Enveloped virus for transfection of exogenous nucleic acids

Abstract

The invention relates to a method for obtaining an enveloped virus comprising an exogenous nucleic acid sequence between at least one of its envelopes and its capsid, characterised in that the method comprises a step of bringing an enveloped virus into contact with a cationic polypeptide / exogenous nucleic acid sequence complex.

Description

Enveloped virus for transfection of exogenous nucleic acids.

[0001] The present invention falls within the field of biotechnology, and more particularly within that of the manipulation of enveloped viruses. It relates to enveloped viruses incorporating exogenous nucleic acids between their membrane and their capsid. This invention also encompasses methods for preparing these viruses as well as their use for the transformation of eukaryotic cells. Furthermore, the invention provides for the use of these viruses for the treatment or prevention of various diseases.

[0002] For about thirty years, methods for treating and preventing diseases based on the injection of genes coding for proteins of interest have been the subject of research and clinical trials. Initially intended to replace a mutated gene, these methods have progressively been used to induce an immune response against specific antigens of infectious diseases or tumor cells.

[0003] To date, the most significant success of these methods is the COVID-19 vaccination, using compositions containing mRNA encoding the SARS-CoV-2 spike protein. More broadly, for infectious diseases, the goal is to replicate the effects of conventional vaccinations by inducing host cells to express one or more pathogen-specific antigens. Compared to vaccinations using attenuated viruses or recombinant proteins, these methods offer the advantage of being simpler to produce and causing fewer side effects.

[0004] Regarding cancer treatment, gene therapy has been explored along three main lines. First, local immune stimulation. This approach involves injecting genes encoding immunostimulatory molecules directly into the tumor to overcome the inhibition of the immune system. For example, genes encoding interleukins have been introduced into tumors, in the hope that the local production of these molecules would reactivate the immune system to destroy cancer cells. However, the results obtained have been disappointing.

[0005] Gene therapy has also been used to immunize against pre-existing cancer cells. This strategy is based on the fact that cancer cells express mutated proteins, aberrantly glycosylated proteins, or viral proteins in the case of tumors of viral origin. In the tumor microenvironment, these proteins do not induce an effective immune response, primarily due to the presence of immune anergy. Therefore, it has been proposed to inject antigens associated with tumor cells into an area where the immune system is active, in order to obtain a more effective response.

[0006] Tumor neoantigens, resulting from accumulations of mutations in cells, can also be distinguished. These mutations can be triggered by exposure to UV radiation, carcinogenic substances in tobacco, or other factors. These mutations alter protein sequences, making the proteins potentially antigenic to the immune system.

[0007] This approach aims to vaccinate patients with nucleic acids encoding these tumor-associated antigens, allowing for better antigen presentation, similar to that observed in cancer cells.

[0008] Finally, gene therapy has also been used to inject cytotoxic genes into cancer cells. This strategy involves introducing into tumor cells genes that code for cytotoxic molecules or genes capable of indirectly inducing the production of such molecules, causing the destruction of cancer cells and the release of antigens, which can stimulate an immune response.

[0009] These different strategies require the use of vectors capable of transporting the nucleic acid of interest to the target cells. Several types of vectors have been developed, adapted to the organism being treated, the target cell, the injection site, and / or the size of the nucleic acid to be transported.

[0010] Viruses, due to their natural ability to inject their genome into cells, have been used in gene therapy for many years. Commonly used virus families include adeno-associated viruses, adenoviruses, retroviruses, and poxviruses. Viruses allow the insertion of large exogenous nucleic acids and offer good protection to these exogenous nucleic acids in the target organism. However, the insertion of an exogenous nucleic acid into the viral genome, followed by the production of viral particles, involves numerous steps, including the culture of eukaryotic cells and the purification of the produced viruses. These procedures are cumbersome and poorly suited to the mass production of personalized batches, which are necessary to specifically treat cancers according to each patient's genetic characteristics.

[0011] Furthermore, there are vectors made up of simple molecules capable of binding to a nucleic acid and facilitating its entry into cells. Examples of these molecules include cationic peptides and lipids.

[0012] Naked nucleic acids, not associated with viral or molecular vectors, can also be used in the form of plasmids, RNA, or single-stranded DNA. These vectors are simple and quick to prepare and are therefore ideal for personalized therapies. However, they exhibit significant susceptibility to degradation in the injected organism and a limited capacity to transform target cells.

[0013] The WO2018191750 document describes delivery systems for large payloads, such as CRISPR proteins, using composite viral particles. These systems involve either the adsorption of a viral particle onto a liposome or the creation of hybrid capsid proteins fused to a non-capsid protein. However, WO2018191750 neither describes nor suggests a structure in which an exogenous nucleic acid would be inserted and localized into the space between the native envelope and the capsid of an intact enveloped virus. The approaches in WO2018191750 rely on the creation of artificial complexes or the genetic engineering of viral proteins, rather than using the native virion structure to house a payload.

[0014] Document WO2022109503 proposes an approach that involves re-engineering the surface of enveloped viruses. The method described in WO2022109503 explicitly involves removing the virus's native envelope to isolate the capsid, followed by the application of a completely artificial coating (e.g., silica-based) to this capsid.

[0015] Therefore, there is a need for a vector that combines the advantages of both viral and non-viral vectors.

[0016] Thus the present invention relates to an enveloped virus characterized in that it comprises an exogenous nucleic acid sequence between at least one of its envelopes and its capsid.

[0017] According to a preferred embodiment of the invention, said envelope is the native envelope of said virus.

[0018] In the context of the present invention, the terms "native envelope," "natively enveloped," or their grammatical variations, refer to the lipid envelope that the virus naturally acquires during its replication cycle by budding through a host cell membrane (such as the plasma membrane, the endoplasmic reticulum membrane, or the Golgi apparatus membrane). This native envelope is characterized by the presence of integrated viral glycoproteins that are essential for the virus's infectivity and natural tropism.

[0019] In contrast, a "non-native" or "artificial" envelope or coating would be a layer added ex vivo after virion production, such as a silica coating, titanium dioxide coating, liposome, or any other synthetic layer that is not biologically acquired during the viral budding process. The invention relies on preserving this native envelope and utilizing the space it defines with the capsid.

[0020] The invention relates to a modified enveloped virus, as well as its method of obtaining and its uses. Unlike prior art approaches which consist of (i) integrating a transgene into the viral genome, (ii) fusing a protein of interest to the viral capsid, or (iii) completely replacing the viral envelope with an artificial coating, the present invention is based on the discovery that the natural space existing between the native envelope and the capsid of an enveloped virus can be used to house an exogenous nucleic acid sequence.

[0021] Thus, the unique structure of the virus according to the invention is that the exogenous nucleic acid is physically contained and sequestered in the natural periplasmic space of the virion, i.e. between the capsid (containing the native viral genome) and the native viral envelope.

[0022] This configuration offers the technical advantage of enabling the co-delivery of two nucleic acid payloads (the viral genome and the exogenous nucleic acid) through a simple and rapid preparation process that does not require complex genetic engineering of the virus. The virus retains its native envelope, thus preserving its infectivity and natural tropism, while carrying an additional payload.

[0023] The enveloped virus allows the transport of the exogenous nucleic acid sequence into a cell to be transfected, while simultaneously enabling the transfer of the virus's genome into that cell. This means that the user can, for example, easily combine a customized sequence of interest, in the form of this exogenous nucleic acid sequence, with a recombinant gene of interest present in the genome of the enveloped virus.

[0024] In the context of the present invention, the term "enveloped virus" refers to a virus whose outer envelope consists of a lipid membrane. The viral envelope is composed of a lipid membrane derived from the host cell's membrane bilayer, into which viral proteins are embedded. Depending on the type of virus, this envelope may originate from the cell's outer membrane or from internal membranes, such as those of the endoplasmic reticulum or the Golgi apparatus within the cell.

[0025] It is important to note that an enveloped virus can comprise several successive envelopes surrounding its capsid (also called a nucleocapsid). In the context of the present invention, the phrase "between at least one of its envelopes and its capsid" is understood to mean that said exogenous nucleic acid sequence can be between two successive envelopes or between the capsid and the first envelope outside the capsid.

[0026] According to a preferred embodiment, said enveloped virus is selected from the group consisting of Herpesvirus, Poxvirus, Hepadnavirus, Asfarvirus, Flavivirus, Alphavirus, Togavirus, Coronavirus, Hepatitis D virus, Orthomyxovirus, Paramyxovirus, Rhabdovirus, Bunyavirus, Filovirus and Retrovirus.

[0027] According to an even more preferred embodiment, said enveloped virus is a Poxvirus (i.e., Poxviridae).

[0028] Poxviruses are a family of enveloped viruses, generally oval or brick-shaped, measuring between 220 and 450 nm in length, 140 to 260 nm in width, and 140 to 260 nm in thickness. Their genome consists of a single linear double-stranded DNA (dsDNA) molecule of 128 to 375 kbp, with covalently closed ends. Poxvirus replication occurs in the cytoplasm of the host cell.

[0029] According to a preferred embodiment, poxvirus is derived from a vaccinia virus, a canaripox or a fowlpox.

[0030] According to an even more preferred embodiment, said poxvirus is derived from a vaccinia virus selected from the Copenhagen, Wyeth and modified Ankara (MVA) strains.

[0031] According to a highly preferred embodiment, said poxvirus is an MVA.

[0032] According to a preferred embodiment, said enveloped virus is a live virus.

[0033] In the context of the present invention, the term "live virus" is meant to refer to a virus capable of infecting a susceptible cell (e.g., an avian cell) and replicating within it.

[0034] In the context of the present invention, the term "exogenous nucleic acid sequence" refers to any nucleic acid sequence that is not naturally present between at least one of the envelopes and the capsid of said virus. For clarity, it is specified that the term "exogenous nucleic acid sequence" does not refer to nucleic acid sequences originating from the cytoplasm or nucleus of the cell in which said virus was produced.

[0035] According to a preferred embodiment of the invention, said exogenous nucleic acid sequence is a recombinant nucleic acid sequence.

[0036] According to a preferred embodiment, said exogenous nucleic acid sequence is DNA or RNA.

[0037] According to a preferred embodiment, said exogenous nucleic acid sequence is a messenger RNA.

[0038] According to a preferred embodiment, said exogenous nucleic acid sequence is in the form of a plasmid, single-stranded DNA, or single-stranded RNA.

[0039] According to a preferred embodiment of the invention, said exogenous nucleic acid sequence is a linear double-stranded nucleic acid and, even more preferably, a closed linear double-stranded nucleic acid. Closed linear nucleic acids, by their production method, leave very few production residues, particularly bacterial ones, thus avoiding cumbersome purification steps and allowing for faster and less expensive use.

[0040] Closed linear nucleic acids are well known to those skilled in the art; their structure, functions, and the processes 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.

[0041] According to a preferred embodiment, said exogenous nucleic acid sequence includes a sequence encoding a polypeptide of interest.

[0042] According to a preferred embodiment, said exogenous nucleic acid sequence further comprises a promoter functionally linked to a sequence encoding a polypeptide of interest.

[0043] In the context of the present invention, the term "functionally linked" is intended to indicate that the binding and activation of an RNA polymerase to said promoter will result in the transcription of the sequence encoding a polypeptide of interest. Generally, to be functionally linked, the promoter is placed upstream of the nucleic acid sequence of the gene of interest.

[0044] According to a preferred embodiment, said promoter is a eukaryotic promoter or a poxvirus promoter.

[0045] According to an even more preferred embodiment, said exogenous nucleic acid sequence further comprises a eukaryotic promoter and a poxvirus promoter functionally linked to a sequence encoding a polypeptide of interest.

[0046] In the context of the present invention, the term "eukaryotic promoter" refers to any sequence of nucleic acids to which the endogenous RNA polymerase of a eukaryotic cell can bind and be activated to induce the initiation of transcription.

[0047] In the context of the present invention, the term "poxvirus promoter" refers to any nucleic acid sequence to which the RNA polymerase encoded by a gene naturally present in the genome of a poxvirus can bind and be activated.

[0048] In the context of the present invention, the expression "sequence encoding a polypeptide of interest" refers to any nucleic acid sequence that can be transcribed into an mRNA and then translated into a polypeptide capable of possessing prophylactic or therapeutic activity. For clarity, it is specified that said prophylactic or therapeutic activity may be induced directly or indirectly by said polypeptide (e.g., by activation of the immune system).

[0049] Preferably, said sequence encoding a polypeptide of interest includes a specific antigen of a tumor cell, pathogen, toxin of interest or microorganism responsible for an infectious disease.

[0050] The present invention also relates to a method for obtaining an enveloped virus comprising an exogenous nucleic acid sequence between at least one of its envelopes and its capsid, characterized in that said method comprises a step of bringing an enveloped virus into contact with a cationic polypeptide / exogenous nucleic acid sequence complex.

[0051] According to a preferred embodiment, said cationic polypeptide is chosen from the group consisting of R8.

[0052] In the context of the present invention, the term "R8" is intended to designate a polypeptide consisting of the sequence RRRRRRRR comprising an N-terminal Acetylation (Ac) and a C-terminal Amidation (NH2).

[0053] According to a preferred embodiment, the cationic polypeptide / nucleic acid ratio (w / w) is between 0.5:1 and 25:1.

[0054] According to a preferred embodiment, the exogenous nucleic acid sequence / virus ratio is between 0.1 and 10 mg / 10 7 pfu.

[0055] The present invention also relates to a composition comprising a virus according to the invention or obtained by a process according to the invention in a pharmaceutically acceptable medium.

[0056] According to a preferred embodiment, said composition further comprises an adjuvant.

[0057] According to a preferred embodiment, said composition further comprises an anti-CTLA4 antibody.

[0058] The present invention also relates to a method of transforming a eukaryotic cell comprising a step of bringing a virus according to the invention or a composition according to the invention into contact with said eukaryotic cell.

[0059] According to one embodiment of the invention, said transformation process is implemented ex vivo.

[0060] According to a preferred embodiment, said eukaryotic cell is a human cell.

[0061] The present invention also relates to an enveloped virus according to the invention or a composition according to the invention for use in the treatment of cancer.

[0062] The present invention also relates to an enveloped virus according to the invention or a composition according to the invention for vaccination against an infectious disease. Brief description of the figures

[0063] presents the results obtained after transfection of eukaryotic cells with varying quantities of virus according to the invention.

[0064] presents the results obtained after transfection of eukaryotic cells with varying amounts of virus according to the invention obtained using different cationic polypeptides. Detailed description of the invention

[0065] In the context of the present invention, the exogenous nucleic acid sequence is preferentially contained in a vector and even more preferentially in a plasmid.

[0066] Vectors for expression in eukaryotic cells are well known to those skilled in the art and are commercially available. Among these, we can mention 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).

[0067] When said exogenous nucleic acid sequence encoding a polypeptide of interest is present, said sequence is preferentially dependent on one promoter, and even more preferentially on a eukaryotic promoter or a poxviral promoter. Most preferably, said sequence is dependent on two promoters, a eukaryotic promoter on the one hand and a poxviral promoter on the other.

[0068] Eukaryotic promoters usable in a vector are well known to those skilled in the art. They can be found, in particular, in the Eukaryotic Promoter Database (http: / / epd.epli.ch / index.php). 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 bactin promoter, the CAG promoter, the TRE promoter, the UAS promoter, the Ac5 promoter, the polyhedrin promoter, the CaMKIIa promoter, the GAL1,10 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.

[0069] Poxviral promoters usable in a vector are known to those skilled in the art and have notably been described by Alharbi et al. ((2019) Poxviral promoters for improving the immunogenicity of MVA delivered vaccines, Human Vaccines & Immunotherapeutics, 15:1, 203-209).

[0070] Within the framework of the present invention, the poxviral promoter is preferably 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.

[0071] The said vector advantageously includes, in addition to the said promoters, the regulatory elements ensuring the expression of the gene(s) of interest in eukaryotic cells.

[0072] Preferably, the regulatory elements ensuring the expression of the gene(s) of interest in eukaryotic cells are chosen from the group including ribosome binding sites, the start codon, the termination codon, Kozak sequences, poly A and introns.

[0073] The vector may comprise several exogenous nucleic acid sequences, each encoding a different polypeptide of interest. In this case, these exogenous nucleic acid sequences may be subject to the same or different regulatory elements.

[0074] According to a preferred embodiment, said exogenous nucleic acid sequence encodes a polypeptide of interest 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, HRV-H and HRV-V virus proteins. Preferably, said exogenous nucleic acid sequence encodes the entirety of said protein. Alternatively, said exogenous nucleic acid sequence encodes a portion of said protein of interest comprising at least one specific epitope of said protein.

[0075] According to another preferred embodiment, said exogenous nucleic acid sequence encodes all or part of at least one antigen selected from the group consisting of antigens of cholera bacillus, Sars-Cov2, dengue, Corynebacterium diphtheriae, 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, tick-borne encephalitis virus, Mycobacterium tuberculosis, Salmonella enterica, varicella virus, enterotoxigenic Escherichia coli, group B streptococci (GBS), herpes simplex virus, HIV, Neisseria gonorrhoeae, non-typhoidal salmonella, norovirus, respiratory syncytial virus (RSV), schistosoma, shigella, and group a streptococcus (GAS). Preferably, said exogenous nucleic acid sequence encodes the entirety of said antigen. Alternatively, said exogenous nucleic acid sequence encodes a portion of said antigen comprising at least one specific epitope of said protein.

[0076] According to another preferred embodiment of the invention, said exogenous nucleic acid sequence codes at least one neo-antigen.

[0077] Indeed, current techniques allow for the complete sequencing of a patient's cancer cells' genetic material and the identification of specific mutations by comparison with the patient's constitutional DNA. If these mutations are non-synonymous, belong to coding sequences expressed by the tumor, and are presumed immunogenic due to their characteristics, then they can be used to induce immunization against the tumor.

[0078] The mutations in the tumor genome and the resulting neoantigens are a unique combination restricted to the tumor of a given patient, or even to the clones that make up that tumor.

[0079] To do this, cancer cells from a patient to be treated are analyzed from a biopsy or surgical specimen. The DNA and RNA of the cancer cells, as well as constitutional DNA from a blood sample, are extracted and sequenced.

[0080] The DNA sequence of healthy cells is compared to the DNA sequence of cancer cells and somatic mutations located in coding and expressed regions are identified.

[0081] The expressed, coding mutated sequences are processed using algorithms to determine, for each patient, which mutations are potentially the most antigenic and can serve as targets. These non-synonymous, expressed, coding mutated sequences, specific to the patient's cancer cells and called neoantigens, are then generated by DNA or RNA synthesis and subsequently formulated into an expression vector (plasmid, closed linear nucleic acid, or RNA). According to a preferred embodiment of the invention, this is achieved using the method described in patent application EP2391731.

[0082] The enveloped virus used in the invention is preferably a poxvirus and even more preferably an MVA virus. The latter is derived from the Ankara strain of vaccinia virus through successive passages through chicken embryo cells. These passages have resulted in the attenuation of this virus, which has thus been able to be used in recent smallpox vaccination campaigns.

[0083] The description of the different MVA strains, the methods for possibly inserting exogenous genes into its genome, as well as the production and purification methods for this virus, are notably available in 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.

[0084] The enveloped virus according to the invention is preferably obtained by transfection of said virus via one of the transfection techniques well known to those skilled in the art. Among these, the use of cationic polypeptides is preferred.

[0085] Cationic polypeptides are a class of molecules characterized by a strong positive charge due to the presence of basic amino acids such as arginine, lysine, or histidine. These peptides exhibit a particular affinity for negatively charged macromolecules, notably nucleic acids (DNA and RNA), and cell membranes, making them especially useful for biotechnological and therapeutic applications.

[0086] Cationic polypeptides, thanks to their positively charged residues, possess several key characteristics that make them suitable for transfection: - Interaction with nucleic acids: They form stable complexes called polyplexes with DNA or RNA, protecting these molecules from enzymatic degradation and facilitating their internalization by cells. - Cell penetration capacity: These peptides interact with the anionic phospholipids of cell membranes, allowing their internalization via mechanisms such as endocytosis or direct translocation. - Biocompatibility and modularity: These peptides are generally biocompatible and can be chemically modified or structured according to the specific needs of the application.

[0087] Among the cationic polypeptides that can be used in the context of the invention, we can mention in particular arginine-rich peptides such as R8, R9, Tat peptide and R6K2, lysine-rich peptides such as Polylysine, K8 and K4H4, hybrid or modified peptides such as Histidine-rich peptides (HRPs), PepFect6, Transportan and CADY peptides, peptide polymers and dendrimers such as polyarginine or polylysine dendrimers, modified PAMAM peptide dendrimers, peptides derived from natural proteins such as Protamine, Melittin, optimized synthetic peptides such as MPG and RGD.

[0088] According to a preferred embodiment of the invention, said cationic polypeptide is an Arginine-rich polypeptide and even more preferably R8 comprising an N-terminal Acetylation (Ac) and a C-terminal Amidation (NH2).

[0089] The applicant was able to demonstrate that the use of R8 made it possible to obtain enveloped viruses according to the invention exhibiting an optimum transfection capacity of the exogenous nucleic acid sequence.

[0090] The R8 polypeptide is a synthetic sequence composed of eight consecutive arginine residues. As a cationic peptide, R8 is distinguished by a high positive charge at physiological pH, conferred by the guanidinium side chains of the arginine residues. This characteristic allows it to interact efficiently with negatively charged surfaces, such as cell membranes, nucleic acids, and other anionic macromolecules. The positive charge of R8 promotes strong electrostatic binding with nucleic acids, enabling its use in gene delivery.

[0091] According to a preferred embodiment of the invention, the enveloped virus according to the invention is obtained by a process comprising a step of bringing an enveloped virus into contact with an exogenous cationic polypeptide / nucleic acid sequence complex.

[0092] According to a further preferred embodiment of the invention, during the step of contacting an enveloped virus and a cationic polypeptide / exogenous nucleic acid sequence complex, the ratio of exogenous nucleic acid sequence to enveloped virus is preferably between 0.1 and 10 mg / 10 7 pfu, even more preferentially between 0.5 and 5 mg / 10 7 pfu and quite preferably between 1 and 3 mg / 10 7 pfu.

[0093] According to a preferred embodiment of the invention, said process includes a preliminary step consisting of preparing said cationic polypeptide / exogenous nucleic acid sequence complex.

[0094] This step is well known to those skilled in the art, who can usefully refer to the available literature on the subject. As a guide, these complexes are preferentially formed by contacting the exogenous nucleic acid sequence with the cationic polypeptide. The relative concentration of these two ingredients is preferably chosen so that the N / P ratio is close to 10:1.

[0095] The nitrogen-to-phosphate (N / P) ratio is a key parameter in the use of cationic polypeptides for nucleic acid transfection. This ratio represents the proportion of cationic groups (nitrogen, N) on the polypeptide relative to the anionic groups (phosphate, P) on the nucleic acids. An appropriate N / P ratio is important to ensure efficient complexation of nucleic acids, their protection against enzymatic degradation, and their intracellular transport. At low N / P ratios, the anionic charges of the nucleic acids may not be completely neutralized, limiting the efficiency of the complex. Conversely, high N / P ratios can lead to an overabundance of cationic charges, increasing cellular toxicity and disrupting cell membranes. In practice, this ratio is determined experimentally and can vary depending on the cationic polypeptide used and the nature of the nucleic acid.

[0096] According to a preferred embodiment, the process according to the invention comprises a step of separating the enveloped virus according to the invention from exogenous nucleic acid sequences that have not integrated into the viral envelope. This latter step can be carried out by treating the enveloped virus according to the invention with a DNase. Alternatively or in addition, the separation step can be carried out by any other means known to those skilled in the art, such as sucrose gradient filtration or ultrafiltration.

[0097] The virus according to the invention can be advantageously included in a composition.

[0098] Preferably, the composition according to the invention further comprises a pharmaceutically acceptable carrier. For the purposes of this invention, the term "pharmaceutically acceptable carrier" means any carrier, solvent, diluent, excipient, adjuvant, dispersion medium, and the like, compatible with pharmaceutical administration.

[0099] The composition according to the invention is advantageously buffered, in order to be suitable for human use, at a physiological or slightly basic pH.

[0100] The composition according to the invention can be administered as a single dose or repeatedly after a certain interval of time ranging from one day to one year. Preferably, administration will take place weekly for seven consecutive times, then once every three weeks.

[0101] The appropriate dosage can be adjusted according to various parameters, in particular the method of administration, the composition used, the age, health, and weight of the host organism, the nature and extent of symptoms, the type of associated treatment, the frequency of treatment, and antibody production.

[0102] A person skilled in the art is able to determine the appropriate quantities of each element within the composition according to the invention. By way of example, the virus according to the invention can be used in a quantity of between 10 4 at 10 9 pfu by injection.

[0103] Preferably, the enveloped virus or 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 a combination of several of these treatments at once.

[0104] The said invention also relates to an enveloped virus or a composition according to the invention, intended to be used for the treatment of cancer or a tumor.

[0105] The said invention also relates to an enveloped virus or a composition according to the invention, intended to be used for the treatment of an infectious disease.

[0106] Preferably said cancer is a carcinoma and said exogenous nucleic acid sequence encodes a polypeptide of interest selected 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.

[0107] Preferably said cancer is a leukemia and said exogenous nucleic acid sequence codes a polypeptide of interest selected from the group consisting of all or part of the aberrant MHC class II proteins, Anti-idiotype, B1, CD19, CD20, CD22, CD25 and CD36.

[0108] Preferably said cancer is a melanoma and said exogenous nucleic acid sequence codes a polypeptide of interest selected from the group consisting of all or part of the MAGE, MART, gp100, Tyrosinase, CD2, CD3, GM2 protein.

[0109] Preferably said cancer is induced by a retrovirus said exogenous nucleic acid sequence codes a protein chosen from the group consisting of all or part of the proteins of the HRV-H and HRV-V viruses.

[0110] Preferably, said exogenous nucleic acid sequence encodes the entirety of said polypeptide of interest. Alternatively, said exogenous nucleic acid sequence encodes a portion of said polypeptide of interest comprising at least one specific epitope of said protein.

[0111] The said invention also relates to an enveloped virus or 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.

[0112] The said invention also relates to an enveloped virus or a composition according to the invention for use in a method for vaccinating against the protein encoded by the gene of interest.

[0113] Preferably, the infectious disease in question is chosen from the group comprising cholera, COVID-19, dengue fever, diphtheria, hepatitis, Haemophilus influenzae type b (Hib), human papillomavirus (HPV), influenza, Japanese encephalitis, measles, meningococcal meningitis, mumps, pertussis (whooping cough), pneumococcal disease, poliomyelitis, rabies, rotavirus diseases, rubella, tetanus, tick-borne encephalitis, tuberculosis, typhoid fever, varicella-zoster virus disease, enterotoxigenic Escherichia coli disease, group B streptococcus (GBS) disease, herpes simplex virus disease, AIDS, malaria, Neisseria gonorrhoeae disease, and diseases caused by non-typhoidal salmonella, norovirus infections, paratyphoid fever, respiratory syncytial virus (RSV) infections, schistosomiasis,Shigella diseases, group A streptococcal (GAS) diseases, tuberculosis, and yellow fever.

[0114] According to a preferred embodiment of the invention, the enveloped virus or the composition according to the invention is intended to be injected subcutaneously, intramuscularly, orally, nasally or intratumorally.

[0115] According to a highly preferred embodiment of the invention, the enveloped virus or the composition according to the invention is intended to be injected subcutaneously.

[0116] Injections can be made with conventional syringes and needles, but preferably via a needle-free injection device such as bioject®.

[0117] Preferably, said composition according to the invention comprises from 1 µg to 10 mg of exogenous nucleic acid sequence.

[0118] Preferably, said composition according to the invention comprises 10 4 at 10 9 pfu of enveloped virus according to the invention.

[0119] According to a preferred embodiment of the invention, said composition further comprises an anti-CTLA4 antibody or antibody fragment.

[0120] The co-administration of an anti-CTLA4 antibody or antibody fragment is likely to enhance the immune response induced by immunotherapy. For this purpose, doses reduced to 1 / 10 and even more preferably 1 / 100 of the usual systemic dose are assumed to be sufficient. At these doses and after co-injection, the concentration in the draining lymph node at the injection site is assumed to be similar to or higher than with systemic administration, 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, and even more preferably ipilimumab. In the context of the present invention, the term antibody also refers to bispecific antibodies comprising at least one CTLA-4-specific paratope.

[0121] The present invention also relates to a vaccine comprising an enveloped virus or a composition according to the invention.

[0122] 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 who has received an identical or different injection directed against the same epitope.

[0123] Thus, the said invention also relates to a set of parts for vaccination comprising:

[0124] - a first vaccine according to the invention for a first injection (bonus),

[0125] - a second vaccine according to the invention for a subsequent injection (boost).

[0126] The enveloped viruses, compositions, part assembly and vaccine according to the invention are preferably contained in a pharmaceutically acceptable medium. Examples Preparation of eukaryotic cells

[0127] BHK21 cells were collected, washed in PBS and diluted to a final concentration of 1.10 5 cells / ml. 100 µl of these cells were distributed into each well of a 96-well plate. 100 µl of GMEM culture medium was added to each well.

[0128] The plates were incubated for 24 hours at 37°C in an atmosphere containing 5% CO₂ 2. Infections and transfections

[0129] In order to evaluate the effectiveness of the compositions and viruses according to the invention, a plasmid comprising the gene encoding GFP under the dependency of the H5 promoter of the Vaccinia virus was used (pDNA-VVH5-eGFP).

[0130] Three types of cationic polypeptides were used:- IR9 with peptide sequence: GLFEAIEGFIENGWEGMIDGWYGRRRRRRRRR- R8 with peptide sequence: Acetyl-RRRRRRRR-amide- Stearyl-R8 with peptide sequence: Stearyl-RRRRRRRR.

[0131] The virus used was MVA.

[0132] 80 µl of DNAp-VVH5-eGFP (10µg / ml) were mixed with 80 µl of cationic polypeptide (100µg / ml), thus obtaining an N / P ratio of 10 / 1. This mixture was incubated for 15 minutes at room temperature with gentle shaking to allow DNA / cationic polypeptide complexation.

[0133] To this mixture were added 20 µL of MVA (1.10 7 pfu / mL or 1.10 8 pfu / mL in PBS). The mixture was incubated for 1 hour at 37°C with gentle stirring to allow complexation of the cationic DNA / polypeptide with the MVA.

[0134] To eliminate uncomplexed plasmids, 20 µl of DNase I (5mg / ml) were added to the reaction medium and incubated for 10 min at room temperature.

[0135] DNase I activity was stopped by adding 800 µl / tube of GMEM. This resulted in each reaction medium having a final volume of 1 ml.

[0136] Each preparation was tested in three different wells (250µl / well).

[0137] The plates were then incubated at 37°C in an atmosphere containing 5% CO2 overnight. Positive transfection controls

[0138] 5.10 5 pfu of MVA and 100 ng of the p-VVH5-eGFP plasmid mixed with lipofectamine 3000 were added to control wells. Negative tests

[0139] To compare the efficiency of the vectors and compositions according to the invention, 200 ng of the p-VVH5-eGFP plasmid alone were added to control wells, cells were treated with p-VVH5-eGFP DNA complexed with each of the cationic polypeptides without MVA, and cells were treated with pDNA-VVH5-eGFP and MVA (5.10 4 pfu / well or 5 10 5 pfu / wells) without cationic polypeptides.

[0140] Untreated cells, cultured in complete GMEM medium, were used as a growth control. Reading the results.

[0141] Before shooting, the culture medium in each well was completely renewed with 200µl of fresh GMEM medium.

[0142] Images of each well were taken via an Incucyte® device for 72 hours. Results

[0143] The results obtained show that the use of MVA viruses having incorporated the pVVH5-eGFP plasmid in their envelope allows for significantly higher expression of GFP compared to the use of plasmid / cationic polypeptide complexes alone.

[0144] This expression is dose-dependent and a maximum expression can be observed at an MVA concentration of 5.10 5 pfu / puit.

[0145] The use of MVA obtained after treatment with plasmid / R8 complexes results in GFP protein expression 2.5 times higher than that obtained with the two other cationic polypeptides used.

[0146] These results show the interest of the viruses according to the invention and more particularly the interest of their preparation via a process using cationic polypeptides and more particularly R8.

Claims

An enveloped virus characterized in that it comprises an exogenous nucleic acid sequence between at least one of its native envelopes and its capsid. Enveloped virus according to the preceding claim characterized in that said virus is a poxvirus. Enveloped virus according to the preceding claim characterized in that said virus is an MVA. Enveloped virus according to any one of the preceding claims characterized in that said virus is a live virus. Enveloped virus according to any one of the preceding claims characterized in that said exogenous nucleic acid is DNA or RNA. Enveloped virus according to any one of claims 1 to 5 characterized in that said exogenous nucleic acid sequence is messenger RNA. Enveloped virus according to any one of claims 1 to 5 characterized in that said exogenous nucleic acid sequence is in the form of a plasmid, single-stranded DNA or single-stranded RNA. Enveloped virus according to any one of the preceding claims characterized in that said exogenous nucleic acid sequence encodes a polypeptide of interest. enveloped virus according to the preceding claim characterized in that said polypeptide of interest comprises an antigen specific to a tumor cell, pathogen, toxin of interest or microorganism responsible for an infectious disease. Enveloped virus according to claim 8 characterized in that said exogenous nucleic acid sequence further comprises a promoter functionally linked to a sequence encoding a polypeptide of interest. Enveloped virus according to claim 10 characterized in that said promoter is a eukaryotic promoter and / or a poxvirus promoter. A method for obtaining an enveloped virus comprising an exogenous nucleic acid sequence between at least one of its envelopes and its capsid, characterized in that said method comprises a step of bringing an enveloped virus into contact with a cationic polypeptide / exogenous nucleic acid sequence complex. A process according to the preceding claim characterized in that said cationic polypeptide is R8. A process according to any one of claims 12 to 13 characterized in that the cationic polypeptide / nucleic acid ratio (w / w) is between 0.5:1 and 25:

1. A method according to any one of claims 12 to 14, characterized in that the ratio of exogenous nucleic acid sequence to virus is between 0.1 and 10 mg / 10 7 pfu. Composition comprising a virus according to any one of claims 1 to 11 or obtained by a process according to any one of claims 12 to 15 in a pharmaceutically acceptable medium. Composition according to the preceding claim characterized in that it further comprises an adjuvant. Composition according to any one of claims 16 to 17 characterized in that it further comprises an anti-CTLA4 antibody. Method for ex vivo transformation of a eukaryotic cell comprising a step of bringing into contact with a virus according to any one of claims 1 to 11 or a composition according to any one of claims 16 to 18 and said eukaryotic cell. A method according to the preceding claim, characterized in that said cell is a human cell. Enveloped virus according to any one of claims 1 to 11 or composition according to any one of claims 16 to 18 for use in the treatment of cancer. Enveloped virus according to any one of claims 1 to 11 or composition according to any one of claims 16 to 18 for vaccination against an infectious disease.

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