Virus-based expression of proteins and genes

Abstract

A nucleic acid molecule in the form of a plasmid comprising an origin of replication, a promoter for the transcription into viral RNA; a 3'NCT triplet upstream of regions for the insertion of gene(s) of interest, separated by Measles virus intergenic regions, and one 5'NCT sharing at least 95% of identity with SEQ ID NO: 11 over its full-length, wherein the 3'NCT triplet shares at least 95% of identity with SEQ ID NO:14 over its full-length, the corresponding helper nucleic acid molecules, a process for the production of virosomes based on these nucleic acid based molecules, the obtained virosomes and their therapeutical uses.

Description

[0001] VIRUS-BASED EXPRESSION OF PROTEINS AND GENES

[0002] Technical Field

[0003] The present invention relates to the production of virosomes from Measle virus (MV) for a safe administration of proteins to a patient, for instance to induce a protective immune response against a pathogen, using virosomes expressing antigens of this pathogen. The proteins are potentially expressed on the surface of the virosome and also encoded on an RNA contained in the virosome and delivered in the target cells.

[0004] Prior art

[0005] Reverse genetics is a molecular biology method for creating a new virus from its cloned cDNA. This technique facilitates the understanding of viral biology and encourages the further development of novel vaccines and vectors for gene delivery. The first recovery of a non-segmented negative-strand RNA virus entirely from cloned cDNA was achieved in 1994 for the rabies virus. Since then, other non-segmented negative-strand RNA viruses have been recovered from cloned cDNA

[0006] The use of attenuated, inactivated or inactive virus for vaccination or gene transfer purposes is known.

[0007] Measles Virus is one of them yet not used in clinical practice. One possible explanation is associated with the natural prevalence of MV-antibodies in Human populations; hence such vectors will be rapidly neutralized by the immune system.

[0008] For instance EP3936517 discloses the use of MV-virus, but this system was not successful in clinical trials, despite numerous attempts. Moreover, this virus cycle and / or mode of action is very complex, hindering its industrial uses for in vitro purposes and / or for clinical developments such as the development of vaccines. The virus comprises 6 important genes, encoding N (nucleocapsid), P (phosphoprotein), M (matrix protein), F (fusion protein), H (hemagglutinin) and L (RNA-dependent RNA polymerase). The first attempt of reverse genetics system was based on the co-transfection of two plasmids (Radecke et al, 1995). Several more recent attempts have been performed. However, those methods remained inefficient. Among them, several attempts were made to increase the efficiency of the so-called MV rescue (artificial reproduction of the viral cycle so as to use it for research or medical purposes). These include modifying the use of other susceptible cells (CHO-hSLAM) by Schneider (1997), following the use of recombinant modified vaccinia virus Ankara (MAV-T7) (Kovacs 2003) or vaccinia virus Lister vaccine strain (Nakatsu 2006) to increase sufficient T7 RNA polymerase expression, or involving the use of host polymerase II promoters (CMV) to drive viral RNA synthesis (Martin 2006, Li 2011). However, the efficiency of the recovery remained relatively low, and there is a need for improvement. Rescueefficiencies in the systems with T7-expressing vaccinia virus are high, though laborious cleaning steps are required.

[0009] For instance, WO 2016 / 110869 describes the use of MV sequences, a construct to allow the production of one single antigen of interest by a patient with (i) one cloning plasmid comprising the entire anti-genome of measles virus, or MV genome like replicon RNA coding for non-MV genes along with a subset of MV genes, (ii) one helper plasmid coding for and expressing N, P, L proteins respectively.

[0010] However, to this day, no vaccines using a rescued viral genome has been successfully developed.

[0011] Brief summary of the invention

[0012] It is the object of the invention to provide a nucleic acid molecule (DNA) in the form of a plasmid comprising:

[0013] - a promoter for the transcription into viral RNA;

[0014] - a 3' NCT region upstream of

[0015] - 1, 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions, or

[0016] 1 ,2 or more cassette(s) for the expression of a protein of interest, and

[0017] - one 5' NCT sharing at least 95% of identity with SEQ ID NO: 11 over its full-length,

[0018] wherein

[0019] the 3' NCT region shares at least 95% of identity with SEQ ID NO:14 over its full-length, and

[0020] the intergenic regions share at least 95% of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19 over their full-length,

[0021] the said nucleic acid molecule being under the control of an RNA Polymerase l / ll and / or comprises the T7 promoter (SEQ ID NO:17) to allow the transcription of the DNA into a single stranded RNA and under the control of the MV RNA-dependent RNA polymerase to replicate the translated RNA and produce a mRNA from the RNA template, the said cassette(s) for the expression of a protein of interest or the said regions for the insertion of genes, preferably after the insertion of the said genes, having a combined size being a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11 .

[0022] Preferably, this nucleic acid molecule is further comprising a hairpin region upstream of the 3' NCT region, sharing preferably at least 95% of identity with SEQ ID NO:15or SEQ ID NO:21, and / or an excision region downstream the 5'NCT, sharing preferably at least 95% of identity with SEQ ID NO:16.

[0023] Preferably, this nucleic acid molecule does not encode a peptide sharing 100% of identity with SEQ ID NO:22 over the full-length of the SEQ ID NO:22.

[0024] Advantageously, this nucleic acid molecule is further comprising one or more cassettes encoding the F protein of measles virus and / or a variant of the H protein of measles virus, preferably a truncated version of the SEQ ID NO:22 fused to a targeting moiety, preferably wherein the truncated version of SEQ ID NO:22 is SEQ ID NO:23.

[0025] Preferably, the targeting moiety is for a binding to a marker selected from CD3, CD8, CD40, CD86 ora tumor antigen.

[0026] Alternatively, or in addition, preferably, the targeting moiety is an affinity molecule, advantageously selected from the group consisting of VHH, scFv, fynomer, mimotope, DARPins, affibodies, monobodies and affinity peptide.

[0027] Preferably, this nucleic acid molecule is further comprising an expression cassette for the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, preferably the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11.

[0028] A related aspect of the present invention is process for the production of a (the above) nucleic acid molecule, preferably an expression vector, in the form of a plasmid, the said process comprising the step of selecting a nucleic acid construct comprising:

[0029] - a promoter for the transcription into viral RNA;

[0030] - a 3' NOT region upstream of

[0031] - 1, 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions,

[0032] - possibly an expression cassette for the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, preferably the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11 , and

[0033] - one 5'NCT region sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO: 11 over its full-length,

[0034] wherein

[0035] the 3'NCT region shares at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:14 over its full-length, and

[0036] the intergenic regions share at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19 over their full-length,the said nucleic acid molecule being under the control of an RNA Polymerase l / ll and / or comprises the T7 promoter (SEQ ID NO:17) to allow the transcription of the DNA into a single stranded RNA and under the control of the MV RNA-dependent RNA polymerase to replicate the translated RNA and produce a mRNA from the RNA template, and of

[0037] inserting one or more expression cassette(s) for one or more gene of interest in the said 1 , 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions,

[0038] wherein the said construct has a combined size being a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11

[0039] Preferably, as for the above nucleic acid molecule (expression vector), the nucleic acid construct to be processed does not encode a peptide sharing 100% of identity with SEQ ID NO:22 over the full-length of the SEQ ID NO:22, and / or is further comprising one or more cassettes encoding a fusion protein, preferably the F protein of measles virus, and a truncated version of the SEQ ID NO:22 fused to a targeting moiety (described above), preferably wherein the truncated version of SEQ ID NO:22 is SEQ ID NO:23.

[0040] A related and complementary aspect of the present invention is a nucleic acid molecule in the form of a plasmid L comprising

[0041] - a promoter for the transcription into RNA

[0042] - a cassette encoding L protein of MV, the said L protein sharing at least 95% of identity with SEQ ID NO:6,

[0043] and a poly A signal.

[0044] Another related and complementary aspect of the present invention relates to a nucleic acid molecule in the form of a plasmid NP comprising

[0045] a promoter for the transcription into RNA, and

[0046] genetic element for the expression of the protein N, the said protein N sharing at least 95% of identity with SEQ ID NO:4 over its full-length, and genetic element for the expression of the protein P, the said protein P sharing at least 95% of identity with SEQ ID NO:2 over its full-length, wherein

[0047] either the genetic elements for the expression of the protein N and the genetic elements for the expression of the protein P are separated by a cleaving site so as to generate two messengers RNA, or wherein

[0048] the said plasmid comprises the genetic elements for the expression of the protein N followed by a poly-adenylation signal and a second promotor for the transcription into RNA of the genetic elements for the expression of the protein P.Preferably, the N and P cassettes are encoded on two separated nucleic acid molecules:

[0049] - a plasmid N comprising a promoter for the transcription into RNA, and genetic element for the expression of the protein N, the said protein N sharing at least 95% of identity with SEQ ID NO:4 over its full-length, and / or

[0050] - a plasmid P comprising a promoter for the transcription into RNA, and genetic element for the expression of the protein P, the said protein P sharing at least 95% of identity with SEQ ID NO:2 over its full-length.

[0051] Preferably, the RNA polymerase II promoter is pActb, pCMV, pCMV+intron, pEFlA, pEFS, pCAG, pCBh, pSFFV, pSV40, phBoVl or phPGK, pUBC, and / or, the RNA polymerase II promoter is shorter than 1000 nucleotides and / or wherein the plasmids are shorter than 10000 base pairs.

[0052] Preferably, the nucleic acid construct(s) of the present invention is (are) further comprising a cassette for eukaryotic and / or prokaryotic selection.

[0053] Another related aspect of the present invention relates to a genetic system comprising the two nucleic acid molecules L and NP described here above, or the three nucleic acid molecules L, N and P as described above, preferably wherein the RNA polymerase promoter of the nucleic acid molecule L is different of the RNA polymerase promoter of the nucleic acid molecule N and / or P.

[0054] In such a genetic system, wherein the nucleic acid molecule L and NP both comprise a cassette for eukaryotic and / or prokaryotic selection, preferably, the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule L is different as compared with the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule NP.

[0055] Another related aspect of the present invention relates to a process for the production of virosomes expressing one or several protein(s) of interest comprising the steps of

[0056] growing a eukaryotic cell line, preferably a mammalian cell line in a cell culture medium;

[0057] engineering the first nucleic acid molecule (comprising the 3'NCT region, gene(s) of interest, intergene, 5'NCT) and

[0058] transfecting the nucleic acid molecule within the said selected eukaryotic cells and

[0059] recovering the said virosomes in the cell culture medium.Preferably, this process is further comprising the step of engineering the genetic system described here above and co-transfecting the three different nucleic acid molecules within the said eukaryotic cells.

[0060] Preferably, the recovering of the virosomes comprises a DNAse treatment to hydrolyze host cell's DNA, filtration and / or precipitation of the virosome.

[0061] Preferably, in this process, the plasmids L and / or N are introduced in a molar excess as compared to the plasmid P, or the plasmid L is introduced in a molar excess as compared to the plasmid NP, the said molar excess being of at least 1.5 (mol plasmid L and / or N: mol plasmid P or plasmid Lplasmid NP, preferably of at least 2.5, more preferably of at least 4, but preferably in a molar excess of less than 10.

[0062] In this process, possibly, the amount of the cloning plasmid is introduced in a molar relative amount, as compared with the plasmids L, N and / or P (or L and NP), this amount being comprised between 0.1 and 10, preferably between 0.3 and 6, for instance at a ratio of the cloning plasmid:plasmid L or plasmid N comprised between 2:1 to 1 :1, and / or ratio of the cloning plasmid:plasmid P comprised between 10:1 and 3:1.

[0063] Another related aspect of the present invention relates to the virosomes obtainable by this process.

[0064] Another related aspect of the present invention is the Measles virus-derived virosomes comprising one, two or more protein(s) of interest, and an RNA sequence comprising;

[0065] - a 3' NCT region upstream of

[0066] - 1,2 or more cassette(s) for the expression of this (these) protein(s) of interest, each cassette being separated by a different intergenic region, and

[0067] - a 5' NCT region sharing at least 95% of identity with SEQ ID NO 11 over its full-length,

[0068] - preferably a cassette for the expression of the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, preferably the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11 ,

[0069] wherein the 3' NCT region shares at least 95% of identity with SEQ ID NO: 14 over its full-length, and

[0070] the intergenic regions share at least 95% of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19;

[0071] the said RNA sequence having a total size being a multiple of 6 nucleotides, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11.Preferably these virosomes do not express a peptide sharing 100% of identity with SEQ ID NO:22 over its full-length.

[0072] Advantageously, these virosomes express a peptide sharing 100% of identity with a part of SEQ ID NO:22, preferably a peptide sharing 100% of identity with SEQ ID NO:23.

[0073] Another related aspect of the present invention is a pharmaceutical composition comprising these virosomes, being preferably for use in vaccination or in gene therapy or in oncology or in the treatment of autoimmune disease.

[0074] Preferably, this pharmaceutical composition is for oral administration, advantageously with encapsulated and / or with a gastric resistance composition.

[0075] Other embodiments according to the present invention are mentioned in the appended claims and the subsequent description of an embodiment of the invention.

[0076] Brief description of the Drawings

[0077] Figure 1 is a FACS analysis of transfection using mCherry virosomes as in the prior art (left panel), or as in the prior art, but refining the culture and transfection protocols (right panel).

[0078] Figure 2 shows the structure of the cloning plasmid according to the present invention.

[0079] Figure 3 is a bar graph of measles RNA expression in producing cells Figure 4 shows the antibody induction.

[0080] Figure 5 shows the T Cell response using the prior art's protocols (lefts panel; light symbols, untreated; dark symbols, treated) or the constructs according to the present invention.

[0081] Figure 6 shows the survival of mice submitted to lethal doses of flu and protected or not by a vaccine.

[0082] Figure 7 shows a cloning vector with a targeting moiety (Anti Her2 VHH). Figure 8 shows the expression of viral genome in the targeted cell.

[0083] Defailed description of an embodiment of fhe invention

[0084] The inventors have firstly attempted to take advantage of the constructs of WO 2016 / 110869 (prior art).

[0085] However, the first experiments have allowed only 0.1% of transfected cells. Even after several rounds of fine-tuning, they obtained only about 7% of cells expressing the protein of interest.

[0086] Moreover, the antigen level was very low, pointing to a low DNA to RNA transcription efficiency, leading to a low translation level.A low amount of transfected cells combined with a low level of expression resulted in unacceptable performances during in vitro and in vivo testing, including undetectable immune response in vivo.

[0087] In completely redesigning the constructs, with two helper plasmids, the inventors have succeeded in expressing 6 different proteins using one cloning plasmid.

[0088] Indeed, the inventors have preferred the complexity of a system with three (or even four) plasmids to be co-transfected in one cell and have succeeded in obtaining high and consistent production of antigens / proteins of interests. In this system, the two (or three) helper plasmids comprise expression cassettes under the control of the cellular RNA polymerase (I or II) and / or under the control of bacterial polymerase, to express the measles' proteins required for the viral cycle (L, N and P), whereas the coding plasmid comprises one expression cassette that will be transcribed by a cellular RNA polymerase (1 or 2) and / or a procaryotic T7, into a single-stranded positive RNA, which will be subsequently processed by the MV proteins to create a negative-stranded RNA so as to create a mRNA encoding the proteins of interest.

[0089] Hence, a first aspect of the present invention relates to a nucleic acid molecule in the form of a plasmid (cloning vector) comprising:

[0090] - an origin of replication,

[0091] - a (dual) promoter (Cellular Polymerase l / ll and / or T7 procaryotic polymerase) for the transcription into viral RNA;

[0092] - a 3' NCT upstream of

[0093] - 1 , 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions, or 1 ,2 or more cassette(s) for the expression of a protein of interest, and

[0094] - one terminator region (5' NCT) sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO 11 over its full-length,

[0095] wherein the 3' NCT triplet shares at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:14 over its full-length, and the intergenic regions share at least 95% (preferably at least 97, 98, 99 or even 100%) with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / orSEQ ID NO:19; SEQ ID NO:19 inducing a lower expression,

[0096] the said nucleic acid molecule being under the control of an RNA Polymerase (RNA polymerase l / ll and / or the T7) promoter to allow the transcription of the DNA into a single stranded RNA and under the control of the MV RNA-dependent RNA polymerase (L protein; see below) to replicate the translated RNA and produce a mRNA from the RNA template, the said cassette(s) for the expression of a protein of interest or the said regions for the insertion of genes, preferably after the insertion of the said genes, having a combined sizebeing a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11.

[0097] In other words, even if the construct comprises the region for the insertion of the gene(s) of interest, rather than the gene(s) of interest, the region starting at the first nucleotide of SEQ ID NO:14 and finishing at the last nucleotide of SEQ ID NO:11 can easily be designed to be a multiple of 6 base pairs after the insertion of the cassette(s), allowing an easy insertion of coding sequences. For instance, 1, 2, 3, 4 or 5 nucleotides (potentially with no coding function) can be inserted together with the expression cassettes to meet the requirements of this "6 nucleotides rule", while keeping the desired functions of the encoded proteins. A related aspect is thus a process to generate such cloning vector with one or several gene(s) of interest, comprising the step of selecting the above vector with the regions for the insertion of one or several gene(s) of interest, of inserting this or these gene(s) of interest and ensuring a multiple of 6 base pairs for the region starting at the first nucleotide of SEQ ID NO:14 and finishing at the last nucleotide of SEQ ID NO:11.

[0098] Indeed, the skilled practitioner, possibly using bioinformatic tools, can readily decide a convenient locus for addition or removal of one triplet or even of one nucleotide, or of two, four or five nucleotides. The skilled practitioner can also test the functionality of the resulting construct.

[0099] Preferably, this construct is devoid of the full-length H protein of MV (SEQ ID NO:22). Indeed, this protein may trigger unwanted immune response.

[0100] On the other hand, when there is a need for specific targeting of cell or tissues of a patient, the construct advantageously comprises a cassette encoding:

[0101] - F (Fusion) protein such as the F protein of MV (but the F protein of other, preferably related, viruses is possible and sometimes preferred to increase the flexibility of the targets) and - a truncated and / or engineered H protein (SEQ ID NO:22), (further) comprising a binding moiety. A preferred truncated H protein is SEQ ID NO:23.

[0102] The remaining part of the H protein allows interaction with the F protein to enter the cellular target, while avoiding immune response. This truncated H protein advantageously further comprises a tag, to monitor correct expression and / or a spacer to avoid folding of the binding moiety to exposed regions of the F and / or H proteins.

[0103] The targeting moiety is preferably any single chain peptide allowing a specific affinity for a target. Among them are antibodies or similar peptides (VHH, scFv, fynomer, mimotope, DARPins, affibodies, monobodies and affinity peptide). In the context of the present invention, the binding moiety is not limited (besides its peptidic nature); yet should be compatible with a correct expression and its binding function: either a specific affinity for the target and low unspecific binding, or an affinity for a bivalent molecule, comprising (i) amoiety for the binding moiety associated to the truncated form of SEQ ID NO:22 and (ii) a moiety having an affinity for a desired target. Such specificity is advantageously determined in vitro in ensuring more specific binding to the desired target than non-specific binding. A useful alternative is thus the truncated H protein (advantageously SEQ ID NO:23) further harboring an affinity moiety for a bivalent molecule comprising, on one hand, an epitope for the affinity moiety and, on the other hand, a targeting element allowing a specific enrichment of the virosome close to a desired target. This allows an increased flexibility since (i) common elements are kept and the specificity is easily designed for different targets, just by contacting, in vitro, the engineered H protein with a different bivalent molecule and / or since (ii) targeting elements can more easily be chosen, including targeting elements being not of peptidic nature (e.g. aptamers), or peptidic targeting elements hardly expressed by the virosome.

[0104] A preferred spacer is a succession of alternating glycine (G) and serine (S) residues, preferably a spacer size comprised between 10 and 30, preferably between 15 and 20. Tags are well-known to the skilled practitioner; possible tags include His, FLAG, cMyc, HA or V5.

[0105] Among the targets are immune cells (antigen presenting cells, hence expressing CD40 and / or CD86, or lymphocytes; hence expressing CD3 or CD8) or cancer cells (general targets or neoepitopes) or cells for gene therapy treatment.

[0106] In the context of the present invention, preferably, the sequence of identity between nucleotides sequences (e.g. at least 95%) is calculated upon a BLASTn alignment of the complete sequence listed in the present patent application and the sequence to be compared.

[0107] Conversely, when the cassettes defined here below are for the translation of proteins, the identity is preferably determined using BLASTp with the default parameter (BLOSUM 62 matrix gap creation; cost 11 , extension cost 1 ; word size of 5) . The full-length of the defined peptide (protein) sequence is aligned by the BLASTp algorithm with the protein to test. In these conditions any identical residue (and also possibly labelled as conserved (+)) is considered. Hence in these conditions, the proteins share at least 95% of identical (or conserved) aminoacids.

[0108] The inventors have considered several options for the promoter:

[0109] RNA polymerase I;

[0110] RNA polymerase II

[0111] The bacterial T7.

[0112] The RNA polymerase I offers the advantage of allowing the transcription of long fragments and the disadvantage of requiring a nuclear localization of the plasmid.On the other hand, the bacterial T7 promoter allows high levels of transcription and a cytoplasmic action, but needs a cell line expressing the bacterial T7 polymerase.

[0113] Preferably, if a T7 promoter is inserted upstream, a T7 terminator is inserted downstream of the 5' NOT region.

[0114] Hence the combination of one nuclear promoter (e.g. the Polymerase I) with the T7 allows a more robust expression.

[0115] The inventors have found that the potential disadvantage associated to the use of the T7 promoter is also rescued upon placing the cassette for the expression of the M (see below) protein downstream this RNA molecule: with this construct, only full-length transcripts can lead to virosomes.

[0116] Other promoters for the RNA Polymerase II are known, for instance those listed here below:

[0117]

[0118] The presence of intergenic regions is essential to allow the expression of the different genes to form different proteins (proteins of interest and, preferably, the viral M protein). One skilled practitioner can select which intergenic region to place upstream which cassette so as to fine tune the relative expression of the different proteins. Possibly an intergenic region is placed only once in the construct, even if the same intergenic region can be placed twice (or more) in the construct. The organization of the intergenic regions in the construct is of interest in vaccination, for instance to control the humoral versus cellular response, but also in gene therapy, with the expression of exposed proteins at a desired level (for instance to target one cell type or one tissue) and the expression of the curative proteins(or genes) at another level and also in the general design with one level of expression of the M protein (if present) and another level of expression of the protein(s) of interests.

[0119] Hence, preferably, this nucleic acid molecule is further comprising an expression cassette for the M protein, the said M protein sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO 13, preferably the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11.

[0120] The inventors do consider that the expression of the M protein by the transfected cell is important for the budding of the viruses.

[0121] The inventors have advantageously placed the sequence encoding the M protein on the cloning plasmid, after having determined that this design ensures the production of complete coding virosomes (especially when a T7 promoter is present on this construct). Moreover, the cassette of the M protein has been advantageously placed at the distal extremity of the promoter for the same purpose.

[0122] More preferably a Poly adenylation sequence is inserted downstream of the 5'NCT region or (if present), downstream of theT7 terminator.

[0123] Preferably, this DNA molecule is further comprising hairpin-forming regions (just) upstream of the 3'NCT region, such as SEQ ID NO:15 or SEQ ID NO:21 (or genetic elements allowing a similar effect for the transcription, preferably sharing motives with SEQ ID NO:15 and / orSEQ ID NO:21 ) and / or excision region(s) (just) downstream the 5'NCT region, being preferably SEQ ID NO:16.

[0124] In other words, in this region, upstream of the viral genome (and the embedded sequences coding for the gene(s) of interest), there is advantageously a genetic element helping the transcription, preferably a sequence forming a hairpin, such as SEQ ID NO:15 or SEQ ID NO:21.

[0125] Conversely, in this region, downstream of the viral genome, there is advantageously a sequence coding for an excision element precisely at the end of SEQ ID NO:15, such as a ribozyme, for instance SEQ ID NO:16.

[0126] This allows the production of the coding RNA by the polymerase L, regardless the associated sequences potentially present, especially when several promoters are placed upstream of the coding sequence and / or terminator, poly A region and one or several selection cassettes are placed downstream the coding sequence.

[0127] A corresponding and related aspect of the present invention is a nucleic acid molecule in the form of a plasmid L comprising:

[0128] - an origin of replication

[0129] - a promoter for the transcription into RNA- a cassette encoding L protein of MV, the said L protein sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:6,

[0130] and a poly A signal.

[0131] This is a part of the "helper system".

[0132] The inventors have chosen to generate a plasmid encoding only the L protein since its large size and despite expected difficulties caused by its separation from the N and P expression cassettes. The inventors have, instead, found a better control of the expression of this protein, when carried on a specific plasmid.

[0133] Another corresponding and related aspect of the present invention is a nucleic acid molecule in the form of a plasmid NP comprising

[0134] an origin of replication,

[0135] a promoter for the transcription into RNA, and

[0136] genetic element for the expression of the protein N, the said protein N sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:4 over its full-length, and genetic element for the expression of the protein P, the said protein P sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:2 over its full-length, wherein either

[0137] the genetic elements for the expression of the protein N and the genetic elements for the expression of the protein P are separated by a cleaving site so as to generate two messengers RNA, or wherein

[0138] the said plasmid comprises the genetic elements for the expression of the protein N followed by a poly-adenylation signal and a second promotor for the transcription into RNA of the genetic elements for the expression of the protein P.

[0139] A preferred cleaving site is P2A or T2A; exemplary sequences share at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NOV or SEQ ID NQ:20.

[0140] This is the second part of the "helper system".

[0141] The inventors have found that the dissociation of the helper system in two distinct molecules allows a more predictable and robust expression.

[0142] The risk of missing one of the three plasmids has been advantageously mitigated upon placing the cassette for the M protein on the cloning plasmid (see above).

[0143] Preferred promoters of these nucleic acid molecules are RNA polymerase II promoter, such as those chosen in the group consisting of pActb, pCMV, pCMV+intron, pEFl A, pEFS, pCAG, pCBh, pSFFV, pSV40, phBoVl or phPGK, pUBC.

[0144] Alternatively, or in addition, a preferred promoter is the T7 (SEQ ID NO:17).Alternatively, or in addition, a preferred promoter is an RNA polymerase I promoter.

[0145] Advantageously, the RNA polymerase promoters are shorter than 2000 base pairs, preferably shorter than 1500 base pairs, more preferably shorter than 1000, 900, 800, 700 or even 600 base pairs.

[0146] This avoids too large plasmid constructs, even if the incorporation of the polymerase (I or II) promoters are much larger than the T7 promoter; in other words, the incorporation of the polymerase (I or II) promoters in the plasmids is against a prejudice since the large size of the plasmids is a problem.

[0147] Preferably, these nucleic acid molecules (especially for the "helper system") further comprising a cassette for eukaryotic and / or prokaryotic selection.

[0148] In a preferred alternative, the N and P cassettes of the helper system are on two different nucleic acid molecules (DNA plasmids). This allows better control of the relative expression of the two encoded proteins; for instance, the inventors have found that more plasmids encoding the L and N proteins, as compared to the plasmid encoding the P proteins, allows increased production of the virosomes.

[0149] A related aspect of the present invention is a genetic ("helper") system comprising the two nucleic acid molecules L and NP described above, wherein preferably the RNA polymerase promoter of the nucleic acid molecule L is different of the RNA polymerase promoter of the nucleic acid molecule NP, or a genetic ("helper") system comprising the three nucleic acid molecules L, N and P described above, wherein preferably the RNA polymerase promoter of the nucleic acid molecule is the same as for the L, N and P proteins.

[0150] This allows to better tune the relative expression levels of the different proteins. Conversely, or in addition, a (the) genetic system comprising the nucleic acids molecule L and NP both comprise a cassette for eukaryotic and / or prokaryotic selection, wherein the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule L is different as compared with the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule NP.

[0151] This allows a specific selection, ensuring the presence of the two plasmids. The same stands true for the system with three plasmids, L, N and P.

[0152] A related aspect of the present invention is a process for the production of virosomes expressing one or several protein(s) of interest comprising the steps of:

[0153] growing a eukaryotic cell line, preferably a mammalian cell line in a cell culture medium;

[0154] engineering the cloning vector as described here above andtransfecting the nucleic acid molecule within the said selected eukaryotic cells and recovering the said virosomes in the cell culture medium.

[0155] Preferably, this process is further comprising the step of engineering the "helper" genetic system described here above (plasmid L and NP) and co-transfecting the three different nucleic acid molecules within the said eukaryotic cells.

[0156] The alternative without the helper system or without the full helper system is based on competent cell lines expressing L, N and / or the P proteins at the required levels.

[0157] Another related aspect of the present invention relates to the virosomes obtainable by this process.

[0158] Conversely, another related aspect of the present invention relates to Measles Virus-derived virosomes comprising one, two or more protein(s) of interest, and an RNA sequence comprising;

[0159] - a 3'NCT region upstream of

[0160] - 1,2 or more cassette(s) for the expression of a protein of interest being separated by an intergenic region, and

[0161] - one 5' NCT region sharing at least 95% ( preferably at least 97, 98, 99 or even 100%) with SEQ ID NO 11 over its full-length,

[0162] wherein the 3' NCT region shares at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:14 over its full-length, and

[0163] the intergenic regions share at least 95% (preferably at least 97, 98, 99 or even 100%) with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19,

[0164] the RNA sequence having a combined size being a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11 .

[0165] Still another related aspect of the present invention relates to a pharmaceutical composition comprising these virosomes.

[0166] Such a pharmaceutical composition is advantageously used for vaccination (cancer, infectious disease, therapeutic vaccination to correct unwanted immune responses) or for gene therapy.

[0167] Indeed, the inventors have found that the technology allows to elicit either a humoral response, a cellular response, or a balance of the two, which provides a significant advantage over mRNA vaccines or over vaccines based on single epitopes.

[0168] The virosomes used for these pharmaceutical compositions are advantageously engineered to express at their surface a targeting protein, as well as therapeutic proteins and / or nucleic acid sequences encoding them.These targeted virosomes, expressing the MV F protein and a truncated H protein fused to a targeting segment are useful for a plurality of clinical applications.

[0169] Among them are gene therapy (including gene replacement, gene reparation and knocking-down of gene expression level), oncology, and treatment of infectious diseases.

[0170] The engineered MV virus for gene therapy is primarily based on the introduction of a mRNA (and possible as well on the direct introduction of the encoded protein). Hence this treatment is of a transitory nature, reducing the risks. If needed, the treatment is repeated upon time.

[0171] Among the gene therapy options are the treatment of the lysosomal diseases, including Fabri, Gaucher, Pompe, Tay-Sachs, Nimann-Pick, Krabbe, Farber, Wolman, 1-Cell diseases, MPS, metachromatic leukodystrophy and neuronal ceroid lipofuscinoses.

[0172] Several coagulopathies are also well-addressed by these constructs, including deficiencies in one of the coagulation Factors, including Factor VIII or Factor IX. For these diseases, preferably, a specific addressing to the liver, pancreas, or bone marrow is grafted on the truncated H protein.

[0173] Such virosome targeted to the brain tissue allows SMN1 or GDNF gene (RNA) supplementation for the treatment of Spinal muscular atrophy and Parkison's Disease, respectively.

[0174] As an alternative, virosomes encoding a CRISPR / CAS system is preferably used to correct genetic defects, where the cloning construct as above allows for a transient expression of (i) the CAS enzyme, (ii) of the guide RNA and, possibly (iii) the virosome is further comprising the repair DNA matrix.

[0175] The gene therapy approach is also useful for knocking down the expression of detrimental genes, including in infectious diseases, in cancer, in the regulation of the immune system (e.g. inflammation including chronic inflammation) and in neurodegenerative diseases upon reducing the expression of factors involved in the disease progression, such as the increased expression in Down's disease (DYRK1A, SOD1, ETS2 and SYNJ1). Knocking down of SYNJ1 in Parkinson's and / or Alzheimer's Disease is also an advantageous option allowed by the targeted virosomes of the present invention.

[0176] The targeted virosome as above also, advantageously, allows a precise treatment of cancer: targeted virosomes preferably encode cassettes for knocking-down the expression of known antigens or of neoantigens (patient specific), or encode (suicide) genes associated to a prodrug therapy, such as cytosine deaminase (associated with F-fluorocytosine) or Herpes virus Thymidine kinase (associated to Ganciclovir) or nitroreductase in association with a CB1954 treatment.The preferred targets for the virosomes are selected from the group consisting of CD34 to treat AML, CD133 to treat brain or colon tumors, preferably glioblastoma are addressed by selecting one or several targets among CD133, CD44, and / or Sox2, CD44 to target prostate or head and neck cancer, CD20 to target metastatic melanoma or B cell lymphoma, EPCAM (CD326), CD44 and / or CD166 to target colorectal cancer; CD24, CD44 and ESA to target pancreatic cancer, Seal, CD45 and / or CD34 to treat pulmonary adenocarcinoma, Strol, CD105 and / or CD44 to target bone sarcoma, orMuCl in epithelial adenocarcinoma (breast, lung, pancreas, prostate), CD19 for B-cell malignancies, CD3, CD4 and / or CD8 for targeting T cell lineages, CD30 for Hodgkin lymphoma. Tumors overexpressing (tyrosine kinase) receptors, such as HER2, EGER, ALK, ROS1, RET, cMET, FGRF are also preferred targets of the present virosomes, as well as tumors over expressing Estrogen progesterone or androgen receptor.

[0177] The neaoantigens for a precise targeting of a patient's tumor are preferably identified by sequencing of a significant portion of the tumor's DNA, as compared to normal tissue (reference genome or, preferably, patients normal tissue).

[0178] Alternatively, known oncogenes are advantageously targeted for downregulation (preferred, advantageously upon expression of a siRNA orshRNA, or an antisense construct) or for "repair".

[0179] For instance established mutations in RAS (e.g. pancreatic, colon, lung cancer), MYC (lymphomas, leukemias), HER2 (breast, gastric cancer), ABL (chronic myeloid leukemia), BRAF (melanoma, thyroid cancer, colorectal cancer, pancreatic cancer), RET (thyroid cancer), AKT (ovary, breast cancer), ALK (lymphoma, lung cancer) and EGFR (lung cancer) oncogenes as well as WNT or ERK are advantageously treated by the above virosomes encoding a cassette for knocking them out.

[0180] Conversely, for diseases associated with immune disorders, the preferred targets addressed by the virosomes are CD3, CD4 CD8, CD45RA or CD45RO, CD25, CD 127, CCR4 as well as CCR6, GDI 61 and IL23R.

[0181] Advantageously, this approach allows a specific quiescence induction in the case of the treatment of an unwanted immune response, preferably a knocking-down of metabolic pathway(s) of the targeted immune cells, preferably glucose uptake, glycolysis and / or mTOR signaling, and / or the introduction of quiescence factors, preferably BACH2, FOXO1 and / or KLF2.

[0182] Preferably, depending on the disease to treat, miRNAs are among the factors to be knocked-down, or overexpressed, and the specificity is obtained through the combination of the cell surface marker with the target miRNA(s).For instance, for Specific Lupus Erythematosus, miR-146a is preferred for cellspecific over-expression, whereas miR-21 and / or miR-148a cell-specific downregulation is preferred.

[0183] Conversely, for Rheumatoid arthritis, miR-146 and / or miR155 are preferred for cell-specific knocking-down, whereas for multiple sclerosis, miR-326, miR-155 and / or miR-21 are preferred targets fora cell-specific downregulation.

[0184] For IBD, the down-regulation of the expression of miR-21 in fibroblasts is preferred.

[0185] In addition, other tissues are advantageously targeted, such as the liver (hepatocytes), for instance ASGPR1 (asialoglycoprotein receptor), SLC10A1 / NTCP (bileacid transporter, hepatocyte-enriched) or MRP2 (ABCC2) (canalicular transporter) targets, or the kidneys, for instance LRP2 / Megalin, CD13, Nephrin (NPHS1), Podocalyxin or AQP2.

[0186] Further characteristics and advantages of the present invention will be derived from the non-limitative following description, and by making reference to the drawings and the examples.

[0187] Examples

[0188] Comparative Example

[0189] A MV-based construct as in the prior art, with a coding vector for the expression of a gene of interest and a helper vector encoding N, P and L proteins of MV has been generated for the expression of mCherry protein in Vero cells.

[0190] Unfortunately, there was almost no expression (Figure 1, left panel).

[0191] The inventors have tried to refine the experimental system, and have invested significant time and energy in adapting the cell type (HEK293 cells), culture medium, the culture conditions and the transfection condition, yet still based on cationic lipids. However, they obtained no significant improvement, only a very marginal expression, as shown in Figure 1 , right panel, up to 6.7% of positive cells and a rather low expression level.

[0192] This might explain why, in practice, no commercial MV-based system is available at large, commercial or clinical, scale.

[0193] Example 1 : design of the new construct

[0194] Against this prejudice, the inventors have reconsidered the whole MV-based system. First of all, the inventors have decided to separate the system for the expression of the L protein from the system for the expression of the proteins N and P: instead of one helper vector, the inventors have designed two helper plasmids acting synergically. This approach, although sharing less features with those of the wild-type MV where there is a naturalregulation for the correct expression levels of these proteins, is, in practice, unexpectedly more potent.

[0195] On the other hand the cloning vector has been simplified and focused on the key MV elements identified by the inventors (Figure 2): the MV 3' NOT region, one or several MV intergenic regions, a cassette for the expression of the MV M protein (downstream) and the MV 5' NOT region (terminator). Moreover, the inventors have adapted the final constructs (after the incorporation of the gene(s) of interest), so that the length from the first nucleotide of the 3' NCT to the last nucleotide of the 5' NCT is a multiple of 6 base pairs.

[0196] The inventors has further completed the design in adding a dual promotor upstream of the 3' NCT, here a Pol 1 promoter and a T7 (the transfected cell line do express the T7 polymerase), separated by a region encoding an hammerhead ribozyme (HH in Figure 2) and, downstream of the 5' NCT, a second ribozyme (sequence from hepatitis delta virus; HDV in Figure 2), a T7 terminator and a poly A region.

[0197] Example 2 - mRNA expression

[0198] Thanks to this design with two helper plasmids and the specific cloning plasmid, engineered to comprise two cassettes for expressing two flu proteins (hemagglutinin and neuraminidase), the transfection of HEK293 cells now allows a high expression of MV RNA (encoding the protein M) and of Flu RNA.

[0199] Example 3 - IgG expression

[0200] The virosomes have been administered to mice and the level of IgG was measured.

[0201] As expected, the WT virus induces a large response (Figure 4).

[0202] On the other hand, the protocol for virosome production, based on the prior art but with refined culture conditions (as in Figure 1 , right panel), yields only a marginal IgG induction.

[0203] The new protocol with two helper plasmids and a redesigned cloning vector allows a very high IgG production.

[0204] Example 4 - T cell activation or IFNa production

[0205] The same system was compared (Figure 5) using the prior art protocol (left panels: upper panel the positive control showing a strong T cell activation and the lower panel being the test with no T cell activation: the treated and untreated lines overlap).Lymph nodes ganglions have been obtained from mice treated or not with the virosomes (or a direct administration of the two Flu antigens), then challenged with the Flu virus.

[0206] Interestingly, in the right panel, the design according to the invention has yielded virosomes expressing Flu proteins allowing a strong activation of both the humoral and the cellular immune response, matching the response obtained by a direct administration of Flu antigens, far above the baseline with empty vectors.

[0207] Example 5 - Additional tests on mice

[0208] The inventors have generated three helper plasmids (SEQ ID NOs: 25-27) and a cloning plasmid encoding the flu antigen HA, NA and NP (SEQ ID NO:24). Then HEK293 cells have been cotransfected using a molar ratio of L:N:P plasmid of 4:4:1 and a molar ratio L:cloning plasmid of approx. 1 :1.5.

[0209] After 72 hours, the virosomes have been recovered and the presence of the desired epitope in the cloning vector has been verified.

[0210] Seven-week-old female C57 / BI mice have been acclimated for one week, then assigned to 5 groups:

[0211] GP1: PR8-IAV;

[0212] GP2: virosomes with HA, NA and NP genes;

[0213] GP3: virosomes with HA, NA and NP + adjuvant (Addavax);

[0214] GP4: empty virosomes;

[0215] GP5: Neg Control.

[0216] Mice of Group 1 have been injected (intramuscular) with the PR8-IAV virus (1000 PFU); Mice of groups 2-4 have been injected with the virosome, virosome+ adjuvant or the empty virosome at days 1, and 22; with a particle number being equivalent to 1000 PFU (PGR quantification with a flu virus as reference).

[0217] At Day 36, mice from all Groups have been challenged (intranasal) with a lethal dose of Mice-adapted Puerto Rico influenza A strain (PR8-IAV). In practice, the "lethal dose" has been determined to cause at least 30% of weight loss in at least 50% of the mice. In these difficult experimental settings, the "lethal dose" was of 10 PFU (intranasal). Survival, weight and temperature has been recorded for two weeks.

[0218] As shown in Figure 6, untreated mice (negative control) or mice treated with empty virosomes have 50% death after 8 days. In these settings, mice (pretreated) with PR8-IAV have a slightly better survival. However, using Gehan-Breslow-Wilcoxon statistical test, this tendency of improved survival is non-significant (P=0.5857).Conversely, the survival for mice treated with virosomes showed a marked improvement (P=0.0027) and the adjuvant has only a marginal additional effect.

[0219] Example 6 - targeted expression

[0220] The inventors have designed a functional cloning vector (matching the 6-nucleotides rule), here, with a cassette encoding a luciferase gene (NanoLuc), the MV F protein and a truncated MV H gene just after the exposed disulfide bridges, fused to a GGGS linker (SEQ ID NO:28, then to a VHH specific for HER2 further comprising a Myc antigen (for control purposes) and the M protein.

[0221] The same construct without the HER2 VHH has been generated as a negative control.

[0222] The SKBR3 cells (expressing Her2) have been transfected with the three helper plasmids as above (L, N and P plasmids; molar ratio, 4:4:1 ) and with either no cloning vector, or with the "non-VHH-HER2" cloning vector encoding NanoLuc, or with the cloning vector sketched at Figure 7 (or briefly explained here above) expressing a functional VHH moiety for a specific binding to the HER2 receptor expressed by the SKBR3 cells.

[0223] Infection protocol:

[0224] The inventors have prepared a 6-well culture plate, seed it with SKBR3 cells, and wait until they reach 80% confluence. The cells have been infected with 50pl of virosome diluted in 450p I of McCoy culture medium 2%FBS for a 3hours incubation.

[0225] Then 1 ,5ml of McCoy culture medium (with 2% FBS) is added, to end up with a total of 2ml of McCoy Medium with 2%FBS for a 72 hour incubation.

[0226] After 72h hours, all culture medium (and the last free virosomes) has been removed, and the culture is washed with PBS.

[0227] The SBKR3 cells have been detached from the well by trypsin treatment for 3 minutes, and McCoy 10% FBS has been added to help the cell recover from the Trypsination.

[0228] A centrifugation is then performed at 400g for 7 minutes, the supernatant is discarded and the pellet is resuspended in 300 I of PBS

[0229] Revelation protocol:

[0230] The inventors have used Promega's Nanoglo kit and a standard plate reader (able to read visible light); lOOpI of cell suspension in a white 96 well-plate has been contacted with lOOpI of nanoglo, for a 3min incubation and the results at read at visible light.

[0231] The results are shown at Figure 8: only the condition with the HER2 targeting has been able to trigger production by the cells of the reporter protein. The inventors havebeen surprised by the large amount of the protein production, reflected by the huge amount of the generated luminescence.

[0232] This shows that, although it can be considered as very detrimental to produce MV virosomes with the H protein, the inventors have shown that a truncated H protein is acceptable. Moreover, the grafting of a tag on this truncated protein, in synergy with the presence of the F protein, allows the infection of any cell, depending on the expressed cell markers and the addressing tag engineered on the virosome. This offers a unique opportunity for gene therapy purposes in its broadest meaning, including CRISPR / Cas (and the like) in situ engineering, immunology, cancer treatment, replacement therapy: even if the expression risks to be transient (the virosome does not introduce DNA, only RNA), the administration is to be easily repeated upon time for low risks of side-effects due to the transient capacities and manageable duration. This also allows a specific cytotoxic approach, for instance in cancer therapy, where a virosome encoding toxic molecules can be selectively targeted on tumor cells.

[0233] It should be understood that the present invention is not limited to the described embodiments and that variations can be applied without going outside of the scope of the claims.

Claims

23CLAIMS1 . A nucleic acid molecule in the form of a plasmid comprising: - a promoter for the transcription into viral RNA;- a 3'NCT region upstream of- 1 , 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions, or1 ,2 or more cassette(s) for the expression of a protein of interest,- one 5'NCT region sharing at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO: 11 over its full-length, and- an expression cassette for the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11 , andwhereinthe 3'NCT region shares at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO: 14 over its full-length, andthe intergenic regions share at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19 over their full-length,the said nucleic acid molecule being under the control of an RNA Polymerase l / ll and / or comprises the T7 promoter (SEQ ID NO:17) to allow the transcription of the DNA into a single stranded RNA and under the control of the MV RNA-dependent RNA polymerase to replicate the translated RNA and produce a mRNA from the RNA template,the said cassette(s) for the expression of a protein of interest, or the said regions for the insertion of genes after the insertion of the said genes, having a combined size being a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11 .

2. The nucleic acid molecule of claim 1, further comprising a hairpin region upstream of the 3'NCT region sharing preferably at least 95% of identity with SEQ ID NO:15 or SEQ ID NO:21 and / or an excision region downstream the 5'NCT, sharing preferably at least 95% of identity with SEQ ID NO:16.

3. The nucleic acid molecule of claim 1 or 2, wherein the nucleic acid molecule does not encode a peptide sharing 100% of identity with SEQ ID NO:22 over its full-length.

4. The nucleic acid molecule according to any one of the preceding claims further comprising one or more cassettes encoding a fusion protein, preferably the F protein of measles virus, and / or a truncated version of the SEQ ID NO:22fused to a targeting moiety, preferably wherein the truncated version of SEQ ID NO:22 is SEQ ID NO:23.

5. The nucleic acid molecule of claim 4, wherein the targeting moiety is an affinity binding molecule, or a molecule with selectivity for a marker selected from CD3, CD8, CD40, CD86, CD34 CD133, CD44, CD20, EPCAM (CD326), CD166, CD24, ESA, Seal, CD45, Strol, CD105, MuCl, CD19, CD30, CD45RA, CD45RO, CD25, CD127, CCR4, CCR6, GDI 61, IL23R, ASGPR1, SLC10A1, MRP2, LRP2, GDI 3, Nephrin, Podocalyxin, AQP2, HER2, EGER, ALK, ROS1 , RET, cMET, FGRF or a tumor antigen, the said affinity molecule or the said molecule with selectivity for the marker being preferably selected from the group consisting of VHH, scFv, fynomer, mimotope, DARPins, affibodies, monobodies and affinity peptide.

6. The nucleic acid molecule according to any one of the preceding claims, wherein the RNA polymerase II promoter is pActb, pCMV, pCMV+intron, pEFl A, pEFS, pCAG, pCBh, pSFFV, pSV40, phBoVl or phPGK, pUBC.

7. A process for the production of a nucleic acid molecule, preferably an expression vector, in the form of a plasmid, the said process comprising the step ofselecting a nucleic acid construct comprising:- a promoter for the transcription into viral RNA;- a 3'NCT region upstream of- 1 , 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions, - one 5' NCT region sharing at least 95% ( preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO: 11 over its full-length, andan expression cassette for the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11 ,whereinthe 3'NCT region shares at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:14 over its full-length, andthe intergenic regions share at least 95% (preferably at least 97, 98, 99 or even 100%) of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19 over their full-length,the said nucleic acid molecule being under the control of an RNA Polymerase l / ll and / or comprises the T7 promoter (SEQ ID NO:17) to allow the transcription of the DNA into a single stranded RNA and under the control of the MV RNA-dependent RNA polymerase to replicate the translated RNA and produce a mRNA from the RNA template,and ofinserting one or more expression cassette(s) for one or more gene of interest in the said 1 , 2 or more regions for the insertion of gene(s) of interest, separated by intergenic regions, wherein the said construct has a combined size being a multiple of 6 base pairs, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11.

8. A nucleic acid molecule in the form of a plasmid L comprising - a promoter for the transcription into RNA- a cassette encoding L protein of MV, the said L protein sharing at least 95% of identity with SEQ ID NO:6,and a poly A signal.

9. A nucleic acid molecule in the form of a plasmid NP comprising a promoter for the transcription into RNA, andgenetic element for the expression of the protein N, the said protein N sharing at least 95% of identity with SEQ ID NO:4 over its full-length, and genetic element for the expression of the protein P, the said protein P sharing at least 95% of identity with SEQ ID NO:2 over its full-length, whereineither the genetic elements for the expression of the protein N and the genetic elements for the expression of the protein P are separated by a cleaving site so as to generate two messengers RNA, or whereinthe said plasmid comprises the genetic elements for the expression of the protein N followed by a poly-adenylation signal and a second promotor for the transcription into RNA of the genetic elements for the expression of the protein P.

10. A nucleic acid molecule in the form of a plasmid N comprising a promoter for the transcription into RNA, and genetic element for the expression of the protein N, the said protein N sharing at least 95% of identity with SEQ ID NO:4 over its full-length.

11. A nucleic acid molecule in the form of a plasmid P comprising a promoter for the transcription into RNA, and genetic element for the expression of the protein P, the said protein P sharing at least 95% of identity with SEQ ID NO:2 over its full-length.

12. The nucleic acid molecule according to any one of the preceding claims 8 to 11, wherein the RNA polymerase II promoter is pActb, pCMV, pCMV+intron, pEFlA, pEFS, pCAG, pCBh, pSFFV, pSV40, phBoVl or phPGK, pUBC, and / or, wherein the RNA polymerase II promoter is shorter than 1000 nucleotides and / or wherein the plasmids are shorter than 10000 base pairs.1613. The nucleic acid molecule according to any one of the preceding claims 8 to 12 having a size of less than 10000 base pairs.

14. The nucleic acid molecule according to any one of the preceding claims 8 to 13, wherein the plasmids are shorter than 10000 base pairs and having a size of less than 10000 base pairs.

15. The nucleic acid molecule according to any one of the preceding claims 8 to 14 further comprising a cassette for eukaryotic and / or prokaryotic selection.

16. A genetic system comprising the two nucleic acid molecules L and NP, or the three nucleic acids L, N and P, according to any one of the preceding claims 8 to 11 or 13-15.

17. The genetic system of claim 16 wherein the RNA polymerase promoter of the nucleic acid molecule L of claim 8 is different of the RNA polymerase promoter of the nucleic acid molecule NP of claim 9, or wherein the RNA polymerase promoter of the nucleic acid molecule L of claim 8 is different of the RNA polymerase promoter of the nucleic acid molecule N of claim 10, and / or of the RNA polymerase promoter of the nucleic acid molecule P of claim 11.

18. A genetic system comprising the nucleic acids according to any one of the preceding claims 8 to 15 or the genetic system of claimsl 6 or 17, wherein the nucleic acid molecule L and NP of claims 8 and 9 both comprise a cassette for eukaryotic and / or prokaryotic selection, wherein the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule L of claim8 is different as compared with the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule NP of claim 9, or wherein the nucleic acid molecule L of claim 8, the nucleic acid molecule N of claim 10 and / or the nucleic acid molecule P of claim 11 comprise a cassette for eukaryotic and / or prokaryotic selection, wherein the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule L of claim 8 is different as compared with the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule N of claim 10, and / or is different as compared with the said cassette for eukaryotic and / or prokaryotic selection of the nucleic acid molecule P of claim 11.

19. A process for the production of virosomes expressing one or several protein(s) of interest comprising the steps ofgrowing a eukaryotic cell line, preferably a mammalian cell line in a cell culture medium;engineering the nucleic acid molecule according to any one of the preceding claims 1 to 6, or using the nucleic acid molecule obtained in claim 7 and27transfecting the nucleic acid molecule within the said selected eukaryotic cells and recovering the said virosomes in the cell culture medium.

20. The process of claim 19, further comprising the step of engineering the genetic system according to any one of the preceding claims 16-18 and co-transfecting the three or four different nucleic acid molecules within the said eukaryotic cells.

21. The process of claims 19 or 20, wherein the recovering of the virosomes comprises a DNAse treatment to hydrolyze host cell's DNA, filtration and / or precipitation of the virosome.

22. The process of claim 19 to 21, wherein the plasmids L and / or N are introduced in a molar excess as compared to the plasmid P, or wherein the plasmid L is introduced in a molar excess as compared to the plasmid NP, the said molar excess being of at least 1.5 (mol plasmid L and / or N: mol plasmid P or plasmid Lplasmid NP), preferably wherein the promoters for the L, N and P proteins are the same.

23. The process of claim 19 to 22, wherein the cloning plasmid is introduced in a molar relative amount as compared to the plasmids L, N, and P, the said molar relative amount being between 0.1 and 9 (mol cloning plasmid: mol sum of plasmids L, N ,P), and wherein the relative ratios of plasmids L an / or N, and P are as defined in claim 22.

24. The virosomes obtainable by the process of claims 19 to 23.

25. Measles virus-derived virosomes comprising one, two or more protein(s) of interest, and an RNA sequence comprising;- a 3' NCT region upstream of viral genomic sequence- 1 ,2 or more cassette(s) for the expression of this (these) protein(s) of interest, each cassette being separated by a different intergenic region, and- a 5' NCT region sharing at least 95% of identity with SEQ ID NO 11 over its full-length, - a cassette for the expression of the M protein, the said M protein sharing at least 95% of identity with SEQ ID NO 13, the said cassette being placed after an intergenic region and just upstream of SEQ ID NO:11 ,wherein the 3' NCT region shares at least 95% of identity with SEQ ID NO:14 over its full-length, andthe intergenic regions share at least 95% of identity with SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:18 and / or SEQ ID NO:19;the said RNA having a total size being a multiple of 6 nucleotides, as calculated starting from the first nucleotide of the SEQ ID NO:14 to the last nucleotide of SEQ ID NO:11 .

26. The virosome of claim 24 or 25 comprising no MV F or H protein.2827. The virosome of claim 24 or 25 comprising the MV F and a truncated MV H protein fused to a targeting moiety, preferably wherein the truncated protein H corresponds to an amino acid segment encoded by SEQ ID NO:23.

28. The virosome of claim 27 wherein the targeting moiety is a peptide chain specifically recognizing a cell surface marker selected from the group consisting of CD34, CD 133, CD44, nestin, Sox2, CD20, EPCAM, CD 166, CD24, ESA, Seal, CD45, Strol, CD105, MuCl, CD19, CD3, CD4, CD8, CD30, HER2, EGFR, ALK, ROS1, RET, cMET, FGRF, CD45RA or CD45RO, CD25, GDI 27.

29. A pharmaceutical composition comprising the virosomes of according to any one of the preceding claims 24 to 28 being for use in vaccination.

30. A pharmaceutical composition comprising the virosomes according to any one of the preceding claims 24 to 28, being for use in gene therapy.

31. A pharmaceutical composition comprising the virosomes according to any one of the preceding claims 24 to 28, being for use in the treatment of cancer.

32. A pharmaceutical composition comprising the virosomes according to any one of the preceding claims 24 to 28, being for use in the treatment of autoimmune disease.

33. The pharmaceutical composition of any one of claims 29 to 32, wherein the administration route is selected from intramuscular, intradermal, oral, intranasal, or transderma I.

34. The pharmaceutical composition of claim 33, being for oral administration and wherein the virosomes have been encapsulated, and / or the composition being gastric resistant.

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