Non-viral vector for enhanced transcription

Abstract

The invention relates to a non-viral vector comprising a gene of interest functionally linked to a eukaryotic promoter and to a promoter of a cytoplasmic replication virus. The invention relates to a composition comprising: - a cytoplasmic replication virus; - a non-viral vector comprising a gene of interest functionally linked to at least one eukaryotic promoter and to at least one viral promoter that is functional in the cytoplasmic replication virus.

Description

NON-VIRAL VECTOR FOR ENHANCED TRANSCRIPTION

[0001] The present invention relates to a non-viral vector comprising a gene of interest functionally linked to a eukaryotic promoter and to a promoter of a cytoplasmic replicating virus and a pharmaceutical composition characterized in that it comprises at least one non-viral vector comprising a gene of interest functionally linked to a eukaryotic promoter and to a native promoter of said cytoplasmic replicating virus.

[0002] Methods of treating and / or preventing diseases based on the injection of a gene encoding a protein of interest have been the subject of studies and clinical trials for around thirty years.

[0003] Initially considered to replace a mutated gene, these methods have been increasingly used to induce an immune response against specific antigens of infectious diseases or tumor cells.

[0004] To date, the greatest success achieved using these methods is vaccination against coronavirus disease 2019 using compositions comprising an mRNA encoding the Sars-CoV2 spike protein. More generally, in the context of infectious diseases, the idea is to reproduce the effect obtained by conventional vaccinations by making the host cells express one or more antigens specific to the pathogen responsible for the disease. Compared to vaccination with an attenuated virus or a recombinant protein, these products have the advantage of being easier to produce and causing fewer side effects.

[0005] In cancer treatment, gene therapy has been used in three main ways. The first involves injecting genes encoding immunostimulatory molecules into the tumor site, capable of overcoming local inhibition of the immune system. For example, genes encoding interleukins have been injected directly into tumors. The local production of interleukins is believed to allow normal immune system activity and the destruction of cancer cells. However, the results obtained with this type of strategy have been disappointing.

[0006] The idea of ​​immunizing against pre-existing cancer cells is based on the observation that cancer cells express mutated proteins, aberrantly glycosylated proteins, or, in the case of tumors induced by viral infection, viral proteins. Within the tumor, the expression of these proteins does not lead to the production of an effective immune response. Indeed, the tumor is notably capable of creating a microenvironment that prevents the initiation of an immune response targeting these antigens and / or the cells that express them. Moreover, even if an immune response is induced, it does not always succeed in being effective due to the anergy that reigns within the tumor. It has therefore been postulated that it is more effective, in terms of the immune response generated, to inject the antigens associated with tumor cells into an area where the immune system is active.

[0007] Among the antigens associated with tumors, we can also cite tumor neoantigens resulting from the accumulation of mutations in a cell population which leads to cancerization. These mutations can result, for example, from exposure to ultraviolet rays or from carcinogenic substances in tobacco or other types. Some are directly involved in carcinogenesis, others are simply in a "passenger" situation, but all, due to the change of amino acids in the sequence of a protein, have the potential to be considered by the immune system as different from the self and therefore antigenic.

[0008] A second line of research involves injecting cancer cells with genes encoding cytotoxic molecules or causing the direct or indirect production of cytotoxic molecules. This strategy is expected to allow the destruction of tumor cells and possibly the release of antigens into the body, thus enabling the activation of the immune system.

[0009] The latest line of research has involved using nucleic acids encoding tumor-associated antigens to vaccinate patients. Indeed, producing the antigen by the patient's cells, rather than injecting them in situ, allows for better presentation of the antigen, more in line with what happens at the level of the cancer cells.

[0010] However, the anti-tumor response observed in the context of these different treatments is not better than the clinical responses observed in the context of conventional treatments.

[0011] It is therefore desirable to have new products and / or new methods for improving the immune response against the protein of interest encoded by the injected gene. Application EP16794685.4 describes compositions comprising a DNA or RNA vector comprising a gene encoding a protein of interest, a poxvirus and an antibody against CTLA4.

[0012] It is also desirable to have vectors, compositions of matter and methods for improving the expression of a gene of interest in eukaryotic cells, whether in vivo or in vitro.

[0013] Thus, the present invention relates to a non-viral vector comprising a gene of interest functionally linked to a eukaryotic promoter and to a promoter of a cytoplasmic replication virus.

[0014] In the context of the present invention, the term "cytoplasmic replication virus" is intended to designate a virus whose replication in eukaryotic cells takes place in whole or in part and preferably completely in the cytoplasm. For the sake of clarity, it is specified that the "cytoplasmic replication virus" according to the invention is a virus specific to eukaryotic cells. Thus, said "cytoplasmic replication virus" is not a bacteriophage and the "promoter of a cytoplasmic replication virus" is not the promoter of a bacteriophage, such as the promoter of bacteriophage T7.

[0015] According to a preferred embodiment of the invention, the cytoplasmic replicating virus is chosen from the group consisting of Megaviricetes, Algavirales, Phycodnaviridae, Imitervirales, Mimiviridae, Pimascovirales, Ascoviridae, Iridoviridae, Marseilleviridae, Pokkesviricetes, Asfuvirales, Asfarviridae, Chitovirales and Poxviridae.

[0016] According to a highly preferred embodiment of the invention, said cytoplasmic replication virus is a poxviridae (also called poxvirus).

[0017] In the context of the present invention, the term "promoter of a cytoplasmic replication virus" refers to a nucleic acid sequence to which an RNA polymerase of a cytoplasmic replication virus can bind and be activated.

[0018] In the context of the present invention, the term "eukaryotic promoter" refers to any nucleic acid sequence to which the endogenous RNA polymerase of a eukaryotic cell can bind and be activated to induce the initiation of transcription. For the sake of clarity, it is specified that a "eukaryotic promoter" is not necessarily a native promoter of a eukaryotic cell. Indeed, there are promoters (e.g. CMV) which are of viral origin and to which the endogenous RNA polymerase of a eukaryotic cell can bind and be activated.

[0019] In the context of the present invention, the term "functionally linked" is intended to indicate that the attachment and activation of an RNA polymerase on said promoter will result in the transcription of said gene of interest. Generally, to be functionally linked, the promoter is placed upstream of the nucleic acid sequence of the gene of interest.

[0020] In the context of the present invention, the term "non-viral vector" is intended to designate a nucleic acid sequence. This nucleic acid sequence is not included in a viral genome, nor encapsulated in a viral particle.

[0021] In the context of the present invention, the term "nucleic acid sequence" refers to a coding nucleic acid sequence of the DNA type.

[0022] In the context of the present invention, the expression "gene of interest" refers to any nucleic acid sequence transcribed into an mRNA that may have prophylactic or therapeutic activity. Preferably, said gene of interest encodes an antigen specific to a tumor cell or a microorganism responsible for an infectious disease.

[0023] According to one embodiment of the invention, said nucleic acid sequence is selected from the group comprising plasmids and linear DNA or RNA vectors.

[0024] According to one embodiment of the invention, said nucleic acid sequence is naked, that is to say that it is not associated with molecules promoting its penetration into eukaryotic cells such as, for example, cationic polymers, viral proteins, lipids and / or liposomes.

[0025] According to a preferred embodiment of the invention, said nucleic acid sequence is a double-stranded closed linear nucleic acid. In addition, closed linear nucleic acids by their mode of production are accompanied by very few production residues, in particular bacterial, which makes it possible to avoid heavy purification steps and allows faster and less expensive use.

[0026] Closed linear nucleic acids are well known to those skilled in the art, their structure, their functions and the methods for their production are described in particular in documents CN103080337, EP2601312, GB201013153, JP2013535210, US2013216562, WO12017210, AU2010209532, CA2751130, CN102301010, DK2391731, EA021069, EA201101141, EP2391731, EP2612925, ES2400890, GB200901593, HK1159693, IL213930, IN05006CN2011, JP2012516147, KR20110107846, MX2011007937, NZ594004, SG173102, US2012282283, US9109250, WO10086626.

[0027] The present invention also relates to a composition comprising:

[0028] - a cytoplasmic replication virus,

[0029] - a non-viral vector comprising a gene of interest functionally linked to at least one eukaryotic promoter and at least one viral promoter functional in said cytoplasmic replicating virus.

[0030] The present invention also relates to a set of parts for sequential or simultaneous use comprising:

[0031] - a cytoplasmic replication virus,

[0032] - a non-viral vector comprising a gene of interest functionally linked to at least one eukaryotic promoter and to at least one viral promoter functional in said cytoplasmic replicating virus.

[0033] In the context of the present invention, the term "functional in said cytoplasmic replicating virus" is intended to designate a promoter to which an RNA polymerase encoded by the genome of said virus can bind and be activated. Preferably, said virus is non-recombinant and the gene encoding said RNA polymerase is naturally present in said virus.

[0034] It is thus understood that the simultaneous injection of the virus and the non-viral vector comprising a eukaryotic promoter and a cytoplasmic replicating virus promoter will allow the expression of the gene of interest in the cytoplasm and in the nucleus of the cell into which they are injected. In a non-viral vector whose gene of interest is functionally linked to a eukaryotic promoter, the transcription of the gene of interest is subject to the prior penetration of the vector into the nucleus of the cell. Indeed, the enzymatic machinery necessary for said transcription is absent from the cytoplasm of the cell.

[0035] Furthermore, the presence of a cytoplasmic replicating virus will allow the transcription of the gene of interest in the cytoplasm, because the virus will provide the elements necessary for the transcription of the gene of interest using the viral promoter functionally linked to said gene of interest.

[0036] This combination makes it possible to significantly increase the expression, in vivo or in vitro, of the gene of interest in the transfected cells and thus to increase the effect produced by this expression. This effect may in particular be an increase in the immune response against the protein expressed from the gene of interest or an increase in the specific activity of said protein.

[0037] According to a preferred embodiment of the invention, said cytoplasmic replication virus is a poxvirus.

[0038] This embodiment is particularly preferred in the case where the gene of interest encodes an immunogenic protein because poxviruses are known to have an adjuvant activity which will add to the overexpression of the immunogen to improve the immune response of the transfected organism.

[0039] According to a preferred embodiment, the cytoplasmic replicating virus does not comprise heterologous sequences.

[0040] According to a preferred embodiment, the poxvirus is derived from a vaccinia virus, a canaripox or a fowlpox.

[0041] According to a preferred embodiment, said poxvirus is derived from a vaccinia virus selected from the Copenhagen, Wyeth and modified Ankara (MVA) strains.

[0042] According to a preferred embodiment, said poxvirus is derived from a vaccinia virus of the Copenhagen strain. According to a preferred embodiment, said poxvirus is derived from a vaccinia virus of the MVA strain.

[0043] In the context of the present invention, the term “drift” is intended to mean that said virus belongs to said strain.

[0044] According to a preferred embodiment of the invention, said eukaryotic promoter is the CMV promoter.

[0045] According to a preferred embodiment of the invention, said non-viral vector further comprises the regulatory elements ensuring the expression of said gene of interest.

[0046] According to a preferred embodiment of the invention, said non-viral vector is chosen from the group consisting of plasmids, closed linear DNA vectors, or even mRNA vectors.

[0047] According to a preferred embodiment of the invention, the regulatory elements ensuring the expression of said gene of interest in eukaryotic cells is a translation initiation region in host cells.

[0048] According to a preferred embodiment of the invention, said cytoplasmic replication virus is non-recombinant.

[0049] According to a preferred embodiment of the invention, the cytoplasmic replicating virus is alive or killed.

[0050] According to a preferred embodiment of the invention, said composition comprises a pharmaceutically acceptable carrier allowing its administration by injection to humans or animals.

[0051] Said invention also relates to a vector, a composition or a set of parts according to the invention, intended to be used for the treatment of a cancer or a tumor.

[0052] The said invention also relates to a vector, a composition or a set of parts according to the invention, intended to be used for the in vitro transfection of a eukaryotic cell.

[0053] The said invention also relates to a method for in vitro transfection of a eukaryotic cell, remarkable in that the said eukaryotic cell is brought into contact with a vector, a composition or a set of parts according to the invention.

[0054] Said invention also relates to a composition according to the invention, intended to be used for the treatment of cervical cancer, ENT cancer or any other tumor induced by the HPV virus, liver cancer linked to the chronic hepatitis C virus, or cancer expressing the MUC1 protein.

[0055] Said invention also relates to a vector, a composition or a set of parts according to the invention, intended to be used for the treatment of an infectious disease.

[0056] Said invention also relates to a composition according to the invention for use in a method for vaccinating against the protein encoded by the gene of interest. Detailed description of the invention

[0057] Non-viral vectors for expression in eukaryotic cells are well known to those skilled in the art and are commercially available. These include, in particular, the plasmids pCI-neo (GenBank® accession number U47120, Promega® 1841), pVIVO2-mcs (Invivogen®, pvivo2-mcs), pVAX1 (ThermoFisher® V26020), pIRES (Clontech®, PT3266-5) and pcDNA 3.1 (ThermoFisher® V79020).

[0058] The eukaryotic promoters that can be used in a non-viral vector are well known to those skilled in the art. They can in particular be found in the Eukaryotic promoter database. In the context of the present invention, the eukaryotic promoter is preferably chosen from the group consisting of the CMV promoter, the EF1a promoter, the SV40 promoter, the PGK1 promoter, the Ubc promoter, the human bacterium promoter, the CAG promoter, the TRE promoter, the UAS promoter, the Ac5 promoter, the polyhedrin promoter, the CaMKIIa promoter, the GAL1 promoter, the TEF1 promoter, the GDS promoter, the ADH1 promoter, the CaMV35S promoter, the Ubi promoter, the H1 promoter and the U6 promoter. Preferably, said eukaryotic promoter is a CMV promoter.

[0059] The poxviral promoters that can be used in the non-viral vector are known to those skilled in the art and have been described in particular by Alharbi et al. ((2019) Poxviral promoters for improving the immunogenicity of MVA delivered vaccines, Human Vaccines & Immunotherapeutics, 15:1, 203-209).

[0060] In the context of the present invention, the poxviral promoter is preferentially chosen from the group consisting of the promoters P11, I1L, p7.5, TK, F7L, H5R, mH5, pSyn, SSP, pHyb, LEO, pB8, pF11, B8R, K6L, A44L, C1IR, B2R, and FP4b.

[0061] Said non-viral vector comprises, in addition to said promoters, the regulatory elements ensuring the expression of the gene(s) of interest in eukaryotic cells.

[0062] Preferably, the regulatory elements ensuring the expression of the gene(s) of interest in eukaryotic cells are chosen from the group comprising ribosome binding sites, the start codon, the termination codon, Kozak sequences, poly A and introns.

[0063] Said non-viral vector may comprise several genes of interest each encoding a different protein. In the case of a plurality of genes of interest, the latter may be placed under the control of identical or different regulatory elements.

[0064] According to a preferred embodiment, said gene of interest encodes a protein selected from the group consisting of all or part of Brachyury, CA125, CEA, EGFR, HER-2 / neu, KSA, Mesothelin, MUC-1, NY-ESO, p53, PAGE-4, PAP, PSA, PSCA, PSMA, Ras, sTn, TARP, VEGF, Aberrant class II protein, Anti-idiotype, B1, CD19, CD20, CD22, CD25, CD36, MAGE protein, MART, gp100, Tyrosinase, CD2, CD3, GM2, proteins of the HRV-H and HRV-V virus. Preferably, said gene of interest encodes the entirety of said protein. Alternatively, said gene of interest encodes a portion of said protein of interest comprising at least one specific epitope of said protein.

[0065] According to another preferred embodiment, said gene of interest codes for all or part of an antigen chosen from the group consisting of the antigens of the cholera bacillus, Sars-Cov2, dengue, Corynebacteriumdiphtheriae, hepatitis virus, haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza virus, Japanese encephalitis virus, plasmodium, measles virus, Neisseria meningitidis, mumps virus, Bordetella pertussis, Bordetella parapertussis., poliovirus, rabies virus, rotavirus, rubivirus, Clostridium tetani bacteria, tick-borne meningoencephalitis virus, Mycobacterium tuberculosis, Salmonella enterica, varicella virus, enterotoxic Escherichia coli, group B Streptococcus (GBS), herpes simplex virus, HIV, Neisseria gonorrhoeae, non-typhoidal salmonella, norovirus, respiratory syncytial virus (RSV), schistosoma, shigella, group A streptococcus (GAS). Preferably, said gene of interest encodes the entirety of said antigen. Alternatively, said gene of interest encodes a portion of said antigen comprising at least one specific epitope of said protein.

[0066] According to another preferred embodiment of the invention, said gene of interest encodes a neoantigen.

[0067] Indeed, current techniques allow the complete sequencing of the genetic heritage of a patient's cancer cells and the identification of specific mutations by comparison with the patient's constitutional characteristics. If these mutations are non-synonymous, belong to coding sequences expressed by the tumor, and are supposedly immunogenic due to their characteristics, then these mutations can be used to induce immunization against the tumor.

[0068] Mutations in the tumor genome and the resulting neoantigens are a unique combination restricted to a given patient's tumor, or even to the clones that compose that tumor.

[0069] To do this, the cancer cells of a patient to be treated are analyzed from a biopsy or surgical specimen. The DNA and RNA of the cancer cells, as well as the constitutional DNA from a blood sample, are extracted and sequenced.

[0070] The DNA sequence of healthy cells is compared to the DNA sequence of cancer cells and somatic mutations in coding and expressed regions are identified.

[0071] The mutated coding and expressed sequences are processed using algorithms to define for each patient which mutations are potentially the most antigenic and can serve as a target. These mutated, non-synonymous, coding, expressed sequences, specific to the patient's cancer cells, called neoantigens, are then generated by DNA or DNA synthesis and then put in the form of an expression vector (plasmid or closed linear nucleic acid or RNA). According to a preferred embodiment of the invention, using the method described in patent application EP2391731.

[0072] The poxvirus used in the composition is preferably an MVA virus. The latter is derived from the Ankara strain of vaccinia virus by successive passages on chicken embryo cells. These different passages led to the attenuation of this virus, which was thus able to be used in the latest smallpox vaccination campaigns.

[0073] The description of the different strains of MVA, the methods for possibly inserting exogenous genes into its genome as well as the methods for producing and purifying this virus are notably accessible in the documents WO0168820, WO0242480, WO03008533, WO03048184, WO03053463, WO03054175, WO03088994, WO03097675, WO03097844, WO03097845, WO03097846, WO04048582, WO04048606, WO05054484, WO06089690, WO08028665, WO08045346, WO08131926, WO08131927, WO08138533, WO09052328, WO09152969, WO10057650, WO10060632, WO10102822, WO11042180, WO11092029, WO12010280, WO12048817, WO12059243, WO13083254, WO13189611, WO14019718, WO14037124, WO14062778, WO14063832, WO9813500, WO9915692.

[0074] Preferably, the composition according to the invention further comprises a pharmaceutically acceptable carrier. In the context of the present invention, the term "pharmaceutically acceptable carrier" is intended to designate all carriers, solvents, diluents, excipients, adjuvants, dispersion media, and the like, compatible with pharmaceutical administration.

[0075] The composition for use in the invention is suitably buffered so as to be suitable for human use at physiological or slightly basic pH.

[0076] The composition according to the invention can be administered to the patient by a variety of administration methods such as, for example, subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, intratumoral, intravascular, intra-arterial routes.

[0077] Injections can be given with conventional syringes and needles, but preferably via a needle-free injection device such as bioject(r).

[0078] The administration of the composition according to the invention can take place in a single dose or repeated after a certain time interval ranging from one day to one year. Preferably, the administration will take place weekly seven times in a row and then once every three weeks.

[0079] The appropriate dosage may be adapted according to various parameters, in particular the mode of administration, the composition used, the age, health, and weight of the host organism, the nature and extent of the symptoms, the type of associated treatment, the frequency of treatment.

[0080] A person skilled in the art is able to determine the appropriate amounts of each element within the composition according to the invention. For example, the poxvirus can be used in an amount of between 10 4 at 10 9 pfu, the nucleic acid sequence in an amount between 1 µg and 10 mg per injection.

[0081] Preferably, the composition according to the invention can be used in conjunction with radiotherapy, chemotherapy, surgery and / or other immunotherapy products such as anti-PD1 and anti-PDL1 antibodies, or the combination of several of these treatments at the same time.

[0082] Said invention also relates to a composition according to the invention, intended to be used for the treatment of a cancer or a tumor.

[0083] Preferably, said cancer is a carcinoma and said gene of interest encodes a protein chosen from the group consisting of all or part of the proteins Brachyury, CA125, CEA, EGFR, HER-2 / neu, KSA, Mesothelin, MUC-1, NY-ESO, p53, PAGE-4, PAP, PSA, PSCA, PSMA, Ras, sTn, TARP and VEGF.

[0084] Preferably, said cancer is a leukemia and said gene of interest encodes a protein chosen from the group consisting of all or part of the aberrant proteins of MHC class II, Anti-idiotype, B1, CD19, CD20, CD22, CD25 and CD36.

[0085] Preferably, said cancer is a melanoma and said gene of interest encodes a protein chosen from the group consisting of all or part of the protein MAGE, MART, gp100, Tyrosinase, CD2, CD3, GM2.

[0086] Preferably, said cancer is induced by a retrovirus and said gene of interest encodes a protein chosen from the group consisting of all or part of the proteins of the HRV-H and HRV-V virus.

[0087] Preferably, said gene of interest codes for the entirety of said protein. Alternatively, said gene of interest codes for a portion of said protein of interest comprising at least one epitope specific to said protein.

[0088] The said invention also relates to a composition or set of parts according to the invention, intended to be used for the treatment of cervical cancer, ENT cancer or any other tumor induced by the HPV virus, liver cancer linked to the chronic hepatitis C virus, or cancer expressing the MUC1 protein.

[0089] Said invention also relates to a composition or set of parts according to the invention for use in a method for vaccinating against the protein encoded by the gene of interest.

[0090] Preferably, said infectious disease is selected from the group comprising cholera, COVID-19, dengue fever, diphtheria, hepatitis, haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza, Japanese encephalitis, malaria, measles, meningococcal meningitis, mumps, whooping cough, pneumococcal disease, poliomyelitis, rabies, diseases caused by rotavirus, rubella, tetanus, tick-borne encephalitis, tuberculosis, typhoid, chickenpox, diseases caused by enterotoxigenic Escherichia coli, diseases caused by group B Streptococcus (GBS), diseases caused by herpes simplex virus, AIDS, malaria, diseases caused by Neisseria gonorrhoeae, non-typhoidal salmonella diseases, norovirus diseases, paratyphoid fever, respiratory syncytial virus (RSV) diseases, schistosomiasis disease,diseases caused by shigella, diseases caused by group A streptococcus (GAS), tuberculosis and yellow fever.

[0091] According to a preferred embodiment of the invention, the composition or the set of parts according to the invention is intended to be injected subcutaneously, intramuscularly, orally, nasally or intratumorally.

[0092] According to a completely preferred embodiment of the invention, the composition or the whole part according to the invention is intended to be injected subcutaneously.

[0093] Preferably, said composition or said set of parts according to the invention comprises from 1 µg to 20 mg µg of non-viral vector according to the invention.

[0094] Preferably, said composition or said set of parts according to the invention comprises 10 4 at 10 9 pfu of cytoplasmic replicating virus.

[0095] According to a preferred embodiment of the invention, said composition or said set of parts further comprises an anti-CTLA4 antibody or antibody fragment.

[0096] The co-administration of an anti-CTLA4 antibody or antibody fragment is likely to strengthen the immune response induced by immunotherapy. For this purpose, doses reduced to 1 / 10 of the usual systemic dose are assumed to be sufficient. At these doses and after co-injection, the concentration in the drainage lymph node of the injection site is assumed to be higher than by systemic route, while the systemic concentration itself is greatly reduced, as is the risk of adverse effects. Finally, the composition according to the invention will preferably comprise an anti-CTLA4 antibody or antibody fragment, even more preferably ipilimumab. In the context of the present invention, the term antibody also designates bispecific antibodies comprising at least one CTLA-4-specific paratope.

[0097] The present invention also relates to a vaccine comprising a composition or set of parts according to the invention.

[0098] The said vaccine may be intended for a first injection (prime) in a naive individual and / or for a subsequent injection (boost) in an individual having received an identical or different injection directed against the same epitope.

[0099] Thus, said invention also relates to a set of parts for vaccination comprising:

[0100] - a first vaccine according to the invention for a first injection (prime),

[0101] - a second vaccine according to the invention for a subsequent injection (boost).

[0102] The compositions, set of parts and vaccine according to the invention are preferably included in a pharmaceutically acceptable medium. Examples Example 1

[0103] The effectiveness of the compositions according to the invention in stimulating the immune system was measured in C57BL / 6 mice.

[0104] The non-viral vectors used include the ovalbumin-encoding gene operably linked to the CMV promoter (pVax1-OVA) or the ovalbumin-encoding gene operably linked to the CMV promoter and the VVH5 promoter (pVax1-VVH5-OVA).

[0105] Four groups each comprising 5 female mice aged 8 weeks received respectively:

[0106] - 200µL comprising 100 µg of the pVax1-OVA vector, 50µg of anti-CTLA4 in PBS.

[0107] - 200µl comprising 100 µg of the pVax1-VVH5-OVA vector, 50µg of anti-CTLA4 in PBS

[0108] - 200µl comprising 100 µg of the vector pVax1-VVH5-OVA 2.5 10 5 pfu of MVA, 50µg of anti-CTLA4 in PBS,

[0109] - 200µL of PBS.

[0110] The different compositions were injected subcutaneously into the left flank of the mice. The injections took place on D0, D5, D10 and D17.

[0111] The animals were sacrificed on day 25 and their spleens were collected. The number of T lymphocytes expressing IFNg, specific for ovalbumin, was measured. An increase in this number was observed in mice receiving the pVax1-VVH5-OVA vector in combination with MVA.

Claims

Non-viral vector comprising a gene of interest functionally linked to a eukaryotic promoter and to a promoter of a virus whose replication in eukaryotic cells takes place in whole or in part in the cytoplasm. Composition comprising:- a virus whose replication in eukaryotic cells takes place in whole or in part in the cytoplasm,- a non-viral vector comprising a gene of interest functionally linked to at least one eukaryotic promoter and to at least one viral promoter functional in said cytoplasmic replication virus. Set of parts for sequential or simultaneous use comprising- a virus whose replication in a eukaryotic cell takes place in whole or in part in the cytoplasm,- a non-viral vector comprising a gene of interest functionally linked to at least one eukaryotic promoter and to at least one viral promoter functional in said cytoplasmic replication virus. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that said virus whose replication in eukaryotic cells takes place in whole or in part in the cytoplasm is a poxvirus and even more preferably an MVA. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that said promoter of a cytoplasmic replication virus is selected from the group consisting of promoters P11, I1L, p7.5, TK, F7L, H5R, mH5, pSyn, SSP, pHyb, LEO, pB8, pF11, B8R, K6L, A44L, C1IR, B2R, and FP4b. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that said eukaryotic promoter is selected from the group consisting of the CMV promoter, the EF1a promoter, the SV40 promoter, the PGK1 promoter, the Ubc promoter, the human b actin promoter, the CAG promoter, the TRE promoter, the UAS promoter, the Ac5 promoter, the polyhedrin promoter, the CaMKIIa promoter, the GAL1 promoter, the TEF1 promoter, the GDS promoter, the ADH1 promoter, the CaMV35S promoter, the Ubi promoter, the H1 promoter and the U6 promoter. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that said non-viral vector further comprises the regulatory elements ensuring the expression of said gene of interest. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that the non-viral vector is chosen from the group consisting of plasmids and linear DNA vectors. Vector according to claim 7, composition according to claim 7 or set of parts according to claim 7, characterized in that the regulatory elements ensuring the expression of said gene of interest in eukaryotic cells is a translation initiation region in host cells. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, characterized in that said cytoplasmic replicating virus is non-recombinant. Composition according to one of the preceding claims, characterized in that it comprises a pharmaceutically acceptable carrier allowing its administration by injection to humans or animals. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3, for its use for the treatment of a cancer or a tumor. Vector, composition or set of parts according to claim 12 characterized in that said cancer is a carcinoma and said gene of interest encodes a protein chosen from the group consisting of all or part of the proteins Brachyury, CA125, CEA, EGFR, HER-2 / neu, KSA, Mesothelin, MUC-1, NY-ESO, p53, PAGE-4, PAP, PSA, PSCA, PSMA, Ras, sTn, TARP and VEGF. Vector, composition or set of parts according to claim 12 characterized in that said cancer is a leukemia and said gene of interest encodes a protein chosen from the group consisting of all or part of the aberrant proteins of MHC class II, Anti-idiotype, B1, CD19, CD20, CD22, CD25 and CD36. Vector, composition or set of parts according to claim 12 characterized in that said cancer is a melanoma and said gene of interest encodes a protein chosen from the group consisting of all or part of the protein MAGE, MART, gp100, Tyrosinase, CD2, CD3, GM2. Vector, composition or set of parts according to claim 12 characterized in that said cancer is induced by a retrovirus and said gene of interest encodes a protein chosen from the group consisting of all or part of the proteins of the HRV-H and HRV-V virus. Vector, composition or set of parts according to claim 12 characterized in that said gene of interest codes for a neo-antigen. Vector according to claim 1, composition according to claim 2 or set of parts according to claim 3 for its use for vaccination against an infectious disease. Vector, composition or set of parts according to claim 18 characterized in that said infectious disease is selected from the group comprising cholera, COVID-19, dengue fever, diphtheria, hepatitis, haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza, Japanese encephalitis, malaria, measles, meningococcal meningitis, mumps, whooping cough, pneumococcal disease, poliomyelitis, rabies, diseases caused by rotavirus, rubella, tetanus, tick-borne encephalitis, tuberculosis, typhoid, chickenpox, diseases caused by enterotoxigenic Escherichia coli, diseases caused by group B Streptococcus (GBS), diseases caused by herpes simplex virus, AIDS, malaria, diseases caused by Neisseria gonorrhoeae, diseases caused by non-typhoidal salmonella, diseases caused by norovirus, paratyphoid fever,diseases caused by respiratory syncytial virus (RSV), schistosomiasis disease, diseases caused by shigella, diseases caused by group A streptococcus (GAS), tuberculosis and yellow fever.