synthetic soluble chemicals with radioisotopes that find their site of action by affinity or biologics such as radiolabeled antibodies to better deliver radiation to the target tumor. A typical example of the latter is iodine-131. While different isotopes may be used for different therapeutic paradigms, isotopes useful in the present invention may preferably be selected from the list consisting of yttrium-90, iodine-131, lutetium-177, iridium-192, astatine-211, bismuth-213, actinium-225, samarium-153, radium-223, phosphorus-32, strontium-89, erbium-169, holmium-166, terbium-161, gallium-68, rhenium-188 or rhenium-186.

[0339] In addition, the second nucleic acid construct has the advantage that it can be applied systemically and that the expression of at least one immunomodulatory gene or protein can be induced by radiation. Thus, radiation has a dual effect, since it not only promotes the transduction of the virus into the irradiated cells, but also induces the expression of at least one immunomodulatory gene or protein.

[0340] The first effect described above relates to an important concept of the present invention, namely that radiotherapy marks the tumor for transgene expression from a gene vector such as an AAV vector. This fact has very important clinical applications because the data of the present application show that when the AAV vector is administered parenterally (e.g., intravenously), tumor irradiation converts the tumor into a target for transgene (e.g., IL-12) expression. Using this method, the inventors were even able to cure subcutaneous tumors when the vector was administered intravenously. After intravenous administration of AAV.ISREx4.IL12miR122T to animals with subcutaneous tumors, the tumor was eliminated if the tumor had been previously irradiated, but not if it had not received any irradiation. In these experiments, tumor irradiation alone (by intravenous administration of a control virus encoding luciferase) could not induce tumor regression.

[0341] It should also be explicitly noted that in the context of the present patent application, indefinite articles and numerical indications, such as "a", "an", "one", "two", etc., are generally understood as "at least"-indications, i.e. "at least one...", "at least two...", etc., unless it is clearly inferred from the respective context or it is obvious or technically mandatory for a person skilled in the art to indicate that there is only "exactly one...", "exactly two...", etc.

[0342] Other advantages, features and details of the invention will become apparent from the following description, in which embodiments of the invention are described in detail with reference to the accompanying drawings. In this regard, the features mentioned in the claims and in the description may each be essential to the invention individually or in any combination.

[0343] The foregoing explanation of the embodiments specifically describes the invention in the context of examples. Of course, without departing from the scope of the invention, the individual features of the embodiments can be freely combined with each other as long as it is technically reasonable.

[0344] Other embodiments of the invention are described below.

[0345] Embodiment 1: A coincident expression system for combined gene and radiation therapy of a disease in a recipient mammal, wherein

[0346] a. The recombinant expression system comprises a first nucleic acid construct encoding a target STING, wherein the target STING responds to a STING agonist that recognizes the target STING but not human STING and its natural variants, wherein the sequence encoding the target STING is operably linked to an element that allows expression and / or translation of the target STING; and / or a second nucleic acid construct comprising a therapeutic gene sequence, wherein the therapeutic gene sequence is operably linked to a STING-responsive promoter; and

[0347] b. administering the recombinant expression system to the recipient mammal after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system with a low dose of radiation; and / or

[0348] c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation and / or administering a STING agonist to the recipient mammal.

[0349] Embodiment 2: A recombinant expression system according to embodiment 1, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases and infectious diseases, preferably a solid cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, neuroendocrine tumor, bile duct cancer or carcinoid tumor.

[0350] Embodiment 3: According to the recombinant expression system of embodiment 1 or 2, the therapeutic gene sequence of the second nucleic acid construct encodes a protein or gene selected from the group consisting of: cytokines such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones; or genes such as shRNAs targeting cytokines, nuclear factors or hormones.

[0351] Embodiment 4: The recombinant expression system according to embodiment 3, wherein the protein encoded by the therapeutic gene of the second nucleic acid construct is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-2 1 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony-stimulating factor (GM-CSF), type I interferon (IFN-α and IFNβ), IFN-γ, TNF-α, FLT3-ligand; immunostimulatory monoclonal antibodies or scFV or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides or shRNAs against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

[0352] Embodiment 5: A recombinant expression system according to any one of embodiments 1 to 4, wherein the STING response promoter second nucleic acid construct is selected from the group consisting of: IFN-β response promoter, CXCL9 response promoter, CXCL10 response promoter, STAT-motif promoter, IRSF3-motif promoter, ISRE-motif promoter, NFκB-motif promoter, STAT 6-motif promoter, preferably wherein the STING response promoter comprises at least one interferon-stimulated response element (ISRE).

[0353] Embodiment 6: The recombinant expression system according to any one of embodiments 1 to 5, wherein the STING-responsive promoter of the second nucleic acid construct is selected from the list consisting of:

[0354] a. A nucleotide sequence comprising 1 to 4 copies of an ISRE as described in SEQ ID NO: 11, preferably comprising four copies of the ISRE;

[0355] b. according to the nucleotide sequence of SEQ ID NO:12;

[0356] c. The nucleotide sequence according to SEQ ID NO:13.

[0357] Embodiment 7: A recombinant expression system according to any one of Embodiments 1 to 6, wherein the second nucleic acid construct of the expression system containing the therapeutic gene further comprises at least one sequence motif that inhibits the transgenic expression of the therapeutic gene sequence in the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

[0358] Embodiment 8: The recombinant expression system of Example 7, wherein the microRNA highly abundant in the liver is selected from the group consisting of miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, preferably, wherein the microRNA abundant in the liver is miR-122.

[0359] Embodiment 9: The recombinant expression system according to any one of embodiments 1 to 8, wherein the low-dose radiation is based on X-rays, gamma rays or particle rays.

[0360] Embodiment 10: The recombinant expression system according to any one of embodiments 1 to 9, wherein the total radiation dose of the low-dose radiation is 40 Gy or less, preferably 8 Gy or less.

[0361] Embodiment 11: The recombinant expression system according to any one of embodiments 1 to 10, wherein the low dose radiation is administered using stereotactic radiosurgery preferably performed by a linear accelerator (LINAC) machine, a gamma knife machine or proton beam therapy.

[0362] Embodiment 12: A recombinant expression system according to any one of embodiments 1 to 11, wherein the time interval between the low-dose irradiation of b. and the subsequent administration of the expression system is between 1 minute and 10 days, preferably between 10 minutes and 2 days, and more preferably between 30 minutes and 24 hours.

[0363] Embodiment 13: The recombinant expression system according to any one of embodiments 1 to 12, wherein the subsequent low-dose irradiation of c. administered after administration of the recombinant expression system is repeated at intervals between 1 day and 30 days, preferably between 5 days and 20 days, more preferably between 7 days and 15 days.

[0364] Embodiment 14: A recombinant expression system according to any one of embodiments 1 to 13, wherein after administering the recombinant expression system comprising the first nucleic acid construct, a human non-responsive STING agonist is administered to the recipient mammal, the agonist being preferably selected from the group consisting of: 5,6-dimethylxanthone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone) and 10-carboxymethyl-9-acridone (CMA), more preferably wherein the STING agonist is DMXAA.

[0365] Embodiment 15: A recombinant expression system comprising a nucleic acid construct, wherein the nucleic acid construct comprises a therapeutic gene sequence operably linked to a STING-responsive promoter, wherein the nucleic acid construct is preferably the second nucleic acid construct according to any one of Embodiments 3 to 8.

[0366] definition

[0367] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena are rarely, if ever, completed and / or carried out to perfection or to achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0368] Approximately: As used herein, the term "approximately" may be used to modify any quantitative comparison, value, measurement, or other representation that could permissibly vary without resulting in a change in the basic function to which it is related.

[0369] A / an: As used in the specification, "a" or "an" may mean one or more. As used herein in one or more claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more.

[0370] Or / and / or: Use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring only to alternatives and "and / or."

[0371] The term "about" generally refers to within the standard deviation of the stated value as determined using standard analytical techniques for measuring the stated value. These terms can also mean plus or minus 5% of the stated value.

[0372] Substantially: The term "substantially" should be understood to mean that the methods or compositions include only the specified steps or materials, as well as those steps or materials that do not materially affect the basic and novel characteristics of such methods and compositions.

[0373] Stereoisomers: In addition, the compounds used in the present invention may contain one or more asymmetric carbon atoms and therefore exist in two or more stereoisomeric forms. When the compound contains an alkenyl or alkenylene group, cis (E) and trans (Z) isomerism may also occur. The present invention includes the use of individual stereoisomers of the compound and (where appropriate) its individual tautomeric forms as well as mixtures thereof.

[0374] Analog: As used herein, the term "analog" refers to a compound that is similar in structure to another compound but has a slightly different composition, such as the replacement of one atom by an atom of a different element or a functional group.

[0375] Isotopes in compounds: The present invention also includes the use of all suitable isotopic variations of the compounds described herein or their pharmaceutically acceptable salts. An isotopic variation of a compound of the present invention or its pharmaceutically acceptable salt is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into compounds and their pharmaceutically acceptable salts include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36Cl, respectively. Certain isotopic variations of compounds and their pharmaceutically acceptable salts, such as those in which radioactive isotopes such as 3H or 14C are incorporated, can be used for drug and / or substrate tissue distribution studies. Tritiated isotopes (i.e., 3H) and carbon-14 isotopes (i.e., 14C) are particularly preferred because of their ease of preparation and detectability. Further substitution with isotopes such as deuterium (i.e. 2H) may provide certain therapeutic advantages resulting from greater metabolic stability (e.g., prolonged in vivo half-life or reduced dosage requirements) and may therefore be preferred in some circumstances. Isotopic variations of the compounds of the invention and pharmaceutically acceptable salts thereof can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

[0376] Cytokine: The term "cytokine" refers to a general term for biomolecules that affect / influence cells of the immune system. This definition is intended to include, but is not limited to, those biomolecules that act locally or can circulate in the blood, and when used in the compositions or methods of the present invention, are used to regulate or modulate an individual's immune response to cancer. Cytokines can be many different substances, such as chemokines, interferons, interleukins, and growth factors. Exemplary cytokines for practicing the present invention include, but are not limited to, interferons (e.g., IFN-α, IFN-β, and IFN-γ), interleukins (e.g., IL-1 to IL-29, particularly IL-2, IL-7, IL-12, IL-15, and IL-18), tumor necrosis factors (e.g., TNF-α and TNF-β), chemokines (e.g., CXCL9, CXCL10), granulocyte-macrophage colony stimulating factor (GM-CSF), TGF-β, FLT3 ligands, and fragments or variants thereof. It is worth noting that exogenously provided cytokines are a type of immunotherapy.

[0377] Immunomodulation: As used herein, the term "immunomodulation" refers to the property of initiating or altering (e.g., enhancing or attenuating) the activity of cells involved in an immune response. An immunomodulatory composition or method can enhance the activity of cells involved in an immune response, for example, by increasing pro-inflammatory or anti-inflammatory markers, and / or can reduce the activity of cells involved in an immune response, for example, by reducing pro-inflammatory or anti-inflammatory markers.

[0378] Agonist / human non-responsive STING agonist: As used herein, the term "agonist" refers to a ligand that stimulates the activity of a protein when bound to it. The term "human non-responsive STING agonist" refers to an agonist that does not substantially bind to human wild-type STING or its natural variants, but binds to mutated human STING or STING from other species, such as STING from a mouse, rat, or monkey. Optionally, the term "human non-responsive STING agonist" refers to an agonist that binds to human wild-type STING or its natural variants without inducing STING activity, while the STING activity of mutated human STING or STING from other species (such as STING from a mouse, rat, or monkey) is induced when the agonist is bound. Thus, the term "human non-responsive" refers to an agonist that does not activate one of the endogenous human STING proteins encoded in the genome of a human subject (according to one of the known haplotypes of human STING) (i.e., hSTING[R] as described in SEQ ID NO:1, hSTING[H] as described in SEQ ID NO:2, hSTING[AQ] as described in SEQ ID NO:3, hSTING[H] as described in SEQ ID NO:4, STING[Q] having the amino acid substitution R293Q, or STING[HAQ] having the amino acid substitutions R71H, G230A, and R293Q), wherein the exogenously provided DMXAA-sensitive human STING responds to the "human non-responsive" agonist.

[0379] Protein / polypeptide: The terms "protein", "peptide" and "polypeptide" are used interchangeably herein to refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to proteins, peptides or polypeptides of any size, structure or function. Typically, a protein, peptide or polypeptide is at least three amino acids in length. A protein, peptide or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide or polypeptide may be modified, for example, by the addition of chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization or other modifications, and the like. A protein, peptide or polypeptide may also be a single molecule or a multimolecular complex. A protein, peptide or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide or polypeptide may be naturally occurring, recombinant or synthetic, or any combination thereof.

[0380] Antibody / monoclonal antibody: As used herein, the term "antibody" refers collectively to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during immune responses in any vertebrate, for example, mammals (such as humans, goats, rabbits, camels and mice) and non-mammalian species, such as shark immunoglobulins. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by producing cells that form hybrid antibodies from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0381] Single-chain variable fragment / scFv: The terms "single-chain variable region" and "scFv" refer to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are continuously connected by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.

[0382] As used herein, the term "nanobody" refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibodies, and is known to those skilled in the art.

[0383] As used herein, the term "blocking peptide" refers to a peptide that is able to interfere with the molecule to which it is targeted in such a way that the interaction of the molecule with its target is weakened. For example, a blocking peptide targeting TGF-β would weaken its immunosuppressive function.

[0384] Nucleic acid: The term "nucleic acid" includes any nucleotide, its analogs and polymers thereof. The terms "polynucleotide" or "nucleotide sequence" as used herein refer to a polymeric form of nucleotides (ribonucleotides (RNA) or deoxyribonucleotides (DNA)) of any length. These terms refer to the primary structure of the molecule and thus include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include, as equivalents, analogs of RNA or DNA prepared from nucleotide analogs and modified polynucleotides (such as but not limited to methylated, protected and / or capped nucleotides or polynucleotides). These terms include polyribonucleotides or oligoribonucleotides (RNA) and polydeoxyribonucleotides or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges. The term encompasses nucleic acids containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. Technicians know that he can replace variable amounts of nucleosides in nucleic acids with modified nucleosides, which may include derivatives of purine and pyrimidine bases, such as pseudouridine, N1-methyl pseudouridine, 5-methyluridine, 5-methylcytosine, 6-methyladenosine, 2-thiouridine to improve translation efficiency. In some embodiments, the prefix poly-refers to nucleic acids containing 2 to about 10,000, 2 to about 50,000 or 2 to about 100,000 nucleotide monomer units. In some embodiments, "oligonucleotide" refers to nucleic acids containing 2 to about 200 nucleotide monomer units.

[0385] Gene: As used herein, the term "gene" means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression. In addition, shRNA is also included in this definition.

[0386] Therapeutic gene sequence: As used herein, the term "therapeutic gene sequence" means any nucleotide sequence encoding a therapeutic molecule for disease treatment. Non-limiting examples of such molecules are proteins, shRNAs, hormones or blocking peptides.

[0387] Immunomodulatory protein: As used herein, the term "immunomodulatory protein" refers to a protein that modulates (eg, by stimulating immunostimulatory proteins or by inhibiting immunosuppressive proteins) the immune system by inducing activation and / or increasing the activity of immune system components.

[0388] Hybridization: As used herein, the term "hybridizing" or "hybridizes" is understood as the formation of hydrogen bonds between base pairs on opposing strands by two nucleic acid strands (eg, an oligonucleotide and a target nucleic acid), thereby forming a duplex.

[0389] Promoter: The term "promoter" is used herein in its ordinary sense to refer to a nucleotide region containing a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene and is capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. It may contain genetic elements to which regulatory proteins and molecules (such as RNA polymerase and other transcription factors) can bind to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned", "operably linked", "under control" and "under transcriptional control" mean that the promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of the sequence.

[0390] Constitutive promoter: The term "constitutive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0391] Inducible promoter: The term "inducible promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0392] STING responsive promoter: As used herein, the term "STING responsive promoter" refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, enables the gene product to be produced in a cell substantially only when a polypeptide regulated by STING activity is present in the cell. Non-limiting examples of such polypeptides are cytokines, particularly interferons such as IFN-β, chemokines such as CXCL9 or CXCL10, transcription factors such as NFκB or STAT, interleukins such as IL-6 and TNF-α.

[0393] Mutation: As used herein, the term "mutation" refers to the substitution of a residue in a sequence (e.g., a nucleic acid or amino acid sequence) with another residue, or the deletion or insertion of one or more residues in a sequence. Mutations are generally described herein by identifying the original residue, followed by the position of the residue in the sequence and the identity of the newly replaced residue. Various methods for generating amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0394] Identity: As used herein, the term "identity" refers to the overall correlation between polymer molecules, such as polypeptide molecules and / or nucleic acid molecules (e.g., DNA molecules and / or RNA molecules). In some embodiments, if the sequence of the polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, then they are considered to be "substantially identical" to each other. For example, the calculation of the identity percentage of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for the purpose of optimal comparison (e.g., for optimal alignment, a gap can be introduced in one or both of the first and second sequences, and non-identical sequences can be ignored for the purpose of comparison). In certain embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or substantially 100% of the length of the reference sequence. Then compare the nucleotide or amino acid at the corresponding position.When the position in the first sequence is occupied by the residue (for example, nucleotide or amino acid) identical to the corresponding position in the second sequence, then the molecule is identical at this position.Considering the number of spaces and the length of each space that need to be introduced for the optimal comparison of two sequences, the identity percentage between the two sequences is a function of the number of identical positions shared by the sequences.The determination of the identity percentage between the comparison of sequences and two sequences can be completed using a mathematical algorithm.For example, the algorithm (CABIOS, 1989, 4:11-17) of Meyers and Miller (which has been incorporated into the ALIGN program (version 2.0)) can be used to determine the identity percentage between two nucleotide sequences.In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Or, the GAP program in the GCG software package can be used to determine the identity percentage between two nucleotide sequences using the NWSgapdna.CMP matrix.

[0395] Hybridization under stringent conditions: In the present description, hybridization under stringent conditions refers to hybridization conditions in which only long sequences with almost perfect complementary matches are guaranteed to anneal. This term is well known in the field of genetic engineering and has been used for many years in patent practice as a quasi-structural feature for defining DNA claims. It is noteworthy that the corresponding claims define their subject matter not only by the ability to hybridize under stringent conditions, but also by a further functional feature, namely that the polypeptide encoded by the claimed DNA sequence has at least the functional properties of STING.

[0396] Connected: As used herein, the term "connected", when used for two or more parts, means that the parts are physically bound or connected to each other, forming a sufficiently stable molecular structure so that the parts remain bound under conditions where the linkage is formed, preferably under conditions (e.g., physiological conditions) where the new molecular structure is used. In certain preferred embodiments of the present invention, the linkage is a covalent linkage. In other embodiments, the linkage is non-covalent. The parts can be directly or indirectly connected. When two parts are directly connected, they are either covalently bonded to each other, or close enough so that the intermolecular forces between the two parts maintain their association. When two parts are indirectly connected, they are each covalently or non-covalently connected to a third part, and the third part maintains the association between the two parts. Generally, when two parts are referred to as being connected by a "joint" or "linking moiety" or "linking portion", the linkage between the two connected parts is indirect, and generally each linking portion is covalently bonded to the joint. The linker can be any suitable moiety that reacts with the two moieties to be joined in sufficient quantity to produce a reasonable yield, within a reasonable period of time, under conditions consistent with the stability of the moiety (which can be appropriately protected as appropriate).

[0397] Operably linked / effectively linked: As used herein, the term "operably linked" or "effectively linked" is a juxtaposition in which the components are in a relationship that allows them to function in their intended manner. A control element such as a promoter that is "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a target coding element; in some embodiments, the control element acts in trans on the target functional element.

[0398] RNA interference / microRNA / shRNA: As used herein, the term "RNA interference" generally refers to a process in which an RNA molecule reduces or inhibits the expression of a nucleic acid sequence that shares substantial or complete homology with the RNA molecule. Without wishing to be bound by any theory, it is believed that in nature, the RNA interference pathway is initiated by a type III endonuclease called Dicer, which cuts long double-stranded RNA (dsRNA) into double-stranded fragments typically having 21-23 base pairs and 3' overhangs of 2 bases (although variations in length and overhangs are also contemplated), referred to as "short interfering RNA" ("siRNA"). Such siRNAs comprise two single-stranded RNAs (ssRNAs): an "antisense strand" or "guide strand" comprising a region that is substantially complementary to the target sequence and a "sense strand" or "passenger strand" comprising a region that is substantially complementary to the region of the antisense strand. One of ordinary skill in the art will appreciate that the guide strand may be fully complementary to the target region of the target RNA, or may be incompletely complementary to the target region of the target RNA. As used herein, the term "microRNA" or "miRNA" or "miR" refers to a specific type of siRNA that is physiologically produced in an organism, rather than synthetically produced. The miRNA binds to the corresponding miRNA target sequence and reduces or inhibits the expression of a nucleic acid sequence that shares basic or complete homology with the miRNA. As used herein, the term "shRNA" or "short hairpin RNA" refers to a single transcript that adopts a stem-loop structure, which is processed into siRNA by the RNAi mechanism. A typical shRNA molecule comprises two inverted repeats containing sense and antisense target sequences separated by a loop sequence. The base-paired segment can be any suitable length that allows in vivo target gene inactivation, wherein one strand of the base-paired stem is complementary to the mRNA of the target gene. The loop of the shRNA stem-loop structure can be any suitable length that allows in vivo target gene inactivation. The base-paired stem can be completely base-paired, or can have 1 or 2 mismatched base pairs.

[0399] Target gene / RNA: As used herein, "target gene" refers to a gene whose expression will be regulated (e.g., upregulated or inhibited). As used herein, the term "target RNA" refers to an RNA to be degraded or translationally inhibited or otherwise inhibited using one or more agents (e.g., one or more miRNAs or siRNAs). Target RNA may also be referred to as a target sequence or target transcript. RNA may be a primary RNA transcript (e.g., pre-mRNA) or a processed transcript transcribed from a target gene, such as an mRNA encoding a polypeptide. As used herein, the term "target portion" or "target region" refers to a continuous portion of the nucleotide sequence of a target RNA. In some embodiments, the target portion of an mRNA is at least long enough to serve as a substrate for RNA interference (RNAi)-mediated cleavage in the presence of a suitable miRNA or siRNA. The length of the target portion may be about 8-36 nucleotides, e.g., about 10-20 or about 15-30 nucleotides. The target portion length may have a specific value or sub-range within the above range. For example, in certain embodiments, the length of the target portion can be about 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19- 29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 nucleotides.

[0400] Recombinant: As used herein, the term "recombinant" refers to a nucleic acid or polypeptide designed, modified, prepared, expressed, produced, manufactured and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or has been otherwise manipulated to express one or more genes or gene components that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof; and / or a polypeptide prepared, expressed, produced or isolated by any other means, including splicing or connecting selected nucleic acid sequence elements to each other, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of a polypeptide or one or more components, parts, elements or domains thereof. In some embodiments, one or more of such selected sequence elements exist in nature. In some embodiments, one or more of such selected sequence elements are designed on a computer. In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of a known sequence element (e.g., from a natural or synthetic source, e.g., in the germline of a source organism of interest (e.g., human, mouse, etc.)).

[0401] Natural variant: The term "natural variant" refers to a variant of a gene or transcript that is derived from the same locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code (which results in a diversity of codons encoding the same amino acid), or due to alternative splicing of pre-mRNA, or the presence of polymorphisms such as single nucleotide polymorphisms (SNPs) and allelic variants. The natural variants exist in nature and are not artificially produced. In contrast, when the term "variant" is used without the word "natural" associated therewith, the variant may include artificially produced variants, such as mutations. With respect to human STING, some known natural variants are truncated forms of STING[R], STING[AQ], STING[H], STING[HAQ], and STING[H].

[0402] Wild type: As used herein, "wild type" refers to a naturally occurring nucleic acid sequence on a genetic locus in an organism's genome, as well as a sequence transcribed or translated from such a nucleic acid. Therefore, the term "wild type" may also refer to an amino acid sequence encoded by a nucleic acid. Since a locus may have more than one sequence or allele in an individual population, the term "wild type" includes all such naturally occurring alleles. As used herein, the term "polymorphism" means that there is a variation (i.e., there are two or more alleles) on a genetic locus of an individual in a population. As used herein, "mutant" refers to a change in the sequence of a nucleic acid or a protein, polypeptide or peptide encoded therein, which is the result of recombinant DNA technology.

[0403] Non-human: As used herein, the term "non-human" refers to a gene or protein that is not substantially derived from a wild-type human gene or protein. In the context of the present invention, targeted mutations of human proteins still make them human, while hybridization of a portion of a human protein with a portion of a homologous protein of another species makes it non-human.

[0404] Hybrid STING: As used herein, the term "hybrid STING" refers to STING in which a portion of the protein sequence is derived from human STING and another portion is derived from a homologous STING of another species.

[0405] Target STING: As used herein, a target STING is a STING that responds to a STING agonist that recognizes the target STING but not human STING and its natural variants. Thus, the target STING may be a mutated human STING, but not the endogenous human STING encoded in the genome of a human subject (according to one of the known haplotypes of human STING), i.e., hSTING[AQ] as described in SEQ ID NO: 1, hSTING[H] as described in SEQ ID NO: 2, hSTING[R] as described in SEQ ID NO: 3, hSTING[H] as described in SEQ ID NO: 4, STING[Q] with the amino acid substitution R293Q, or STING[HAQ] with the amino acid substitutions R71H, G230A, and R293Q. Thus, the responsiveness to a STING agonist is determined in the "in vitro-DMXAA assay test". Non-limiting examples of target STING are murine STING or a mutated human STING hSTING[R]-S162A / G230I / Q266I.

[0406] STING responsive promoter: As used herein, the term "STING responsive promoter" refers to a promoter activated by transcription factors of the STING signaling pathway, such as interferon regulatory factor 3 (IRF3) or nuclear factor κB (NFκB). Examples of STING promoters are interferon stimulated response elements (ISRE) or κB DNA response elements.

[0407] Cistron / monocistron / bicistron / polycistronic: As used herein, the term "cistron" refers to a segment of a DNA or RNA molecule that encodes a single protein or a single functional RNA molecule. The terms "monocistron", "bicistron" or "polycistronic" refer to the number of cistrons on a DNA or RNA molecule. For example, a bicistronic vector comprising two cistrons under the control of one promoter results in the translation of two different proteins.

[0408] Exogenous / foreign: The term "exogenous" or "foreign" when applied to a protein, gene, nucleic acid or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid or polynucleotide that has been introduced into a cell or organism by artificial or natural means; or with respect to a cell, the term refers to a cell that has been isolated and subsequently introduced into other cells or organisms by artificial or natural means. An exogenous nucleic acid can be from a different organism or cell, or can be one or more additional copies of a nucleic acid that occurs naturally in an organism or cell. An exogenous cell can be from a different organism or from the same organism. As non-limiting examples, an exogenous nucleic acid is a nucleic acid that is located in a chromosomal location different from the chromosomal location in which it is located in a natural cell, or alternatively is a nucleic acid that is flanked by a nucleic acid sequence that is different from a naturally occurring nucleic acid sequence.

[0409] Plasmid: A "plasmid" is a common type of vector, an extrachromosomal DNA molecule that is capable of replication independently of chromosomal DNA. In some cases, it is circular and double-stranded.

[0410] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA or RNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of directing the expression of genes operatively connected thereto. Such vectors are referred to herein as "expression vectors". It is understood by those of ordinary skill in the art that the "viral vectors" described herein include viral components, such as capsid proteins, in addition to the transgenes described herein.

[0411] Expression system: "Expression system" or "expression construct" means one or more nucleic acid molecules capable of directing transcription. An expression system includes at least one or more transcription control elements (such as promoters, enhancers or their structural and functional equivalents) that direct gene expression in one or more desired cell types, tissues or organs. Additional elements, such as transcription termination signals, may also be included. Therefore, the terms "expression system" and "recombinant expression system" encompass DNA or RNA nucleic acid molecules capable of directing transcription. In the specification of the present invention, the corresponding DNA or RNA nucleic acid molecules are also referred to as "nucleic acid constructs".

[0412] AAV: The term "AAV" refers to adeno-associated virus and can be used to refer to the naturally occurring wild-type virus itself or its derivatives. Unless otherwise required, the term covers all subtypes, serotypes and pseudotypes, as well as naturally occurring and recombinant forms. The AAV genome consists of single-stranded DNA and contains inverted terminal repeats (ITRs) at both ends of the DNA chain and two open reading frames: rep and cap, encoding replication proteins and capsid proteins, respectively. Foreign polynucleotides can replace the natural rep and cap genes. AAV can be made with a variety of different serotypes of capsids, which have different transduction spectra, or as used herein, have "tropism" for different tissue types. As used herein, the term "serotype" refers to an AAV that is recognized and distinguished from other AAVs based on the reactivity of the capsid protein with a determined antiserum, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAVrh10. For example, serotype AAV2 is used to refer to an AAV containing a capsid protein encoded by the cap gene of AAV2 and a genome containing 5' and 3' ITR sequences from the same AAV2 serotype. Pseudotyped AAV refers to an AAV containing a capsid protein from one serotype and a viral genome including 5'-3' ITRs of a second serotype. Pseudotyped rAAV is expected to have the cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotyped rAAV is produced using standard techniques described in the art.

[0413] Adenovirus: The term "adenovirus" refers to any virus in the genus Adenovirus (including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenus). Typically, adenoviral vectors are generated by introducing one or more mutations (e.g., deletions, insertions or substitutions) into the adenoviral genome of an adenovirus to accommodate the insertion of non-native nucleic acid sequences (e.g., for gene transfer) into the adenovirus.

[0414] Lentivirus: As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesna-Medivirus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0415] Virus particle: As used herein, the term "virus particle" refers to all or part of a virion. For example, a virus particle comprises a recombinant genome and may further comprise a capsid. The virus particle may be a gene therapy vector. In this article, the terms "virus particle" and "vector" are used interchangeably. For the purposes of this application, a "gene therapy" vector is a virus particle that can be used for gene therapy, i.e., a virus particle that contains all the required functional elements for expressing a transgene (such as a CFI nucleotide sequence) in a host cell after administration. Suitable virus particles of the present invention include parvoviruses, retroviruses, lentiviruses, or herpes simplex viruses. The parvovirus may be an adeno-associated virus (AAV). Optionally, the virus particle is an AAV or adenovirus particle.

[0416] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art who read this disclosure will understand that such terms refer not only to the specific subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, MC38 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes.

[0417] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and / or worms. In some embodiments, animals can be transgenic animals, genetically engineered animals, and / or clones.

[0418] Treatment / treating: "Treatment" or "treating" includes (1) inhibiting a disease, disorder or condition in a subject or patient who is experiencing or exhibiting pathology or symptomology of the disease (e.g., arresting further development of the pathology and / or symptomology), (2) ameliorating a disease, disorder or condition in a subject or patient who is experiencing or exhibiting pathology or symptomology of the disease (e.g., reversing the pathology and / or symptomology), and / or (3) achieving any measurable reduction in a disease, disorder or condition in a subject or patient who is experiencing or exhibiting the disease, disorder or condition.

[0419] Prophylactic treatment: "Prophylactic treatment" includes: (1) reducing or alleviating the risk of disease in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all pathology or symptoms of the disease, and / or (2) slowing the onset of disease pathology or symptoms in a subject or patient who may be at risk and / or susceptible to the disease but does not yet experience or display any or all pathology or symptoms of the disease.

[0420] Local administration / local delivery: As used herein, the term "local administration" or "local delivery" refers to delivery that is independent of the transport of the product to its intended target tissue or site through the vascular system when it relates to the delivery of the recombinant expression system, expression vector, viral particle or pharmaceutical composition described herein. The product described herein can be delivered directly to its intended target tissue or site, or near it, for example, in close proximity to the intended target tissue or site. For example, the product can be delivered by injection or implantation of a composition or compound or by injection or implantation of a device containing a composition or compound. After local administration to the target tissue or near the target tissue or site, the product described herein or one or more components thereof can diffuse to the intended target tissue or site. It should be understood that once it has been locally delivered, a portion of the product described herein (usually only a small portion of the administered dose) can enter the vascular system and be transported to another location, including back to its intended target tissue or site. As used herein, the term "local administration" or "local delivery" when referring to the delivery of the viral particles or pharmaceutical compositions described herein, refers to delivery that may rely on transport of the viral vector or pharmaceutical composition through the vascular system to its intended target tissue or site.

[0421] Simultaneous administration: As used herein, the term "simultaneous administration" with respect to two or more compounds (e.g., therapeutic agents) is an administration performed using a dosage and a time interval, which allows the administered compounds to be present together in the body in a non-negligible amount over a period of time, such as at one or more sites of action in the body. The time interval may be a few minutes (e.g., at least 1 minute, 1-30 minutes, 30-60 minutes), a few hours (e.g., at least 1 hour, 1-2 hours, 2-6 hours, 6-12 hours, 12-24 hours), a few days (e.g., at least 1 day, 1-2 days, 2-4 days, 4-7 days, etc.), a few weeks (e.g., at least 1 week, 2 weeks, or 3 weeks, etc.). Therefore, the compounds may, but need not, be administered together as part of a single composition. In addition, the compounds may, but need not, be administered substantially simultaneously (e.g., less than 5 minutes apart, or less than 1 minute apart) or within a short time of each other (e.g., less than 1 hour apart, less than 30 minutes apart, less than 10 minutes apart, about 5 minutes apart). According to various embodiments of the present disclosure, the compounds administered within such a time interval may be considered to be administered substantially simultaneously. In certain embodiments of the present disclosure, the compounds administered simultaneously are present in vivo (e.g., in the blood and / or at sites of local complement activation) at effective concentrations over a period of time. When administered simultaneously, the effective concentration of each compound required to elicit a specific biological response may be lower than the effective concentration of each compound when administered alone, thereby reducing the dose required for one or more of the compounds relative to the compound when administered as a single compound. The effects of multiple compounds may be, but not necessarily, additive or synergistic. The compounds may be administered multiple times. A non-negligible concentration of a compound may be, for example, less than about 5% of the concentration required to elicit a specific biological response (e.g., a desired biological response).

[0422] Sequential administration: As used herein, the term "sequential administration" of two or more compounds refers to administering two or more compounds to a subject so that the compounds are not present together in the subject's body or at relevant active sites in the body at concentrations greater than non-negligible. Administration of the compounds may be alternating, but need not be. Each compound may be administered multiple times.

[0423] Systemic administration: As used herein, the term "systemic administration" and similar terms are used herein in accordance with their usage in the art and refer to the administration of a compound (e.g., a therapeutic agent, an expression vector, a viral particle, a pharmaceutical composition, and / or a formulation) such that the compound is widely distributed in the body in significant amounts and has a biological effect, such as its desired effect, in the blood, and / or reaches its desired site of action through the vascular system. Typical systemic administration routes include (i) administration by direct introduction of the compound into the vascular system, or (ii) subcutaneous, oral, pulmonary, or intramuscular administration, wherein the compound is absorbed, enters the vascular system, and is transported to one or more desired sites of action through the blood.

[0424] Intratumoral administration: The term "intratumoral administration" refers to the administration of a compound (e.g., a therapeutic agent, an expression vector, a viral particle, a pharmaceutical composition, and / or a formulation) such that the compound is present within a tumor after administration. One of ordinary skill in the art will appreciate that administration can occur directly within a tumor or in close proximity to a tumor.

[0425] Subject: As used herein, the term "subject," "test subject," or "patient" refers to any organism to which a provided compound or composition is administered according to the invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may suffer from and / or be susceptible to a disease, disorder, and / or condition.

[0426] Effective / Therapeutically Effective Amount: As used herein, the term "effective amount" or "therapeutically effective amount" means at least the minimum amount of a compound (e.g., a therapeutic agent, expression vector, viral particle, pharmaceutical composition, and / or formulation) that causes a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to a disease, condition, and / or disorder. As will be understood by one of ordinary skill in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, and the like. For example, an effective amount of a compound for treating a disease, condition, and / or condition in a formulation is an amount that alleviates, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or signs of a disease, condition, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the treatment are outweighed by the therapeutically beneficial effects. In the case of cancer or tumors, an effective amount of a compound may have the effect of reducing the number of cancer cells, reducing the size of a tumor, inhibiting (i.e., slowing down or ideally preventing to some extent) the infiltration of cancer cells into peripheral organs, inhibiting (i.e., slowing down and ideally stopping to some extent) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more symptoms associated with the disease.

[0427] Combination of expression vectors / combination of viral particles: The phrase "combination of expression vectors" or "combination of viral particles" means herein that a first expression vector or a first viral particle is administered to a patient before, during (including simultaneously - preferably co-formulated) and / or after treatment of the individual with a second expression vector or a second viral particle, respectively. The combined expression vectors or viral particles may be used for simultaneous, sequential or separate administration. In all cases, it is preferred to administer any expression vector and viral particle mentioned herein in a pharmaceutically effective amount, i.e., administration involving the total amount of each component of the combination sufficient to show a meaningful patient benefit. The formulations may be conveniently provided in unit dosage form by methods known to those skilled in the art.

[0428] Kit: As used herein, a "kit" provides a combination of a first and a second expression vector or a combination of a first and a second viral particle for combined administration. Preferably, the kit may contain the vectors / particles, for example, in a dosage form for administration. The dosage form contains a sufficient amount of one or more vectors / particles so that the desired effect can be obtained when administered to a subject. Thus, preferably the medical package contains a certain amount of dosage units corresponding to the relevant dosage regimen. Preferably, the kit contains instructions for using the dosage form to achieve the desired effect and the amount of the dosage form to be taken within a specified time period.

[0429] Immunotherapy: As used herein, the term "immunotherapy" refers to a therapy by which regulation or stimulation of the immune system is used to treat a disease, preferably a proliferative disease such as cancer, preferably using an immunomodulatory gene or protein. Immunotherapy can cause stimulation of the immune system, which is typically used for immunotherapy against cancer. However, other diseases such as autoimmune diseases, diabetes, cardiovascular disease, and inflammation can also be treated with immunotherapy. For example, IFN-β is used to treat multiple sclerosis. In addition, IL-2 has been shown to regulate the balance of regulatory T cells and effector T cells, thereby maintaining the function of β cells. Immunotherapy can be used to treat non-cancer diseases such as lupus, rheumatoid arthritis, and diabetes.

[0430] Immune checkpoint inhibitors: As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a compound that inhibits a protein in a checkpoint signaling pathway. Proteins in the checkpoint signaling pathway include, for example, CD27, CD28, CD40, CD 122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB or VISTA. Immune checkpoint inhibitors are known in the art. For example, an immune checkpoint inhibitor can be a small molecule. As used herein, "small molecule" means a composition having a molecular weight of less than about 5 kDa to 50 Da, such as less than about 4 kDa, less than about 3.5 kDa, less than about 3 kDa, less than about 2.5 kDa, less than about 2 kDa, less than about 1.5 kDa, less than about 1 kDa, less than 750 Da, less than about 500 Da, less than about 450 Da, less than about 400 Da, less than about 350 Da, less than 300 Da, less than 250 Da, less than about 200 Da, less than about 150 Da, less than about 100 Da. Small molecules can be, for example, nucleic acids, peptides, polypeptides, peptide mimetics, carbohydrates, lipids, or other organic or inorganic molecules. Alternatively, immune checkpoint inhibitors are antibodies or fragments thereof. For example, the antibody or fragment thereof is specific for a protein in a checkpoint signaling pathway, such as CD27, CD28, CD40, CD 122, CD137, OX40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, PD-L1, PD-L2, TIM-3, TIGIT, Lair1, CD244, HAVCR2, CD200, CD200R1, CD200R2, CD200R4, LILRB4, PILRA, ICOSL, 4-1BB, or VISTA. Exemplary anti-immune checkpoint antibodies include, e.g., ipilimumab (anti-CTLA-4), penbrolizumab (anti-PD-L1), nivolumab (anti-PD-L1), atezolizumab (anti-PD-L1), and duralumab (anti-PD-L1), nivolumab (anti-PD1), relalizumab (anti-LAG-3).

[0431] Adoptive cell therapy: As used herein, the term "adoptive cell therapy" refers to a therapy in which autologous or allogeneic immune cells are infused into a patient to treat certain diseases, such as cancer. Various cell types can be used for this purpose, such as dendritic cells, natural killer (NK) cells, or T cells.

[0432] Adoptive T cell therapy: As used herein, the term "adoptive T cell therapy" refers to a therapy in which autologous or allogeneic T cells are infused into a patient to treat certain diseases such as cancer. One type of adoptive T cell therapy is to separate tumor infiltrating leukocytes (TILs) from a tumor, optionally expand these cells outside the patient's body and infuse them back into the patient. Another adoptive T cell therapy is chimeric antigen receptor (CAR) T cell therapy. CAR-T cells can be designed in a variety of ways to enhance tumor cell toxicity and specificity, avoid tumor immunosuppression, avoid host rejection, and extend their therapeutic half-life.

[0433] Cancer vaccine: As used herein, the term "cancer vaccine" refers to a vaccine that treats an existing cancer or prevents cancer from developing.

[0434] Combination therapy: As used herein, the term "combination therapy" refers to the situation where two or more different compounds (e.g., therapeutic agents, expression vectors, viral particles, pharmaceutical compositions, and / or formulations) are administered in overlapping schemes to expose the subject to two compounds at the same time. When used for combination therapy, two or more different compounds can be administered simultaneously or separately. This combination can include simultaneous administration of two or more compounds in the same dosage form, simultaneous administration in separated dosage forms, and separated administration. That is, two or more compounds can be formulated together in the same dosage form and administered simultaneously. Alternatively, two or more compounds can be administered simultaneously, wherein the compounds are present in separated preparations. In another alternative, a first compound can be administered, followed by one or more additional compounds. In a separated administration scheme, two or more compounds can be administered at intervals of minutes, hours, days, or weeks. In some embodiments, two or more compounds can be administered at intervals of 1-2 weeks.

[0435] Radiotherapy: The term "radiotherapy" refers hereinafter to the medical use of ionizing radiation, usually as part of cancer treatment to control or kill malignant cells. Ionizing radiation can be based on photon rays, such as X-rays or gamma rays, or particle rays, wherein the particles of the particle rays are preferably selected from the group consisting of beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions. It can also be used as part of adjuvant therapy to prevent tumor recurrence after surgery to remove primary malignant tumors. Radiotherapy can work synergistically with other anticancer therapies and can be used before, during and after other anticancer therapies for susceptible cancers. The term "low-dose radiotherapy" refers hereinafter to radiotherapy with an ionizing radiation dose of less than 40Gy, preferably less than 8Gy. Low dose radiation therapy can be any radiation therapy with a dose of ionizing radiation less than 40 Gy, 38 Gy, 36 Gy, 34 Gy, 32 Gy, 30 Gy, 28 Gy, 26 Gy, 24 Gy, 22 Gy, 20 Gy, 19 Gy, 18 Gy, 17 Gy, 16 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, 2.5 Gy, 2.0 Gy, 1.5 Gy, 1 Gy, 0.5 Gy, 0.25 Gy, 0.2 Gy, 0.15 Gy, 0.1 Gy, 0.05 Gy or 0.01 Gy.

[0436] Proliferative disease: As used herein, the term "proliferative disease" refers to a condition in which the unregulated or abnormal growth of cells, or both, can lead to the development of an unwanted condition or disease, which may or may not be cancerous. Exemplary cell proliferative disorders of the present invention encompass a variety of conditions in which cell division is dysregulated. Exemplary cell proliferative disorders include, but are not limited to, tumors, benign tumors, malignant tumors, precancerous conditions, tumors in situ, encapsulated tumors, metastatic tumors, liquid tumors, solid tumors, immune tumors, blood tumors, cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidly dividing cells.

[0437] Cancer / tumor: The term "cancer" or "tumor" refers to the presence of cells with typical characteristics of cancer cells (such as uncontrolled proliferation, immortality, invasion or metastasis potential, rapid growth, and certain characteristic morphological features). In some embodiments, such cells partially or completely exhibit such characteristics due to the expression and activity of immune checkpoint proteins such as PD-1, PD-L1, PD-L2 and / or CTLA-4. Cancer cells usually exist in the form of tumors, but such cells can exist alone in animals, or can be non-tumorigenic cancer cells, such as leukemia cells. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers.Cancer includes, but is not limited to, various cancers, including bladder cancer (including accelerated and metastatic bladder cancer), breast cancer, colon cancer (including colorectal cancer), kidney cancer, liver cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer and lung adenocarcinoma), ovarian cancer, prostate cancer, testicular cancer, genitourinary tract cancer, lymphatic system cancer, rectal cancer, laryngeal cancer, pancreatic cancer (including exocrine pancreatic cancer), esophageal cancer, gastric cancer, gallbladder cancer, cervical cancer, thyroid cancer and skin cancer (including squamous cell carcinoma); including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma Hematopoietic neoplasms of the lymphoid lineage including myeloid leukemia, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, histiocytic lymphoma and Burkitt's lymphoma; myeloid hematopoietic neoplasms including acute and chronic myeloid leukemia, myelodysplastic syndrome, myeloid leukemia and promyelocytic leukemia; central and peripheral nervous system tumors including astrocytoma, neuroblastoma, glioma and schwannoma; thymic carcinoma, mesenchymal tumors including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; mesenchymal tumors including melanoma, peridermia pigmentosa, keratoacanthoma, seminoma Other tumors including ovarian cancer, thyroid follicular cancer and teratoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer, gastric cancer, germ cell tumor, bone cancer, bone tumor, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone , sarcoma, pediatric sarcoma, sinonasal natural killer cells, tumors, plasma cell neoplasms; myelodysplastic syndrome; neuroblastoma; testicular germ cell tumors, intraocular melanoma, myelodysplastic syndrome; myelodysplastic / myeloproliferative disorders, synovial sarcoma, chronic myelogenous leukemia, acute lymphocytic leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), multiple myeloma, acute myeloid leukemia, chronic lymphocytic leukemia, mastocytosis and any symptoms associated with mastocytosis and any metastases thereof. In addition, diseases include urticaria pigmentosa, mastocytosis such as diffuse cutaneous mastocytosis, solitary mastocytoma in humans, and some rare subtypes such as bullous, erythrodermic, and telangiectatic mastocytosis, mastocytosis with associated blood disorders such as myeloproliferative or myelodysplastic syndrome, or acute leukemia, myeloproliferative disorders associated with mastocytosis, mast cell leukemia, and other cancers.Other cancers are also included within the scope of the disorder, including, but not limited to, the following diseases: cancers, including bladder cancer, urothelial cancer, hepatocellular carcinoma, hepatoblastoma, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, testicular cancer, particularly testicular seminoma, and skin cancer; including squamous cell carcinoma; gastrointestinal stromal tumor ("GIST"); hematopoietic tumors of the lymphoid lineage, including leukemias, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, hairy cell lymphoma, and Burkitt lymphoma; myeloid hematopoietic tumors , including acute and chronic myeloid leukemias and promyelocytic leukemias; mesenchymal tumors, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratoma, neuroblastoma and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma and schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, pigmented xenoderma, keratoacanthoma, seminoma, follicular thyroid carcinoma, teratoma, chemotherapy-refractory non-seminomatous germ cell tumors and Kaposi's sarcoma and any metastases thereof. Other non-limiting examples of cancer types suitable for the methods contemplated by the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, bone cancer, brain tumor, lung cancer (including lung adenocarcinoma), small cell lung cancer, bladder cancer, upper Skin cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; Leukemia, such as acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); Chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); And polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease. In some embodiments, cancer is epithelial in nature, including but not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, gynecological cancer, kidney cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer or skin cancer.

[0438] Anti-cancer: An "anti-cancer" agent is capable of negatively affecting cancer cells / tumors in a subject, for example, by promoting the killing of cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to tumors or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the lifespan of a subject suffering from cancer.

[0439] Pharmaceutical composition: The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain additional components that are unacceptably toxic to the subject to which the preparation is to be administered. Such preparations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient used.

[0440] Synergy / Synergistic effect: The term "synergy" or "synergistic effect" refers to the combined effect of two or more compounds that is greater than the sum of the individual effects of the individual cancer compounds / therapies.

[0441] Abscopal effect / direct treatment: As used herein, the term "direct treatment" refers to the administration of a compound, expression system, expression vector, viral vector, viral particle, pharmaceutical composition, or any therapy directly to a targeted location. For example, direct treatment of a tumor according to the present invention refers to administration that targets the tumor but does not target other tumors or metastases. As used herein, the term "abscopal effect" describes a reduction in tumor size by directly treating another tumor. More specifically, direct treatment of a tumor may allow the organism to also target another tumor that has not been contacted with the compound, expression system, expression vector, viral vector, viral particle, pharmaceutical composition, or any therapy.

[0442] In vitro agonist assay test / in vitro DMXAA assay test: The term "in vitro agonist assay test" refers to an assay that allows identification of human non-responsive STING agonists, wherein the agonist activates human STING or a variant of non-human STING, but does not activate human wild-type STING or a naturally occurring variant thereof. The term "in vitro DMXAA assay test" refers to an assay that allows identification of STING that responds to DMXAA.

[0443] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reaction and purification techniques can be performed according to the manufacturer's instructions or as usually completed in the art or as described herein. The aforementioned techniques and procedures can be performed generally according to conventional methods well known in the art, and are described in various general and more specific references as quoted and discussed in this specification in full. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0444] Figure 1, including Figures AG, illustrates A) IFN-β expression in MC38 cells after lentiviral transduction with hSTINGwt or hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test); B) IFN-β expression in Hep2G cells after lentiviral transduction with hSTINGwt or hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test ); C) IFN-β expression in Hep2G cells after lentiviral transduction with mSTINGwt and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 9, **P < 0.01, two-way ANOVA followed by Sidak's multiple comparison test); D) STING-derived cytokine expression in MC38 cells after lentiviral transduction with luciferase, hSTINGwt or hSTINGmut or no transduction and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, one sample at a time by one-sample T test E) STING-derived cytokine expression in Hep2G cells after lentiviral transduction with luciferase, hSTINGwt or hSTINGmut or without transduction and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, one sample at a time by one sample T test); F) Time course of STING-derived cytokine expression in MC38 cells after lentiviral transduction with hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0,05, **P < 0.01, ***P < 0,001, two-way ANOVA followed by Fisher's LSD test); and G) Time course of STING-derived cytokine expression in MC38 cells after lentiviral transduction with hSTINGmut and subsequent DMXAA treatment relative to vehicle-treated cells; data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA followed by Fisher's LSD test).

[0445] Figure 2 Depicted are IFN-β serum levels in mice injected intravenously with AAV8 vectors encoding mSTING or GFP and treated twice weekly with different doses of DMXAA (5, 10 or 20 mg / kg body weight). Data shown are mean ± SEM (n = 4-12 mice / group) **P < 0.01, ***P < 0.001, one-way ANOVA followed by Dunnett's multiple comparison test.

[0446] Figure 3, including panels AH, illustrates A) luciferase activity of MC38 cells transduced by lentivirus with vectors encoding luciferase under the control of different modified IFN-β promoters in the absence or presence of DMXAA; data are mean ± SEM (n = 2 wells / condition), **p < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test (vehicle-DMXAA); B) luciferase activity of MC38 cells transduced by lentivirus with vectors encoding luciferase under the control of different modified chemokine promoters in the absence or presence of DMXAA. Luciferase activity of MC38 cells transduced with vectors encoding IL-12 under the control of IFN-β promoter, CXCL9 promoter or CXCL10 promoter by lentiviral transduction at different time points after DMXAA treatment; data shown are mean ± SEM (n = 3, *P < 0.05, two-way ANOVA, followed by Kruskal-K ... kal-Wallis test; D) IL-12 protein levels in the supernatants of MC38 cells transduced by lentiviral transduction with vectors encoding IL-12 under the control of the IFN-β promoter, CXCL9 promoter, or CXCL10 promoter at different time points after DMXAA treatment; data are mean ± SEM (n = 2-8 wells / condition), ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test; E) and F) MC38 cells transfected with plasmids encoding luciferase under the control of different synthetic promoters in the absence or presence of DMXAA. Luciferase activity of C38 cells; data are mean ± SEM (n = 2 wells / condition), *P < 0.05, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test; and G) and H) luciferase activity of HEK293T cells transfected with plasmids encoding luciferase under the control of different synthetic promoters in the absence or presence of DMXAA; data are mean ± SEM (n = 2 wells / condition), ****P < 0.0001, two-way ANOVA followed by Sidak's multiple comparison test.

[0447] Figure 4, including panels AC, illustrates A) IL-12 protein levels in mouse serum after intravenous injection with AAV8 encoding IL-12 under the control of the IFN-β promoter or the ISRE promoter in the absence of DMXAA; data are mean ± SEM (n = 2-3 animals / condition), *P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test; B) IL-12 protein levels in mouse serum after intravenous injection with AAV8 encoding single-chain (sc) IL-12 operably linked to a miR122 binding site under the control of the ISRE promoter in the absence of DMXAA compared to the AAV8 vector lacking the miR122 binding site. -12 protein levels were reduced; data are mean ± SEM (n = 4 animals / condition), ****P < 0.0001, Student's t-test; and C) IL-12 protein levels were reduced in mouse serum after intravenous injection with AAV8 encoding hSTINGmut under the control of a constitutively active promoter and AAV8 encoding scIL-12 operably linked to a miR122 binding site under the control of an ISRE promoter or without the miR122 binding site in the presence or absence of DMXAA; data are mean ± SEM (n = 4-8 animals / condition), *P < 0.05, two-way ANOVA followed by Tukey's multiple comparison test.

[0448] Figure 5 Figure 2 shows the volume of subcutaneous MC38 tumors implanted in STING KO mice that had been co-injected intratumorally with an AAV8 vector encoding hSTINGmut under the control of a constitutive promoter (AAV8-hSTINGmut) (dose of 1×10 11 vg / mouse) + AAV8 vector encoding scIL-12 operably linked to the miR122 binding site and placed under the control of the ISRE promoter (AAV8-ISREx4IL12miR122T) (dose 0.5x10 11 vg / mouse). These animals received DMXAA (solid triangles) or vehicle (solid circles) at the indicated time intervals. In an additional control group, tumors were injected with AAV8-hSTINGmut (1x10 11 vg / mouse) + AAV8 vector encoding GFP (0.5x10 11 vg / mouse) followed by vehicle injection (open circles). Data are mean ± SEM (n = 4-6 animals / condition) *P < 0.05, **P < 0.01, ****P < 0.0001, mixed effects analysis followed by Tukey's multiple comparison test.

[0449] Figure 6, including Fig. 6A and Figure 6B , illustrates A) subcutaneous MC38 tumor sections immunohistochemically stained with anti-GFP antibody 3 days after intratumoral injection of AAV8-GFP. The transduction efficiency of tumors injected with AAV8-GFP without radiation pretreatment (left) was significantly reduced compared with tumors pretreated with 8Gy radiation (right). B) The figure shows the percentage of strongly GFP-labeled pixels associated with tumor area. Data are mean ± SEM (n = 3-6 animals / condition) ***P < 0.001, unpaired Student's t-test.

[0450] Figure 7 Shown are quantified IFN-β protein levels in supernatants of MC38 cells irradiated and / or transduced with vectors encoding hSTINGmut at the indicated time points after irradiation; data are mean ± SEM (n = 2-6 samples / condition), *P < 0.05, mixed effects followed by Tukey's multiple comparison test (individual effects). #P < 0.05, ####P < 0.0001 vs. MC38 8Gy, two-way ANOVA followed by Tukey's multiple comparison test (comparison of values ​​at given time points).

[0451] Figure 8, comprising panels AD, illustrates A) the growth of MC38 subcutaneous tumors in mm in C57BL / 6 wild-type mice following irradiation or non-irradiation as indicated, followed by intratumoral injection of AAV8-GFP, AAV8-hSTINGmut, and / or AAV8-ISREx4-IL12miR122T. 3The time course of tumor volume progression is shown in Table 1. Data are mean ± SEM (n = 4-5 mice / group), and significance is assessed using a two-way analysis of variance (ANOVA) test and Tukey post hoc test analysis. Simple main effects analysis shows that treatment has a statistically significant effect on tumor growth (***p < 0.001). B) Serum levels of IL-12 measured by ELISA on the 17th day after treatment of animals in A). The dots represent individual values ​​for each mouse, the horizontal line in the middle represents the median, and the error bars represent the standard error of the mean (SEM). As determined by a one-way ANOVA (F (2,7) = 0.4375, p = 0.6622), there are no statistically significant differences between the groups. C) and D) refer to C57BL / 6 wild-type mice with two subcutaneous MC38 tumors, one in the right abdomen and the other in the left abdomen; the former is treated, while the contralateral tumor is not treated in order to analyze the remote effect. Right flank tumors were treated with 8 Gy of local irradiation followed by intratumoral injection of AAV8-GFP, AAV8-hSTINGmut and / or AAV8-ISREx4-IL12miR122T as indicated. C) shows the growth of treated tumors, and D) shows the growth of untreated tumors. Data are mean ± SEM (n = 6 mice / group). Significance was assessed using a two-way analysis of variance (ANOVA) test and Tukey post hoc test analysis. Simple main effects analysis showed that treatment had a statistically significant effect on the growth of treated tumors (***p<0.001) on day 15 after treatment, while having a statistically significant anti-tumor effect on contralateral tumors (*p<0.01).

[0452] Figure 9, including Figures AF, illustrates A) IFN-β mRNA levels in MC38 cells upon stimulation with DMXAA or vehicle in non-transduced cells or cells transduced with AAV8-hSTINGmut after 8 Gy of irradiation or without irradiation (0 Gy); data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, one-way ANOVA followed by Kruskal-Wallis test); B) IFN-β mRNA levels in MC38 cells upon stimulation with DMXAA or vehicle in non-transduced cells or cells transduced with AAV8-hSTINGmut after 8 Gy of irradiation or without irradiation (0 Gy); data shown are mean ± SEM (n = 3, *P < 0.05, **P < 0.01, one-way ANOVA followed by Kruskal-Wallis test); IL-12 mRNA levels in MC38 cells upon stimulation with AAV8-hSTINGmut+AAV8-GFP, AAV8-GFP+AAV-ISREx4-IL12miR122T, or AAV8-hSTINGmut+AAV-ISREx4-IL12miR122T after 8 Gy of irradiation or without irradiation (0 Gy) IFN-β mRNA levels (in C) or IL-12 mRNA levels (in D) in HEK293 cells upon stimulation; data shown are mean ± SEM (n = 3, **P < 0.01, ***P < 0,001, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test); E) IFN-β protein levels (pg / ml) obtained by ELISA in the supernatants of cultured non-transduced MC38 cells treated with different doses of DMXAA (0, 5, 10, 20 and 50 μg / mL) 24 hours after irradiation (0, 2 or 8 Gy) and collected 6, 24 or 48 hours after DMXAA administration; data Mean ± SEM (n = 2-5 samples / condition), ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test; and F) IFN-β protein levels (pg / ml) obtained by ELISA in supernatants of cultured non-transduced MC38 cells treated with DMXAA (50 μg / mL) or vehicle 24 hours after irradiation (8 Gy) or without irradiation (0 Gy) and collected 24 h after DMXAA administration; Data are mean ± SEM (n = 3-4 samples / condition), ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test.

[0453] Figure 10, including Figures AD, shows the results obtained in STING KO mice with subcutaneous MC38 tumors that had been treated with 0 or 8 Gy irradiation as indicated, followed by intratumoral injection of AAV vectors encoding GFP (control) or hSTINGmut and / or IL12miR122T. Two days later, they received intraperitoneal DMXAA or vehicle. The figure illustrates serum levels of A) IFN-β, B) IL-12, C) IFN-γ at the indicated time points. Figure D represents IL-12 levels in tumor extracts obtained 6 h after DMXAA or vehicle treatment values ​​obtained by ELISA. Data are mean ± SEM (n = 4-5 mice per group), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test.

[0454] FIG. 11 , including panels AB, shows the expression of AAV8 encoding GFP under the control of a constitutive promoter (CAG) (AAV8.CAG.GFP) or AAV8 encoding IL-12 coupled to miR122T under the control of the ISRex4 promoter (AAV8-ISREx4-IL12miR122T) (MOI of 10 in both cases). 5 ) transduced mouse cells. Panel A shows a transduction efficiency of approximately 70% for AAV8-CAG. GFP-treated MC38 cells were independent of prior irradiation. Panel B illustrates that MC38 cells irradiated prior to AAV8-ISREx4-IL12miR122T transduction significantly enhanced IL-12 production. Data are mean ± SEM (n = 3 samples / condition). ***P < 0.005, unpaired t-test.

[0455] FIG. 12 , comprising panels AC, shows the effects of AAV8 encoding luciferase coupled to miR122T under the control of the ISREX4 promoter (AAV8-ISREx4-LucmiR122T) (MOI 10 10 μg / ml) in 4% paraformaldehyde (P < 0.05) mice treated with irradiation (8 Gy) or without irradiation (0 Gy) in combination with IFN-α, IFN-β, IFN-γ, or vehicle. 5 ) transduced B16 cells. The figure illustrates that luciferase activity was induced when incubated with IFN-α (A), IFN-β (B) or IFN-γ (C) even in the absence of irradiation, while additional irradiation resulted in a synergistic increase in luciferase activity. Data are mean ± SEM (n = 3 samples / condition), *p < 0.01, **P < 0.01, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test.

[0456] FIG. 13 , including FIGS. AO , shows radiation therapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (0.5×10 11vg) antitumor effect of the combined treatment on melanoma mouse model (A+B), hepatocellular carcinoma mouse model (CH) and subcutaneous glioblastoma mouse model (I+J), as well as the intratumoral and serum levels of IL-12 (K+L) and IFN-γ (M+N), and finally the efficiency of the combined method compared with the treatment with only one component of the combined method or without its component. A) In the melanoma mouse model (B16 cells), tumor volume evaluation at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) or AAV8-ISREx4-LucmiR122T combined with radiotherapy (circles). B) Hematoxylin-eosin staining shows a large number of lymphocytes infiltrating into tumor tissues three months after AAV8-ISREx4-IL12miR122T combined with radiotherapy. C) and D) Evaluation of tumor volume (C) or relative tumor volume (D) at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiotherapy (triangles) and AAV8-ISREx4-LucmiR122T combined with radiotherapy (circles) in a subcutaneous hepatocellular carcinoma mouse model generated by subcutaneous injection of PM299L hepatoma cells. E) Evaluation of survival showed complete survival of AAV8-ISREx4-IL12miR122T treated animals (dashed line) throughout the study period, compared with early death of all AAV8-ISREx4-LucmiR122T treated animals (solid line). F) and G) Evaluation of tumor volume (F) or relative tumor volume (G) at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiation therapy (triangles, dashed line) and AAV8-ISREx4-LucmiR122T combined with radiation therapy (circles, solid line) in a murine model of hepatocellular carcinoma generated by subcutaneous injection of NM53 G03 hepatoma cells. H) Evaluation of survival rate shows that AAV8-ISREx4-IL12miR122T treated animals survived completely throughout the study period (dashed line) compared to the early death of all AAV8-ISREx4-LucmiR122T treated animals (solid line). I) and J) Evaluation of tumor volume at the indicated time points after treatment with AAV8-ISREx4-IL12miR122T combined with radiation therapy (I) and AAV8-ISREx4-LucmiR122T combined with radiation therapy (J) in a subcutaneous murine glioblastoma model. Lines represent changes in tumor volume in individual animals during the study period. K)-N) Irradiation with 0 or 8 Gy followed by intratumoral injection of AAV8-ISREx4-IL12miR122T (0.5x10 11vg) Intratumoral values ​​of IL-12 (K), serum concentrations of IL-12 (L), intratumoral values ​​of IFN-γ (M), and serum concentrations of IFN-γ (N) in mice with subcutaneous B16 (melanoma) tumors. O) Evaluation of tumor volume at indicated time points after no treatment (black circles), treatment with AAV8-ISREx4-LucmiR122T combined with radiotherapy (white circles), treatment with AAV8-ISREx4-IL12miR122T but without radiotherapy (squares), and treatment with AAV8-ISREx4-IL12miR122T combined with radiotherapy (downward triangles) in a murine model of melanoma (B16 cells). Arrows indicate the start of the corresponding treatment. A), C), D), F), G) and O) data are mean ± SEM (n = 4-6 samples / condition) **P < 0.01, ****P < 0.0001, two-way ANOVA followed by Tukey's multiple comparison test (A and O) or Sidak's multiple comparison test (C, D, F, G). KN) data are mean ± SEM (4-8 samples per condition). Two-way ANOVA followed by Tukey's multiple comparison *P < 0.05, **P < 0.01, ***P < 0.001.

[0457] Figure 14, including Figures AB, shows the anti-tumor effect of combined treatment with radiation therapy and intratumoral injection of AAV8-ISREx4-IL12miR122T (AAV-IL12T) compared to corresponding controls injected with AAV8-ISREx4-LucmiR122T (AAV.Luc) in a murine melanoma model, in which some tumor cells lack MHC class I expression (B2Mko, A) or IFN-γ receptor (B16-IFNRGR-KO) or JAK1 (B16-JAK1-KO) (B). Data are mean ± SEM (n = 5-6 samples / sample). **P < 0.01, ****P < 0.0001, two-way ANOVA, followed by Tukey's multiple comparison test.

[0458] Figure 15, including Figures AP, shows the expression of IL-12 or IFN-β in different tumor cell lines, including melanoma (B16, Figures A, B, and KP), glioblastoma (GL261, Figures C and D), pancreatic cancer (KPC Figures E and F), colorectal cancer (MC38 Figures G and H), and breast cancer (TSA Figures I and J). Cultured cells (85,000 / well) received 0 or 8 Gy and were then incubated in the presence or absence of bicistronic vectors containing (A–J) scIL-12 and IFN-β separated by a P2A element under the control of the ISREx4 promoter (AAV8-ISREx4-IL12-IFNbmiR122T), (K–L) scIL-12 and IFN-β separated by a F2A element preceded by a linker domain consisting of the amino acid sequence GGSG (SEQ ID NO: 82) under the control of the ISREx4 promoter (AAV8-ISREx4-IL12-GGSGF2A-IFNbmiR122T), (M–N) scIL-12 and IFN-β separated by a short IRES element (IRES Gtx9, SEQ ID NO: 83) under the control of the ISREx4 promoter. Figure 5. ScIL-12 and IFN-β separated by IRES2 element (SEQ ID NO:83) (AAV8-ISREx4-IRESGtx9-IL12-IFNbmiR122T) or (O–P) scIL-12 and IFN-β separated by IRES2 element (SEQ ID NO:84) under the control of ISRex4 promoter (AAV8-ISREx4-IL12-IRES2-IFNbmiR122T). After 48 h, supernatants were collected and IL-12 and IFN-β were measured by ELISA. Both proteins were detectable only when incubated with vector, while the expression of both proteins was greatly increased after irradiation treatment. Data are mean ± SEM (n = 3 samples / condition). *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, one-way ANOVA followed by Sidak's multiple comparison test.

[0459] Figure 16, including Figures AB, shows serum levels of IL-12 (A) and IFN-β (B) in mice injected intravenously with bicistronic AAV8-ISREx4-IL12-IFNbmiR122T (black circles), monocistronic AAV8-ISREx4-IL12miR122T (grey circles), or monocistronic vector AAV8-ISREx4-IL12 lacking the miRNA122 target sequence (white circles). Data are mean ± SEM (n = 5-7 samples / condition). *p < 0.01, ****P < 0.0001, ns, not significant, two-way ANOVA, followed by Tukey's multiple comparison test.

[0460] Figure 17, including panels AB, shows the anti-tumor effects of combined treatment with radiation therapy and intratumoral injection of bicistronic AAV8-ISREx4-IL12-IFNbmiR122T (rectangles in A, downward triangles in B), monocistronic AAV8-ISREx4-IL12miR122T (upward triangles in A and B), and control vector AAV8-ISREx4-LucmiR122T (circles in A and B) on a melanoma mouse model in which tumor cells express MHC class I (A) or lack MHC class I expression (B). Data are mean ± SEM (n = 6 samples / condition). ****P < 0.0001, two-way ANOVA, followed by Tukey's multiple comparison test.

[0461] Figure 18, including Figures AD, shows transgene expression in irradiated subcutaneous tumors (NM53 liver cancer cells) compared to non-irradiated tumors at the opposite site when AAV8-ISREx4-LucmiR122T is administered by systemic route (intravenous administration). Figure B is a repeat experiment of the experiment in Figure A. Figure C shows luciferase activity in four mice with orthotopic NM53 liver cancer. Two mice were irradiated with 20Gy, and the other two were not irradiated, and then all mice were injected intravenously with AAV8-ISREx4-LucmiR122T. Luciferase activity was measured 6 days after vector injection. It can be seen that luciferase activity only appears in the liver area where those tumors have been previously irradiated with 20Gy. In addition, Figure D shows the size of MC38 subcutaneous tumors in mm after irradiation (20 Gy) or without irradiation as indicated, followed by intravenous injection of AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T). 3 Data are mean ± SEM (n = 5-6 mice per group), ****P < 0.0001, two-way ANOVA followed by Tukey post hoc test.

[0462] Figure 19, including Figures AD, shows the effects of different radiation doses on transgene expression from AAV8-ISREx4-LucmiR122T when the vector is administered by intratumoral injection. Figure A shows luciferase activity when the vector is injected into the tumor after irradiation with 15Gy, and then receives 3 additional doses of 15Gy (total dose = 60Gy). Figure B shows a tumor that receives 4 daily doses of 15Gy and then injects the vector (total dose = 60Gy). In Figure C, the tumor receives 60Gy, followed by intratumoral vehicle injection and two subsequent daily doses of 20Gy (total dose = 100Gy). In all cases, luciferase activity was assessed in the following days after vector injection (as shown). Therefore, Figures AC show different irradiation and treatment paradigms, and the data set is shown in Figure D. *P<0.05. After one-way ANOVA, it was followed by Sidak's multiple comparison test.

[0463] Fig. 20 Figure 2 shows the growth of B16 subcutaneous tumors in mm after irradiation (8 Gy) and intratumoral injection of AAV8-ISREx4-LucmiR122T (AAV.LucT) or AAV8-ISREx4-IL12miR122T (AAV.IL12T) as indicated, followed by administration of the immune checkpoint inhibitor anti-PD1 (αPD1) or the immune checkpoint inhibitor anti-CTLA-4 (αCTLA-4) or no further treatment. 3 Data are mean ± SEM (n = 5-6 mice per group), ns: not significant, ****P < 0.0001, two-way ANOVA followed by Tukey post hoc test.

[0464] Specific implementation form

[0465] Example 1: STING response to DMXAA in vitro

[0466] In the first set of three experiments, the responses of different STINGs to DMXAA were investigated.

[0467] Experiment 1: DMXAA dose response to different STING

[0468] The aim of this experiment was to examine which STINGs were sensitive to the STING agonist DMXAA and whether there was a dose-dependency to DMXAA. As a readout, the expression of the downstream target IFN-β was measured by quantitative real-time PCR (qPCR).

[0469] Materials and methods

[0470] To this end, cells from the murine cell line MC38 or cells from the human cell line Hep2G were transduced in vitro with lentivirus encoding wild-type murine STING (mSTINGwt), wild-type human STING (hSTINGwt) or human triple mutant STING-S162A / G230I / Q266I (hSTINGmut). Transduced cells were sorted by flow cytometry based on the expression of the reporter protein to obtain stable cell lines. Individual stable cells were cultured at 750 cells / mm 2 The cells were seeded at a density of 100 μg / mL in growth medium in a 24-well plate. 24 h after seeding, the cells were incubated with different doses of DMXAA for another 3 h (MC38 cells) or 6 h (Hep2G cells). Thereaft...

Claims

1. A recombinant expression system for combined gene and radiation therapy of a disease in a recipient mammal, wherein a. The recombinant expression system comprises a first nucleic acid construct encoding a target STING, wherein the target STING is responsive to a STING agonist that recognizes the target STING but not human STING and its natural variants, wherein the sequence encoding the target STING is operably linked to an element that allows expression and / or translation of the target STING; and / or a second nucleic acid construct comprising a therapeutic gene sequence, wherein the therapeutic gene sequence is operably linked to a STING-responsive promoter or a radiation-inducible promoter; and b. administering the recombinant expression system to the recipient mammal before, simultaneously with, or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system with low-dose radiation; and / or c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation and / or administering a STING agonist that recognizes the target STING but not human STING and its natural variants to the recipient mammal.

2. The recombinant expression system for use according to claim 1, wherein at least one other additional therapy selected from the group consisting of: a. a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR, more preferably selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TIL; and / or c. a therapy comprising at least one cancer vaccine; and / or d. A therapy comprising at least one exogenous cytokine, And wherein the at least one other additional therapy is applied before, simultaneously and / or after the administration according to step b.

3. The recombinant expression system for use according to claim 1 or 2, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases and infectious diseases, preferably a solid cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin's lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, urothelial carcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, neuroendocrine tumor, bile duct cancer or carcinoid tumor.

4. The recombinant expression system for use according to any one of claims 1 to 3, wherein the therapeutic gene sequence of the second nucleic acid construct encodes a protein or gene selected from the group consisting of: cytokines such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones; or genes such as shRNAs against cytokines, nuclear factors or hormones.

5. The recombinant expression system for use according to claim 4, wherein the protein encoded by the therapeutic gene sequence of the second nucleic acid construct is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), single-chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony-stimulating factor (GM-CSF), type I interferons (IFN-α and IFNβ), IFN-γ, TNF-α, CXCL10, FLT 3-ligands; immunostimulatory monoclonal antibodies or scFVs or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

6. The recombinant expression system for use according to any one of the preceding claims, wherein the second nucleic acid construct comprises at least two therapeutic gene sequences, each encoding an immunomodulatory protein, wherein The first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and The second immunomodulatory protein is an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β; or IL-15-sushi comprising or consisting of the amino acid sequence of SEQ ID NO:80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO:80, wherein the polypeptide has the biological activity of IL-15-sushi; or CXCL10, comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

7. The recombinant expression system for use according to any one of the preceding claims, wherein the second nucleic acid construct is a bicistronic or multicistronic expression cassette comprising two or more therapeutic gene sequences, each of which encodes a different immunomodulatory protein or a protein or RNA with anti-cancer properties, wherein a. the therapeutic gene sequence is separated by a sequence encoding an IRES; and / or b. The therapeutic gene is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide.

8. The recombinant expression system for use according to any one of the above claims, wherein the STING-responsive promoter of the second nucleic acid construct is selected from the group consisting of an IFN-β-responsive promoter, a CXCL9-responsive promoter, a CXCL10-responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFκB motif promoter, and a STAT6 motif promoter, preferably, wherein the STING-responsive promoter comprises at least one interferon-stimulated response element (ISRE).

9. The recombinant expression system for use according to any one of the preceding claims, wherein the STING-responsive promoter of the second nucleic acid construct is selected from the list consisting of: a. a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence having at least 75%, 80%, 85%, 90% or at least 95% sequence identity to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11; b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart; c. the nucleotide sequence according to SEQ ID NO: 12; d. The nucleotide sequence according to SEQ ID NO:

13.

10. A recombinant expression system for use according to any one of the above claims, wherein the second nucleic acid construct of the expression system comprising a therapeutic gene sequence further comprises at least one sequence motif, which inhibits the transgenic expression of the therapeutic gene sequence in tissues or cells that are not intended to express the protein, and the tissue is preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

11. The recombinant expression system for use according to claim 10, wherein the microRNA highly abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, preferably, wherein the microRNA abundant in the liver is miR-122.

12. The recombinant expression system for use according to any one of the preceding claims, wherein the low-dose radiation is based on photon radiation, wherein the photon radiation is preferably selected from the group consisting of: X-rays or gamma rays or particle radiation, wherein the particles of the particle radiation are preferably selected from the group consisting of: beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions.

13. The recombinant expression system for use according to any one of the preceding claims, wherein the total radiation dose of the low-dose radiation is 100 Gy or less, preferably 40 Gy or less, more preferably 8 Gy or less.

14. The recombinant expression system for use according to any one of the preceding claims, wherein the low dose radiation is administered in the form of stereotactic radiosurgery (SRS), intraoperative radiotherapy (IORT), stereotactic body radiotherapy (SBRT), single dose radiotherapy (SDRT), intensity modulated radiotherapy (IMRT), image guided radiotherapy (IGRT), stereotactic ablative radiotherapy (SABR), or hypofractionated radiotherapy preferably delivered by a linear accelerator (LINAC) machine, a gamma knife machine, or proton beam therapy.

15. The recombinant expression system for use according to any one of the preceding claims, wherein the time interval between the low-dose radiation of b. and the subsequent administration of the expression system is between 1 minute and 20 days, preferably between 10 minutes and 2 days, more preferably between 30 minutes and 24 hours.

16. The recombinant expression system for use according to any one of the preceding claims, wherein the subsequent low-dose radiation of c. administered after administration of the recombinant expression system is repeated at intervals of between 1 and 30 days, preferably between 1 and 5 days, more preferably between 1 and 3 days.

17. The recombinant expression system for use according to any one of the preceding claims, wherein after administration of the recombinant expression system comprising the first nucleic acid construct, a human non-responsive STING agonist is administered to the recipient mammal, preferably selected from the group consisting of 5,6-dimethylxanthone-4-acetic acid (DMXAA), 7-bromo-DMXAA, 7-iodo-DMXAA, 7-hydroxy-DMXAA, 7-formyl-DMXAA, 7-hydroxymethyl-DMXAA, 7-(2-hydroxyethyl)-DMXAA, flavone-8-acetic acid (FAA), 2,7-bis(2-dimethylaminoethoxy)fluoren-9-one (tilorone) and 10-carboxymethyl-9-acridone (CMA), more preferably wherein the STING agonist is DMXAA.

18. A recombinant expression system for combined gene and radiation therapy of a disease in a recipient mammal, wherein a. The recombinant expression system consists of a nucleic acid construct encoding at least two therapeutic gene sequences, which are operably linked to a STING-responsive promoter or a radiation-inducible promoter; and b. administering the recombinant expression system to the recipient mammal before, simultaneously with, or after irradiating the tissue or organ (target tissue or organ) of the recipient mammal to be transfected or transduced with the recombinant expression system with low-dose radiation; and / or c. After administering the recombinant expression system in step b), subjecting the target tissue or organ of the recipient mammal to low-dose radiation.

19. The recombinant expression system for use according to claim 18, wherein at least one other additional therapy selected from the group consisting of: a. a therapy comprising at least one checkpoint inhibitor, preferably selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, CTLA-4, LAG3, BTLA, B7H3, B7H4, TIM3, KIR or A2aR, more preferably selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2 or CTLA-4; and / or b. a therapy comprising at least one adoptive cell therapy, preferably selected from adoptive NK cell therapy, adoptive dendritic cell therapy or adoptive T cell therapy, wherein the adoptive T cell therapy is preferably CAR-T cell therapy or a therapy using TIL; and / or c. a therapy comprising at least one cancer vaccine; and / or d. A therapy comprising at least one exogenous cytokine, And wherein the at least one other additional therapy is applied before, simultaneously and / or after the administration according to step b.

20. The recombinant expression system for use according to claim 18 or 19, wherein the disease is selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases and infectious diseases, preferably a solid cancer selected from the group consisting of bladder cancer, bone cancer, brain cancer, breast cancer, thymic cancer, cervical cancer, colon cancer, esophageal cancer, gastric cancer, glioblastoma, head and neck cancer, Hodgkin lymphoma, primary liver cancer, metastatic liver cancer, gallbladder cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, Merkel cell carcinoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, urothelial carcinoma, kidney cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, uterine cancer, plasmacytoma, neuroendocrine tumor, bile duct cancer or carcinoid tumor.

21. A recombinant expression system for use according to any one of claims 18 to 20, wherein the therapeutic gene sequence of the nucleic acid construct encodes a protein selected from the group consisting of: cytokines such as chemokines, interferons, interleukins, lymphokines and tumor necrosis factors; hormones; growth factors; cell surface receptors; enzymes; transcription factors; tumor suppressor proteins; nanobodies; monoclonal antibodies; single-chain variable fragments (scFv); peptides that block the activity of cytokines, nuclear factors or hormones.

22. The recombinant expression system for use according to claim 21, wherein the protein encoded by the therapeutic gene sequence of the nucleic acid construct is selected from the group consisting of interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-10 (IL-10), interleukin-12 (IL-12), single-chain interleukin-12 (scIL-12), interleukin-15 (IL-15), interleukin-15-sushi (IL-15-sushi), interleukin-21 (IL-21), interleukin-23 (IL-23), granulocyte-macrophage colony stimulating factor (GM-CSF), type I interferons (IFN-α and IFNβ), IFN-γ, TNF-α, CXCL10, FLT 3-ligands; immunostimulatory monoclonal antibodies or scFVs or nanobodies that neutralize PD1, PDL1, CTLA4, CD137, TIM3, LAG3; blocking peptides against immunosuppressive factors such as TGF-β or IL-10 or FoxP3.

23. The recombinant expression system for use according to any one of claims 18 to 22, wherein the nucleic acid construct comprises at least two therapeutic gene sequences, each encoding an immunomodulatory protein, wherein The first immunomodulatory protein is a single-chain IL-12 comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 9 or 10, wherein the polypeptide has the biological activity of interleukin-12, and The second immunomodulatory protein is an IFN-β comprising or consisting of the amino acid sequence of SEQ ID NO: 79, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 79, wherein the polypeptide has the biological activity of IFN-β; or IL-15-sushi, comprising or consisting of the amino acid sequence of SEQ ID NO: 80, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 80, wherein the polypeptide has the biological activity of IL-15-sushi or CXCL10, comprising or consisting of the amino acid sequence of SEQ ID NO: 81, or a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 81, wherein the polypeptide has the biological activity of CXCL10.

24. The recombinant expression system for use according to any one of claims 18 to 23, wherein the nucleic acid construct is a bicistronic or multicistronic expression cassette comprising two or more therapeutic gene sequences, each of which encodes a different immunomodulatory protein or a protein or RNA with anti-cancer properties, wherein a. the therapeutic gene sequence is separated by a sequence encoding an IRES; and / or b. The therapeutic gene sequence is separated by a sequence encoding a 2A peptide, preferably a sequence encoding a P2A peptide.

25. The recombinant expression system for use according to any one of claims 18 to 24, wherein the STING-responsive promoter of the nucleic acid construct is selected from the group consisting of an IFN-β-responsive promoter, a CXCL9-responsive promoter, a CXCL10-responsive promoter, a STAT motif promoter, an IRSF3 motif promoter, an ISRE motif promoter, an NFκB motif promoter, and a STAT6 motif promoter, preferably, wherein the STING-responsive promoter comprises at least one interferon-stimulated response element (ISRE).

26. The recombinant expression system for use according to any one of claims 18 to 25, wherein the STING-responsive promoter of the nucleic acid construct is selected from the list consisting of: a. a nucleotide sequence comprising 1 to 10 copies, preferably 2 to 7 copies, more preferably 3 to 5 copies, and particularly preferably 4 copies of a sequence having at least 75%, 80%, 85%, 90% or at least 95% sequence identity to SEQ ID NO: 11 or a sequence identical to SEQ ID NO: 11; b. The nucleotide sequence according to a., wherein the copies are spaced up to 40 nucleotides apart, preferably 4 to 35 nucleotides, more preferably 10 to 30 nucleotides, particularly preferably 15 to 25 nucleotides apart; c. the nucleotide sequence according to SEQ ID NO: 12; d. The nucleotide sequence according to SEQ ID NO:

13.

27. The recombinant expression system according to any one of claims 18 to 26, wherein the nucleic acid construct of the expression system comprising two therapeutic gene sequences further comprises at least one sequence motif that inhibits transgenic expression of the therapeutic gene sequence in tissues or cells that are not intended to express the protein, the tissue being preferably the liver, wherein the sequence motif is preferably a target sequence of a microRNA that is highly abundant in the liver.

28. The recombinant expression system for use according to claim 27, wherein the microRNA highly abundant in the liver is selected from the group consisting of: miR-122, miR-192, miR-199a, miR-101, miR-99a, let7a, let7b, let7c, let7f, preferably, wherein the microRNA abundant in the liver is miR-122.

29. The recombinant expression system for use according to any one of claims 18 to 28, wherein the low-dose radiation is based on photon radiation, wherein the photon radiation is preferably selected from the group consisting of: X-rays or gamma rays or particle radiation, wherein the particles of the particle radiation are preferably selected from the group consisting of: beta particles, alpha particles, neutrons, muons, pions, protons or other heavier positive ions.

30. The recombinant expression system for use according to any one of claims 18 to 29, wherein the total radiation dose of the low-dose radiation is 100 Gy or less, preferably 40 Gy or less, more preferably 8 Gy or less.

31. The recombinant expression system of any one of claims 18 to 30, wherein the low dose radiation is administered in the form of stereotactic radiosurgery (SRS), intraoperative radiotherapy (IORT), stereotactic body radiotherapy (SBRT), single dose radiotherapy (SDRT), intensity modulated radiotherapy (IMRT), image guided radiotherapy (IGRT), stereotactic ablative radiotherapy (SABR), or hypofractionated radiotherapy preferably delivered by a linear accelerator (LINAC) machine, a gamma knife machine, or proton beam therapy.

32. The recombinant expression system for use according to any one of claims 18 to 31, wherein the time interval between the low-dose radiation of b. and the subsequent administration of the expression system is between 1 minute and 20 days, preferably between 10 minutes and 2 days, more preferably between 30 minutes and 24 hours.

33. The recombinant expression system for use according to any one of claims 18 to 32, wherein the subsequent low-dose radiation of c. administered after administration of the recombinant expression system is repeated at intervals of between 1 and 30 days, preferably between 1 and 5 days, more preferably between 1 and 3 days after administration of the recombinant expression system.

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