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  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32211—Cardiovirus, e.g. encephalomyocarditis virus
  • C12N2770/32222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2770/00011—Details
  • C12N2770/32011—Picornaviridae
  • C12N2770/32211—Cardiovirus, e.g. encephalomyocarditis virus
  • C12N2770/32271—Demonstrated in vivo effect
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  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

• the present invention relates to the field of ribonucleic acids, and more particularly to the in vitro synthesis of messenger ribonucleic acid (mRNA), to its stability and to its translation into a polypeptide, in particular into transfected cells.
• mRNA messenger ribonucleic acid
• MRNA is an essential molecule in the production of polypeptides on an industrial scale. Its stability and the efficiency of its transcription and translation strongly influence the yield of downstream polypeptides and therefore their cost.
• MRNA is also a molecule of choice in pharmaceutical compositions used in gene therapy and in genetic vaccination. Indeed, the integration of an mRNA molecule into the genome of a transfected cell has never been demonstrated, unlike DNA molecules. However, it is not stable in solution because it is sensitive to degradation by ribonucleases. The synthesis of stable RNA molecules in vitro and in vivo is therefore critical to reduce the amounts of mRNA required for optimum therapeutic efficacy and thereby reduce their cost. In addition, the use of smaller amounts of more stable mRNA reduces the risk of treatment-related side effects.
• the widely used large-scale mRNA synthesis method is an acellular in vitro transcription system.
• This method uses only a few purified compounds (ie a reaction buffer, a gene-bearing DNA molecule, a recombinant RNA polymerase and four ribonucleotide triphosphates); and the only RNA to be produced is the mRNA that it is desired to synthesize.
• a reaction buffer ie.g., a gene-bearing DNA molecule, a recombinant RNA polymerase and four ribonucleotide triphosphates
• the only RNA to be produced is the mRNA that it is desired to synthesize.
• the end of the reaction there are no degradation products of the mRNA due to the absence of RNase in the reaction mixture.
• There are also no other RNA species which is a major advantage compared to mRNA synthesis by cells in culture. The purification of the mRNA is thus greatly simplified.
• an mRNA is provided with a cap molecule at its 5 'end, which protects the latter from exoribonucleases.
• the cap is a complex molecule composed of two guanosines linked by their 5 'carbon by a chain of three phosphate groups. Terminal guanosine is methylated at position 7 of guanine.
• the mRNA is stabilized thanks to the resistance that the cap gives it to a progressive enzymatic degradation of 5 'to 3' carried out by the exoribonuclease Xrn1.
• the cap performs other functions, including ribosome recruitment (Cowling, 2010). For this, the cap starts by recruiting translation initiation factors, then they recruit ribosomes. These latter then translate the mRNA into proteins.
• a cap molecule On an industrial scale, to enhance the stability of the mRNA and allow efficient translation of the mRNA into polypeptides in the transfected cells, a cap molecule must be incorporated at the 5 'end.
• the capping molecule may be added at a later stage of transcription using a styling enzyme, such as Vaccina virus 2'-O-methyltransferase (IVIartin et al., 1975). .
• Vaccina virus 2'-O-methyltransferase IVIartin et al., 1975.
• this additional step increases the complexity of the synthesis, requires the purification of the styling enzyme and is not very effective (Contreas et al., 1982).
• this enzyme requires S-adenosyl-L-methionine, which is an unstable molecule in aqueous solution.
• analogs of the cap have been developed in the prior art, which, however, are unsatisfactory.
• analogs of the cap have been developed in the prior art, which, however, are unsatisfactory.
• These analogs allow co-transcriptional styling mediated by phage RNA polymerase, thereby avoiding the additional step of cap synthesis, and may also enhance the stability of the mRNA.
• RNA molecule having high stability and translation efficiency, preferably having a stability and translation efficiency at least as high as a capped mRNA molecule.
• RNA molecule having a simple production method and whose manufacturing cost is significantly reduced.
• the present invention relates to a stable mRNA molecule that can be translated with high efficiency and without a cap.
• This mRNA is particularly advantageous because its production cost is much lower than that of a conventional mRNA comprising a cap molecule or an analogue thereof.
• the subject of the present invention is also an mRNA molecule whose efficiency of its transcription in vitro is improved compared with that of a conventional mRNA comprising a cap molecule. or an analogue thereof.
• the mRNA of the invention is also advantageous because, despite the significant decrease in the cost of its production and / or the increase in the yield of its synthesis, said mRNA is at least as efficient as a conventional mRNA with a cap during the transfection of cultured cells and tissues.
• the inventors have demonstrated very surprisingly that the levels and duration of expression obtained after in vivo transfection are at least as high as those of a mRNA with a cap.
• the kinetics of expression in Caco-2 cells of a reporter protein is identical, whether the mRNA of the invention or a control capped mRNA are transfected (FIGS. 2 and 3).
• the inventors have very surprisingly demonstrated that the expression of a reporter protein is 2-fold to about 10-fold higher when transfected with the mRNA of the invention in vivo, in the dermis or muscle of the invention. a mouse than when transfected control capped mRNA ( Figure 5 and Figure 15).
• the reaction is simplified because the additional styling step is not necessary.
• mRNA molecule of the invention is therefore clearly advantageous in terms of reduced costs and facilitated production compared to a mRNA of the prior art, for an expression at least as high.
• mRNA molecule is intended to mean any linear sequence of ribonucleotides. In the present application, these sequences are expressed in the 5 'to 3' direction starting from the 5'-UTR region.
• ribonucleotide any natural ribonucleotide (eg guanine, cytidine, uridine, adenosine), as well as analogues of these nucleotides as well as nucleotides having chemically or biologically modified bases (eg by methylation, alkylation, acylation, thiolation etc.), intercalated bases, modified ribose groups and / or modified phosphate groups.
• natural ribonucleotide eg guanine, cytidine, uridine, adenosine
• analogues of these nucleotides as well as nucleotides having chemically or biologically modified bases (eg by methylation, alkylation, acylation, thiolation etc.), intercalated bases, modified ribose groups and / or modified phosphate groups.
• the cap at the 5 'end of an mRNA molecule can be replaced by at least one copy of an Xrn1 exoribonuclease resistant sequence (xrRNA), originating from the region. 3'-UTR of a virus of the genus Flavivirus, preferably accompanied by an internal ribosome entry sequence (IRES) per open reading phase.
• xrRNA exoribonuclease resistant sequence
• IRS internal ribosome entry sequence
• the cost of in vitro transcription production of such an mRNA molecule is decreased by about 30 fold, relative to a capped mRNA molecule, and its yield is improved.
• the mRNA molecule of the invention is particularly stable in cells transfected, while being translated efficiently, even though it does not have a cap.
• cap is meant the nucleoside 7-methylguanosine (N7-methyl guanosine or m7G) as well as any mutant, variant, analog or fragment thereof which can be connected to the first transcript nucleotide of a mRNA by a 5 'bond -5 'triphosphate.
• the capped analogs include non-methylated analogs (eg P 1 - (Guanosyl) P 3 - (5 '- (guanosyl)) triphosphate), monomethylated (eg P 1 - (5'-7 -methyl-guanosyl) P 3 - (5 '- (guanosyl)) triphosphate), trimethylated (eg P 1 - (5'-2,2,7-trimethylguanosyl) P 3 - (5'- (guanosyl)) triphosphate), or having a substitution of the 3'-OH group of the guanine half m 7 by a 3'-O-methyl group (eg ARCA P 1 - (5 '- (3'-O-methyl) -7 methyl guanosyl) P 3 - (5 '- (guanosyl)) triphosphate).
• non-methylated analogs eg P 1 - (Guanosyl) P 3 - (5 '- (guanosyl) tri
• the subject of the invention is therefore a messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule comprising:
• a 5'-UTR region comprising at least one copy of a consensus sequence GUCAGRYC (N 7 -19) GCCA (N 12 -19) UGCNRYCUG (xrRNA);
• IVS internal ribosome entry RNA sequence
• an IRES sequence is upstream of each open reading phase.
• the subject of the invention is a messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule comprising from 5 'to 3':
• mRNA messenger ribonucleic acid
• a 5'-UTR region comprising at least one copy of a consensus sequence GUCAGRYC (N 7-19 ) GCCA (N 12 -19) UGCNRYCUG (xrRNA);
• IVS internal ribosome entry RNA sequence
• the mRNA molecule also comprises a 3'-UTR region.
• the mRNA molecule also comprises at least one aptamer RNA promoting the penetration of the mRNA molecule into the cells, preferably into muscle cells.
• aptamer RNA can promote penetration directly or indirectly via a peptide.
• the mRNA molecule further comprises a stem-loop at the 5 'end.
• the subject of the invention is a messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule consisting of: 5 'to 3': ⁇ a 5'-UTR region comprising at least one copy a consensus sequence GUCAGRYC (7-19 N) GCCA (N 12 -19) UGCNRYCUG (xrRNA);
• mRNA messenger ribonucleic acid
• IVS internal ribosome entry RNA sequence
• the 3'-UTR untranslated region of Flavivirus has the property of blocking Xrn1 and thus protecting the downstream sequence (Chapman et al., 2014).
• This RNA region is composed of at least one sequence that folds on itself forming a complex three-dimensional structure, which inhibits the progression of Xrn1 and thus the degradation of viral subgenomic RNAs.
• sequences have hitherto never been inserted in 5 'of mRNA.
• these three-dimensional structures may not fold back correctly when they are no longer surrounded by the sequences of the flaviviral 3'UTR region.
• xrRNA sequences inhibit translation
• the inventors have surprisingly demonstrated here that the protective and translation initiation functions of the cap can be successfully replaced by at least one copy of an xrRNA sequence and at least one copy of an IRES sequence, preferably of the EMCV virus without affecting the efficiency of the translation.
• the use of xrRNA and IRES sequences makes it possible to improve the efficiency of the translation in a very important way.
• Flavivirus means any virus of the genus Flavivirus, including yellow fever, dengue fever, West Nile (WNV), Zika, Japanese encephalitis, Rocio, Murray Valley, Bagaza, Kokobera, Ntaya, Kedougou, Sepik, Saint Louis, Usutu, Alfuy, Wesselbron, Ilheus, Bussuquara, Tembusu, Chaoyang, Yokose, Donngang, and so on. virus of the genus Flavivirus.
• the subject of the invention is thus a stable mRNA molecule comprising at least one copy of an xrRNA sequence in the 5 'region of said mRNA. Said mRNA is efficiently translated into protein at levels and times similar to those of a capped molecule. According to a preferred embodiment of the invention, the mRNA molecule comprises two copies of xrRNA.
• Xrn1-resistant RNA sequence or "xrRNA” is meant any polynucleotide sequence which makes it possible to reduce, slow down or prevent the degradation of an mRNA by the exoribonuclease Xrn1.
• the xrRNA sequence comprises a consensus sequence.
• the term "consensus sequence” is meant any sequence comprising at least the sequence represented by 5'-GUCAGRYC (7-19 N) GCCA (N 12 -i 9) UGCNRYCUG-3 ', wherein each N may represent independently a nucleotide selected from A, C, T, G, and U or an analogue of one thereof.
• Each R represents a purine and each Y represents a pyrimidine.
• the number of N nucleotides between the conserved bases may vary in the consensus sequence, from 5 'to 3', from 7 to 19 bases and from 12 to 19 bases, as indicated. Said sequence forms a three-dimensional stem-loop structure.
• the mRNA molecule comprises at least one copy of an xrRNA sequence selected from the sequences represented by: SEQ ID NO: 1 to SEQ ID NO: 44.
• the mRNA molecule comprises more than one copy of said sequences, these copies may be identical or different.
• the mRNA molecule comprises at least one copy of one of the sequences represented by SEQ ID NO: 1 1 and SEQ ID NO: 26.
• the mRNA molecule comprises one copy of the sequence represented by SEQ ID NO: 11 and a copy of the sequence represented by SEQ ID NO: 26.
• the spacer sequence between two xrRNA sequences corresponds to SEQ ID NO: 47.
• the spacer sequence between the xrRNA sequence and the IRES sequence corresponds to SEQ ID NO: 48. Spacing may also be present between two open reading phases or between an open reading phase and a TIRES sequence, when the mRNA molecule comprises at least two open reading phases.
• spacer sequence is meant any noncoding polynucleotide sequence for physically separating the sequence upstream from said downstream sequence spacing sequence.
• the mRNA molecule according to the invention may in particular comprise one or more spacer sequences.
• the spacer sequence may be from 2 to 300 nucleotides in length. Preferably it is 2 and 10 nucleotides, even more preferably between 2 and 5 nucleotides. As an alternative, it may be between 10 and 150 nucleotides and even more preferably between 15 and 40 nucleotides. Preferably, the spacer sequence does not generate secondary structures.
• the mRNA of the invention further comprises at least one internal ribosome entry RNA (IRES) sequence.
• IRS internal ribosome entry RNA
• IRES internal ribosome entry site
• EMCV encephalomyocarditis virus
• IRES sequences are usually located in the 5'-UTR region. They can also be located between two open reading phases, which makes it possible to initiate a bi- or polycistronic translation from a single mRNA.
• the mRNA of the invention comprises a single copy of an IRES sequence in the 5 'region of said mRNA.
• a second copy of an IRES sequence is located between two open reading phases.
• the mRNA of the invention comprises a copy of an IRES sequence in the 5 'region and a copy of an IRES sequence between two open reading phases.
• said IRES sequence (s) is / are located (s) downstream of (s) sequence (s) xrRNA.
• the molecule mRNA comprises at least two IRES sequences
• said sequences may be identical or different.
• one or more IRES sequences can be selected according to their translation initiation efficiency.
• the selection of two different IRES sequences is particularly advantageous when the mRNA molecule comprises at least two different open reading phases and the translation efficiency desired for the first open reading phase differs from that desired for the second.
• IRES elements also have complex three-dimensional structures.
• reciprocal interference between xrRNA and IRES sequences is possible by formation of pairings between RNA sequences of each of the two regions. Such interference could prevent the formation of the correct structures of the xrRNA and IRES sequences respectively.
• xrRNA structures could in particular prevent the recruitment of translation initiation factors and ribosomes performed by IRES.
• the inventors have shown that the presence of xrRNA and IRES sequences makes it possible to obtain protein expression yields in transfected cells, which are at least similar to those obtained with a capped mRNA molecule.
• the xrRNA and IRES sequences together can therefore be substituted for a capping or cap-like molecule. They are essential to ensure the translation of an open reading phase contained in the mRNA of the invention and the stability of the mRNA.
• the mRNA of the invention is thus at least as stable as mRNA with a cap, while being at least as efficiently translated.
• the cost of its synthesis by in vitro transcription is greatly reduced compared to that of a capped mRNA.
• the mRNA of the invention further comprises a stem-loop in the 5'-UTR upstream of the consensus sequence GUCAGRYC (7- N 1 9) GCCA (N 12 -19) UGCNRYCUG (xrRNA), preferably at the 5 'end of the mRNA molecule.
• stem-loop is meant any polynucleotide sequence forming a double-helical structure in which the 5 'end of one strand is physically linked to the 3' end of the other strand through an unpaired loop .
• the stem-loop consists of a double-stranded rod and an unpaired single-stranded loop.
• Said physical bond may be either covalent or non-covalent.
• said physical link is a covalent bond.
• the size of the RNA loop can be for example between 3 and 30 nucleotides.
• the size of the loop is preferably at least 3 nucleotides, preferably at least 4 nucleotides.
• the length of the double-stranded stem may for example be between 5 and 50 nucleotides.
• the length of the stem is preferably between 5 and 50, 5 and 40, 5 and 30, 5 and 25, or more preferably between 5 and 10 nucleotides. Even more preferably, the length of the stem is 6, 7 or 8 nucleotides.
• a stem-loop is preferentially formed at the 5 'end of the mRNA molecule.
• the stem-loop with the sequence of SEQ ID NO: 87.
• the stem-loop is preferentially separated from the xrRNA sequence by a spacer sequence.
• said spacer sequence has a length equal to or less than 5 nucleotides (i.e. of 5, 4, 3, or 2 nucleotides).
• stem-loop structure at the 5 'end of the mRNA molecule also called 5'-SL here, for "stem-loop" at the 5 'end
• 5'-SL stem-loop structure at the 5 'end
• the inventors have not observed any advantageous effect when the stem-loop placed at the 5 'end is separated from the xrRNA sequence by a spacer sequence having a length of approximately 70 nucleotides (see FIG. 15).
• the xrRNA sequence masks the 5 'end with respect to phosphatases and Xrn1, at least when this sequence is in the form of a loop rod and at proximity.
• the subject of the invention is therefore a messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule comprising from 5 'to 3':
• mRNA messenger ribonucleic acid
• a 5'-UTR region comprising at least one copy of a consensus sequence GUCAGRYC (N 7-19 ) GCCA (N 12 -19) UGCNRYCUG (xrRNA);
• IVS internal ribosome entry RNA sequence
• the mRNA molecule may further comprise a second IRES sequence followed by a second open reading phase.
• the mRNA molecule may further comprise an aptamer RNA as defined herein, located between the stem-loop and the xrRNA sequence (s) or, preferably, between the xrRNA sequence (s) and the IRES sequence. .
• the mRNA molecule may further comprise a 3'-UTR region as defined herein.
• the subject of the invention is a messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule comprising from 5 'to 3':
• 5'-UTR comprising a stem loop at the 5 'end followed by at least one copy of a sequence consensus GUCAGRYC (7-19 N) GCCA (N 12- I9) UGCNRYCUG (xrRNA);
• an aptamer RNA is optionally, an aptamer RNA
• IVS internal ribosome entry RNA sequence
• the subject of the invention is a messenger ribonucleic acid (mRNA) molecule devoid of a 5 'to 3' capcompound molecule:
• 5'-UTR comprising a stem loop at the 5 'end followed by at least one copy of a sequence consensus GUCAGRYC (7-19 N) GCCA (N 12- I9) UGCNRYCUG (xrRNA);
• an aptamer RNA is optionally, an aptamer RNA
• IVS internal ribosome entry RNA sequence
• aptamer any nucleic acid having recognition and specificity properties related to its ability to adopt particular three-dimensional structures, in particular similar to monoclonal antibodies (see for example Dunn et al., 2017).
• An aptamer can be composed of modified DNA, RNA and / or RNA, preferably RNA.
• Said aptamer may be composed of 6 to 50 nucleotides, such as ribonucleotides as defined above.
• the aptamer can be isolated by various techniques which are well known to those skilled in the art, such as the identification of an aptamer in vitro by one or more cycles of in vitro selection or from combinatorial libraries of a large number of random sequence compounds by an iterative selection method ("SELEX" technique).
• SELEX iterative selection method
• the identification of an aptamer in vitro by selection advantageously makes it possible to obtain aptamers having a precise effect or function, without however having to know the target against which said aptamer is directed.
• the manufacture or selection of aptamers is, eg, described in European Patent Application EP0533838.
• aptamer RNAs have been identified in the context of this according to their ability to penetrate cells of interest, more particularly tissue cells, preferably muscle cells (eg muscle fiber cells).
• the aptamer according to the invention is capable of passing through a cell membrane, more preferably the plasma membrane and / or the endosomal membrane of a mammalian cell.
• the aptamer according to the invention advantageously comprises 6 to 50 nucleotides, more preferably 10 to 45 nucleotides, 20 to 40 nucleotides, still more advantageously 30 to 40 nucleotides.
• the aptamer is composed of ribonucleotides, as defined above.
• the aptamer according to the invention has at least 70% identity, more advantageously at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity.
• identity at least 97% identity, at least 98% identity, still more preferably at least 99% identity with aptamer A having the sequence of SEQ ID NO: 64, aptamer B having the sequence of SEQ ID NO: 65, or aptamer C having the sequence of SEQ ID NO: 66.
• the percentages of identity referred to in the context of the disclosure of the present invention are determined on the basis of an overall alignment of the sequences to be compared, that is to say on an alignment of the sequences taken in their entirety over the entire length using any algorithm well known to those skilled in the art such as the Needleman and Wunsch algorithm, 1970. This sequence comparison can be done using any logic it is well known to those skilled in the art, for example the needle software using the "open Gap" parameter equal to 10.0, the "Gap extend” parameter equal to 0.5 and a "Blosum 62" matrix.
• the aptamer according to the invention is chosen from aptamer A having the sequence of SEQ ID NO: 64, aptamer B having the sequence of SEQ ID NO: 65, and aptamer C having the sequence of SEQ. ID NO: 66, still more preferably selected from aptamer A having the sequence of SEQ ID NO: 64 and aptamer C having the sequence of SEQ ID NO: 66.
• the mRNA molecule according to the invention comprises at least one copy of an aptamer capable of crossing a membrane, preferably a plasma membrane and / or an endosomal membrane of a mammal.
• an aptamer capable of crossing a membrane, preferably a plasma membrane and / or an endosomal membrane of a mammal.
• said aptamer promotes its penetration into cells, preferably into muscle cells or skin.
• the mRNA molecule according to the invention comprises at least one copy of aptamer A having the sequence of SEQ ID NO: 64, aptamer B having the sequence of SEQ ID NO: 65, and / or aptamer C having the sequence of SEQ ID NO: 66.
• the mRNA molecule comprises the aptamer in the 5'-UTR region upstream of the one or more consensus sequence (s) GUCAGRYC (N 7-1 9) GCCA (N 1 2-19) UGCNRYCUG (xrRNA).
• the aptamer is placed in the 5'-UTR region downstream of one or more sequence (s) consensus GUCAGRYC (N 7-1g) GCCA (N 12- 19) UGCNRYCUG (xrRNA) but upstream of IRES sequences and open reading phases.
• sequence consensus GUCAGRYC (N 7-1g) GCCA (N 12- 19) UGCNRYCUG (xrRNA) but upstream of IRES sequences and open reading phases.
• mRNA messenger ribonucleic acid
• a 5'-UTR region comprising at least one aptamer capable of traversing a cell membrane, preferably a plasma and / or endosomal membrane, preferably a mammalian cell, preferably at least one aptamer selected from the group consisting of aptamers a, B, and C as described herein, and at least one copy of a sequence consensus GUCAGRYC (7-19 N) GCCA (N 12- I9) UGCNRYCUG (xrRNA);
• IRES internal ribosome entry RNA sequence
• the mRNA molecule comprises the aptamer in the 5'-UTR region upstream of consensus sequence (s) GUCAGRYC (N 7-1 9) GCCA (N 12 -19) UGCNRYCUG (xrRNA) ( see for example Figure 1F).
• the RNA molecule may also comprise downstream aptamer (s) sequence (s) consensus GUCAGRYC (N 7- 19) GCCA (N 12-1 9) UGCNRYCUG (xrRNA), e.g., between / the xRRNA consensus sequences and the IRES sequence (s).
• the subject of the invention is a mRNA molecule without a capping molecule comprising from 5 'to 3':
• 5'-UTR comprising a stem loop at the 5 'end followed by at least one copy of a sequence consensus GUCAGRYC (7-19 N) GCCA (N 12- I9) UGCNRYCUG (xrRNA);
• At least one aptamer capable of crossing a cell membrane A copy of an internal ribosome entry RNA sequence (IRES); and
• aptamer A corresponds to the "shot47" aptamer, identified by Tsuji et al., 2013. aptamer A binds favorably to a peptide motif of poly-histidine type with high affinity.
• a aptamer can thus bind any molecule (e.g., peptide or protein) comprising a poly-histidine tag (e.g., the HHHHHH motif).
• the mRNA molecule according to the invention can be linked via the aptamer A to a molecule allowing it to penetrate better cells, such as a "peptide penetrating the cells". or "CPP", said CPP comprising a poly-histidine tag.
• cell penetrating peptide or “CPP” is meant any peptide, polypeptide, or protein that is capable of traversing a cell membrane, more preferably the plasma membrane and the endosomal membrane, of a mammalian cell.
• the CPP retains this property when it is linked to another molecule, in particular an mRNA molecule, thereby causing the membrane to pass through the latter.
• any possible mechanism of membrane traversal is contemplated including, for example, energy dependent (i.e., active, eg endocytosis) transport mechanisms. energy-independent transport mechanisms (eg diffusion).
• CPPs are cationic peptides (Poillot and De Waard, 2011).
• the mRNA molecule can be non-covalently bound in view of its negative charge to the CPP, taking advantage of electrostatic interactions and / or hydrophobicity.
• the mRNA molecule can be covalently bound to the CPP.
• CPPs can form oligomers consisting of at least two identical or different peptide molecules.
• a CPP preferably binds non-covalently to an aptamer RNA. Indeed, this type of bond is advantageous because of its simplicity, by simple mixing of the CPP and mRNA molecules, its low cost and the fact that these molecules are fully biodegradable. Indeed, no non-natural and non-biodegradable chemical group is needed.
• the length of the CPPs according to the present invention is preferably from about 8 amino acid residues to about 60 amino acid residues. More preferably, the length is 8 to 40 amino acid residues, more preferably 8 to 30 amino acid residues, still more preferably 10 to 25 amino acid residues (e.g. or 20 amino acid residues).
• the length of the CPPs is not necessarily limited to those described above. Derivatives of the CPPs described herein, for example having different lengths, can in particular be created by those skilled in the art in view of his general knowledge.
• the CPP of the invention may be the CPP "M12" as described by Gao X.
• said CPP comprises a poly-histidine unit (eg hexahistidine), preferentially linked to said CPP by a spacer.
• the spacer is a hydrophilic spacer, advantageously unstructured and unfilled, more advantageously consisting of glycines and serines.
• the spacer has a length of about 21 amino acids, preferably 21 amino acids.
• the CPPs according to the invention have at least 70% identity, more advantageously at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity. at least 97% identity, at least 98% identity, still more preferably at least 99% identity with the M12-H6 peptide having the sequence of SEQ ID NO: 75, the CPP1-H6 peptide having the sequence of SEQ ID NO:
• the CPP according to the invention is chosen from: M12 -H6 having the sequence of SEQ ID NO: 75, CPP1-H6 having the sequence of SEQ ID NO: 76, CPP2-H6 having the sequence of SEQ ID NO:
• the CPP is bound to the mRNA molecule non-covalently or covalently, preferably non-covalently.
• the CPP is bound to an aptamer RNA included in the mRNA molecule (ie aptamer A when the CPP comprises a poly-histidine tag).
• the CPPs of the present invention do not exert significant cytotoxic and / or immunogenic effects on their target cells after crossing the plasma membrane, i.e. they do not interfere with viability cell, cell transfection and / or penetration.
• the term "not significant” as used in this context means that less than 50%, preferably less than 40% or 30%, of preferably less than 20% or 10% and in particular less than 5% of the target cells are killed after the CPP-bound mRNA molecule has passed through the plasma membrane, and has thus been internalized by the cell.
• the potential intrinsic cytotoxic and / or immunogenic effects of a CPP of the invention may be "masked" by introducing one or more modifications into the peptide, for example by means of chemical synthesis or Recombinant DNA. Such modifications may include, for example, the addition, removal or substitution of functional groups or the variation of the positions of these functional groups. Those skilled in the art are well aware of how such "masking" can be accomplished for a given peptide.
• the subject of the invention is a mRNA molecule without a capping molecule comprising from 5 'to 3':
• a 5'-UTR region comprising at least the aptamer A RNA of SEQ ID NO: 64 and at least one copy of a consensus sequence GUCAGRYC (N 7-1 9) GCCA (N 12 -I 9 ) UGCNRYCUG (xrRNA );
• IVS internal ribosome entry RNA sequence
• mRNA molecule is bound to a cell-penetrating peptide (CPP) fused to a poly-histidine tag, said CPP being preferably selected from: M12-H6 having the sequence of SEQ ID NO: 75, CPP1-H6 having the sequence of SEQ ID NO: 76, CPP2-H6 having the sequence of SEQ ID NO: 77, and CPP3-H6 having the sequence of
• said CPP is noncovalently bound to the aptamer.
• open reading phase is meant any polynucleotide sequence that can be translated into a polypeptide of interest.
• An open reading phase is read in blocks of three successive nucleotides, called codons, each codon representing an amino acid.
• the polypeptide is synthesized by translating the codons of said open reading phase with ribosomes.
• the first amino acid of the polypeptide is generally indicated on the mRNA molecule by the AUG codon, which therefore indicates the beginning of the open reading phase.
• Other start codons are known, such as AUN, or NUG, where N is A, C, U or G.
• the end of the polypeptide is indicated on the mRNA molecule in the form of a stop codon UAA, UGA or UAG.
• the stop codon indicates the end of the open reading phase on the mRNA molecule.
• the open reading phases according to the invention are more particularly open reading phases whose translation into the transfected cell generates products having a therapeutic or vaccinal interest for applications in human or veterinary medicine.
• the products of therapeutic interest are proteins.
• proteins of therapeutic interest mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 92 03120), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc., apolipoproteins: ApoAl , ApoAIV, ApoE, etc. (FR 93 05125), dystrophin or a minidystrophin (FR 91 11947), tumor suppressor proteins: p53, Rb, Rap1A, DCC, k-rev, etc.
• trophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc.
• apolipoproteins ApoAl , ApoAIV, ApoE, etc.
• FR 93 04745 the factors involved in coagulation: Factors VII, VIII, IX, etc., pro-apoptotic proteins: thymidine kinase, cytosine deaminase, etc. ; or all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc., see for example WO 201 1/089527), a ligand RNA (WO 91/19813), etc.
• the protein of interest encoded by the mRNA can also be an antigen, capable of generating in humans or animals an immune response, for the production of vaccines. It may be in particular antigenic proteins specific for Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, or even specific for tumors (EP 259 212). Finally, the protein of interest may be an adjunctive protein, which stimulates the immune response, to improve the effectiveness of a vaccine.
• the protein of interest encoded by the mRNA may be a protein that has a favorable effect on a mRNA molecule without a cap or the protein expressed by said molecule. This favorable effect could result from different mechanisms.
• a protein encoded by mRNA lacking a cap may increase the stability of a mRNA molecule without a cap by binding to said molecule or degrading at least one protein having RNase activity .
• a protein encoded by mRNA lacking a cap may increase the translation of said molecule by promoting the recruitment of the initiation factors or by binding the capped cellular mRNAs in order to inhibit their translation.
• the protein of interest encoded by the mRNA is 2Apro protein derived from a picornavirus, such as the human rhinovirus type 2 (HRV2).
• the 2Apro protein increases the expression of mRNA without a cap in view of its protease activity, cleaving the N-terminal end of the elF4G initiation factor and thus preventing said factor initiation to recognize a capped mRNA.
• This cleavage could make it possible to reduce the competition between the mRNA of the invention and the capped mRNAs in vivo.
• the inventors have surprisingly shown that co-transfection of cells with a first mRNA according to the invention coding for 2Apro and a second mRNA according to the invention coding for a reporter protein makes it possible to increase the expression of the reporter protein. .
• the presence of the 2Apro protein is therefore particularly advantageous because it makes it possible to increase the specific expression of the protein encoded by an mRNA of the invention devoid of a cap (FIG. 7).
• the mRNA of the invention encodes a 2Apro protein, preferably a 2Apro protein derived from a picornavirus, even more preferably the HRV2 virus.
• the 2Apro protein has the sequence of SEQ ID NO: 81
• the 2Apro protein is encoded by an mRNA having the sequence of SEQ ID NO: 80.
• the open reading phase can also encode a therapeutic mRNA.
• This may be, for example, an antisense sequence whose expression in the target cell makes it possible to control the transcription or translation of cellular mRNAs.
• Such sequences may for example be transcribed in the target cell to RNA complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
• the mRNA of the invention further comprises the XRRNA sequence (s) and the IRES sequence, an open reading phase encoding a polypeptide of interest.
• this open reading phase is located downstream of the xrRNA sequence (s).
• the mRNA may comprise several open reading phases.
• the mRNA can be monocistronic, bicistronic or polycistronic.
• the mRNA is monocistronic when it comprises only one open reading phase. It is bicistronic when it comprises two open reading phases and polycistronic when it comprises at least two open reading phases.
• the mRNA of the invention may also comprise one or more non-coding regions. These non-coding regions may especially be regions between two open reading phases. In this case, IRES sequences are advantageously in these non-coding regions located between two open reading phases.
• the messenger ribonucleic acid (mRNA) molecule devoid of a capping molecule or a capping analogue comprises from 5 'to 3':
• a 5'-UTR region comprising at least one copy of a GUCAGRYC (N 7-19 ) GCCA (N 12 -19) UGCNRYCUG (xrRNA) consensus sequence, the one or more copies of said sequence being followed by a single copy of an internal ribosome entry RNA sequence (IRES);
• a 3'-UTR region comprising a poly (A) sequence.
• said mRNA molecule also comprises at least one aptamer RNA promoting the penetration of said molecule into target cells, preferably muscle cells.
• said mRNA molecule comprises at least one aptamer selected from aptamer A having the sequence of SEQ ID NO: 64, aptamer B having the sequence of SEQ ID NO: 65, and aptamer C having the sequence SEQ ID NO: 66.
• the messenger ribonucleic acid (mRNA) molecule devoid of a capping or cap-like molecule consists of from 5 'to 3': a 5'-region.
• UTR comprising at least one copy of a sequence consensus GUCAGRYC (7-19 N) GCCA (N 12 -19) UGCNRYCUG (xrRNA), or the said copies of said sequence being followed by a single copy of an RNA sequence internal ribosome entry (IRES);
• a 3'-UTR region comprising a poly (A) sequence.
• 5'-UTR region any nucleic acid region which is upstream of the translation initiation codon. This region is not coding but may include elements regulating mRNA expression downstream. In addition to the elements xrRNA and IRES, this region may include other elements such as "riboswitch" and / or "T-box". In some embodiments, the 5'-UTR region may be from 10 to 2000 nucleotides in length. Preferably it is between 50 and 1500 nucleotides, and more preferably from 200 to 1000 nucleotides.
• 3'-UTR region is meant any nucleic acid region which is downstream of the termination codon of the translation. This region can influence the expression and / or stability of the mRNA, its location in a cell, or contain binding sites for proteins or small interfering RNAs or microRNAs.
• This region may comprise, for example, elements such as a polyadenylated tail (poly (A)), a stem-loop structure of histone, and / or a region rich in pyrimidines or purines. It may contain coding or non-coding sequences.
• the 3'-UTR region may be from 50 to 500 nucleotides in length.
• the mRNA comprises a polyadenylated tail (poly (A)), comprising a nucleotide sequence of adenine or an analog or variant thereof, of 10 to 300 nucleotides, and preferably between 50 and 100 nucleotides.
• poly (A) polyadenylated tail
• the subject of the invention is a deoxyribonucleic acid (DNA) molecule comprising a polynucleotide which can be transcribed into the mRNA molecule of the invention.
• the DNA molecule comprises the 5'-UTR region of SEQ ID NO: 50, 71, 72, 73, 85, or 86.
• said DNA molecule is included in an expression cassette.
• "Expression cassette” here means a DNA fragment comprising a polynucleotide of interest, for example a polynucleotide that can be transcribed in the mRNA molecule of the invention, operably linked to one or more elements. regulators controlling the expression of gene sequences, such as, for example, promoter sequences and enhancer sequences.
• a polynucleotide is "operably linked" to regulatory elements when these different nucleic acid sequences are associated on a single nucleic acid fragment such that the function of one is affected by the others.
• a regulatory DNA sequence is "operably linked" to a DNA sequence encoding an RNA or a protein if both sequences are located such that the regulatory DNA sequence affects expression of the DNA coding sequence (that is, that the DNA coding sequence is under the transcriptional control of the promoter).
• the coding sequences may be operably linked to regulatory sequences in both sense orientation and antisense orientation.
• the coding sequences of the invention are operably linked to the regulatory sequences in the sense orientation.
• regulatory sequences or “regulatory elements” is meant herein polynucleotide sequences which are necessary to affect the expression and processing of the coding sequences to which they are ligated.
• regulatory sequences include transcription initiation and termination sequences, promoter sequences and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNAs; sequences improving translation efficiency (eg, Kozak sequences); sequences that increase the stability of proteins; and, if necessary, sequences that increase the secretion of proteins.
• the regulatory sequences of the invention comprise promoter sequences, that is to say that the gene coding for the mRNA of the invention is preferably operably linked to a promoter that allows the expression of said mRNA. corresponding.
• a gene encoding the mRNA of the invention is preferably operably linked to a promoter when it is located downstream of the promoter, i.e. 3 'thereof, thereby forming a cassette. 'expression.
• promoter is intended to mean a nucleotide sequence, most often located upstream (5 ') of the coding sequence, which is recognized by the RNA polymerase and the other factors necessary for transcription, and thus controls the expression of said coding sequence.
• a “promoter” as it is understood here comprises in particular the minimal promoters, that is to say short DNA sequences composed of a TATA box and other sequences which make it possible to specify the site of starting the transcription.
• a “promoter” within the meaning of the invention also comprises nucleotide sequences including a minimal promoter and regulatory elements capable of controlling the expression of a coding sequence.
• the promoter sequences of the invention may contain regulatory sequences such as "enhancer" sequences that can influence the level of expression of a gene.
• the promoters according to the invention are those which work with an RNA polymerase used in an acellular transcription system.
• promoters recognized by phage RNA polymerases SP6 and T7 are widely known to those skilled in the art.
• the pMBx-luc2 vectors which carry promoters recognized by the T7 RNA polymerase were used in the experimental part below. Vectors containing such promoters are also available commercially.
• the invention comprises a DNA molecule encoding the mRNA of the invention which is operably linked to at least one regulatory element.
• the DNA molecule encoding the mRNA of the invention is linked to operatively to a promoter sequence, located upstream, thereby forming an expression cassette.
• the invention consists of a DNA molecule encoding the mRNA of the invention which is operably linked to a promoter sequence, located upstream, thereby forming an expression cassette.
• the DNA molecule of the invention comprises:
• a promoter recognized by the T7 RNA polymerase comprising a sequence represented by SEQ ID NO: 46;
• a 5'-UTR region comprising a sequence represented by SEQ ID NO: 50, 71, 72, 73, 85, or 86;
• a 3'-UTR region comprising a sequence chosen from the sequences represented by SEQ ID NOs: 53, 54, 55, and 56.
• the regulatory sequences of the invention comprise transcription terminator sequences, i.e., the gene encoding the mRNA of the invention is preferably operably linked to a transcription terminator.
• transcription terminator here designates a sequence of the genome that marks the end of the transcription of a gene or operon, into messenger RNA.
• the mechanism of transcription termination is different in prokaryotes and eukaryotes. Those skilled in the art know the signals to be used according to the different cell types.
• Rho-independent terminator inverted repeat sequence followed by a series of T (uracils on the transcribed RNA) or a terminator Rho- dependent (consisting of a consensus sequence recognized by the Rho protein)
• a gene encoding the mRNA of the invention is preferably operably linked to a terminator when it is located upstream thereof, that is, say 5 'of it, thus forming an expression cassette.
• the terminators according to the invention are those that work with an RNA polymerase used in an acellular transcription system.
• terminators recognized by the phage RNA polymerases SP6 and T7 are widely known to those skilled in the art.
• Vectors containing such terminators are commercially available.
• the invention comprises a DNA molecule encoding the mRNA of the invention which is operably linked to at least one regulatory element and at least one transcription terminator.
• the invention also provides a vector comprising at least the DNA or mRNA molecule according to the invention.
• vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
• plasmid refers to a double circular loop of single-stranded DNA in which additional DNA segments can be ligated.
• viral vector in which additional DNA segments may be ligated into the viral genome.
• the viral vector may comprise mRNA in a viral genome (such as, for example, retroviruses or RNA viruses).
• Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• Other vectors (such as integrative mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated with the host genome.
• nucleic acid molecule of interest in which a nucleic acid molecule of interest can be inserted in order to introduce and maintain it in a eukaryotic or prokaryotic host cell are known to those skilled in the art.
• the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of this sequence in extrachromosomal form or integration into the chromosomal material of the host), as well as the nature of the host cell (for example, plasmids are preferably introduced into bacterial cells, while YACs are preferably used in yeasts).
• the vector used to code the mRNA of the invention is a vector that can be propagated in the bacterium. More preferably, this plasmid comprises a promoter recognized by an RNA polymerase used in an acellular transcription system, such as those of phages SP6 and T7. Even more preferred, this promoter carried by this plasmid is capable of directing the expression of the mRNA of the invention in the presence of at least said RNA polymerase.
• the vector of the invention comprises an origin of replication to allow the multiplication of said vector in the host cell.
• origin of replication also called ori
• ori is a unique DNA sequence that allows the initiation of replication. It is from this sequence that unidirectional or bidirectional replication begins.
• the person skilled in the art knows that the structure of the origin of replication varies from one species to another; it is therefore specific although they all have certain characteristics. A protein complex is formed at the level of this sequence and allows the opening of the DNA and the start of replication.
• the vector containing the expression cassette of the invention further comprises a selection marker, in order to facilitate the identification of the cells containing said vector, in particular after transformation.
• a "selection marker” according to the invention is a polynucleotide sequence carried by a vector, which allows the identification and selection of cells possessing said vector. Selection markers are well known to those skilled in the art. Preferably, it is a gene encoding a protein conferring resistance to an antibiotic.
• the vectors of the invention comprising the nucleic acid (s) of interest of the invention, are prepared by methods commonly used by those skilled in the art.
• the resulting clones can be introduced into a suitable host by standard methods known to those skilled in the art to introduce polynucleotides into a host cell.
• Such methods can be dextran transformation, calcium phosphate precipitation, polybrene transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, Biolistic injection and direct microinjection of DNA into the nucleus.
• the vector of the invention comprises a DNA molecule encoding the mRNA of the invention.
• the vector of the invention is a plasmid.
• the vector of the invention comprises a an expression cassette, a transcription terminator, an origin of replication, and a selection marker.
• the vector consists of an expression cassette.
• the vector comprises an mRNA molecule of the invention.
• the vector of the invention is a virus or a synthetic RNA vector.
• the subject of the invention is a host cell comprising said vector.
• host cell as used herein is intended to refer to a cell in which a recombinant expression vector has been introduced to express the mRNA of the invention. This term should be understood as encompassing not only said particular host cell but also the offspring thereof. It is understood that some changes may occur over generations because of changes or environmental influences. The offspring may, therefore, not be exactly the same as the parent cell, but is nevertheless also included in the term "host cell” as used herein.
• the DNA and / or vectors described above are particularly useful for producing large amounts of the mRNA of the invention.
• the subject of the invention is a method for producing the mRNA of the invention using the DNA or vector described above.
• the mRNA can thus be produced by any method known to those skilled in the art. This can be done, for example, by chemical synthesis, in vivo expression or in vitro expression.
• the mRNA is expressed in vitro using an acellular mRNA expression system.
• acellular mRNA expression system is meant in the sense of the invention a biochemical system for the synthesis of the mRNA of the invention in the absence of a cell.
• the acellular system has as its basic principle the use of the transcriptional machinery of an organism to produce a specific mRNA from an exogenous genetic information.
• the acellular system for expressing mRNA within the meaning of the invention therefore contains all the elements necessary for the production of mRNA in the absence of a cell.
• the organisms from which this machinery is extracted are many and varied, and come from prokaryotic and eukaryotic organisms.
• this system includes, among other things, the transcriptional machinery coming from the cell. More particularly, this system comprises an RNA polymerase capable of recognizing the promoter of the expression cassette described above. In the presence of the appropriate nucleotides and under the appropriate ionic conditions, this RNA polymerase is thus capable of directing the transcription of the gene coding for the mRNA of the invention.
• RNA polymerase capable of recognizing the promoter of the expression cassette described above. In the presence of the appropriate nucleotides and under the appropriate ionic conditions, this RNA polymerase is thus capable of directing the transcription of the gene coding for the mRNA of the invention.
• Such systems are well known to those skilled in the art for several decades (for a review, see for example: Beckert and Masquida, 201 1). Many methods are available to transcribe DNA into RNA in acellular systems.
• the acellular in vitro transcription system comprises a reaction buffer.
• said system comprises each of the four ribonucleotide triphosphates.
• these four ribonucleotide triphosphates are present at identical concentrations.
• the concentration of GTP is identical to that of the other three ribonucleotide triphosphates.
• the in vitro transcription yield of the mRNA of the invention is thus much higher than that of a mRNA with a cap resulting from an in vitro reaction, which greatly reduces the cost of the synthesis of the mRNA. mRNA.
• the production method comprises a step of purifying said mRNA.
• plasmid DNA comprising a promoter recognized by a phage RNA polymerase and followed by a DNA sequence coding for the mRNA of interest, is brought into contact with a phage RNA polymerase in a cell-free system. in vitro transcription.
• the mRNA synthesized by said method can then be purified.
• said plasmid DNA is linearized before being contacted with the RNA polymerase in the acellular system of transcription in vitro. More preferably, said DNA is linearized by enzymatic digestion downstream of the 3'-UTR region. Even more preferably, said DNA is linearized by enzymatic digestion with SspI or Eco53kI.
• the RNA polymerase is bacteriophage T7 RNA polymerase.
• the mRNA molecule is capable of directing the production of a polypeptide of interest in a eukaryotic organism into which it is introduced. Because of this, it is particularly suitable for gene therapy or gene vaccination.
• the mRNA molecule of the present invention can thus be used as a drug or as a vaccine.
• the invention also relates to a pharmaceutical composition or a vaccine composition.
• the present invention therefore relates to a pharmaceutical or vaccine composition
• a pharmaceutical or vaccine composition comprising the mRNA of the invention.
• the mRNAs included in the composition transfect the cells, which can then translate them into proteins.
• these proteins have a prophylactic activity or a therapeutic activity.
• the inventors have surprisingly demonstrated that the mRNA according to the invention is more efficient than a conventional mRNA with a cap during tissue transfection. Indeed, the inventors have demonstrated very surprisingly that the levels and duration of expression obtained after in vivo transfection are at least as high as those of a mRNA with a cap. In particular, the expression of the mRNA of the invention in vivo, in the dermis or muscle of a mouse, is greater than that of a control capped mRNA (FIG. 5, FIG. 15). In addition, the inventors have very surprisingly demonstrated that the mRNA according to the invention persists in long-term cells (i.e., more than 7 weeks).
• the inventors have surprisingly demonstrated that the co-transfection of the cells with a first mRNA according to the invention coding for the 2Apro protein and a second mRNA according to the invention coding for a second protein makes it possible to increase the translation of this second protein. .
• the pharmaceutical composition comprises the 2Apro protein or an mRNA encoding said protein.
• the 2Apro protein has the sequence of SEQ ID NO: 81.
• the mRNA encoding said protein has the sequence of SEQ ID NO: 80.
• the pharmaceutical composition comprises at least two different molecules of messenger ribonucleic acid (mRNA) lacking a capping molecule comprising from 5 'to 3':
• a 5'-UTR region comprising at least one copy of a consensus sequence GUCAGRYC (N 7-19 ) GCCA (N 12 -19) UGCNRYCUG (xrRNA);
• IRES internal ribosome entry RNA sequence
• an open reading phase according to which at least one of the mRNA molecules comprises an open reading phase encoding the 2Apro protein.
• said pharmaceutical composition comprises a first mRNA molecule, said first mRNA molecule comprising a reading phrase encoding the 2Apro protein, and at least one second mRNA molecule, said second molecule mRNA comprising an open reading phase encoding a second protein of interest. More preferably, said second protein of interest is not 2Apro protein. Even more preferably, said second molecule of interest is an antigen or a therapeutic protein as defined above. According to a particular embodiment of the invention, the molar ratio between the first mRNA and the second mRNA is between 540/1 and 240/1, preferentially between 540/1 and 315/1, even more preferably 465/1. .
• composition will be supplemented with an excipient and / or a pharmaceutically acceptable vehicle.
• pharmaceutically acceptable carrier is intended to denote a compound or combination of compounds used in a pharmaceutical composition that does not cause side reactions and that makes it possible, for example, to facilitate the administration of the active compound or compounds. increase its life and / or effectiveness in the body, increase its solubility in solution or improve its conservation.
• pharmaceutically acceptable vehicles are well known and will be adapted by those skilled in the art depending on the nature and mode of administration of the selected active compound (s).
• the term "pharmaceutically acceptable excipient” is intended to mean a compound or a combination of compounds used in a pharmaceutical composition which does not cause side reactions and which makes it possible, for example, to facilitate the administration of the active compound or compounds, increasing its lifespan and / or its effectiveness in the body, increasing its solubility in solution or even improving its conservation.
• Said excipient may in particular be added to the composition just before administration, for example if the RNA is preserved in the form of a lyophilizate.
• the pharmaceutically acceptable excipients / vehicles are well known and will be adapted by those skilled in the art depending on the nature and mode of administration of the selected active compound (s).
• the pharmaceutically acceptable composition comprises sterile water and / or chloroquine as an excipient.
• the "chloroquine" excipient also includes any variant or analog thereof, such as primaquine.
• the pharmaceutical composition further comprises chloroquine.
• the pharmaceutical composition comprises a weight ratio of between 1 / 0.5 to 1/4 mRNA / chloroquine, and more particularly a weight ratio of 1/1 mRNA / chloroquine.
• the pharmaceutical composition further comprises 5 ⁇ g RNA / 2.5 to 20 ⁇ g chloroquine.
• the pharmaceutical composition further comprises at least one CPP as described herein, non-covalently linked to the mRNA molecule according to the invention.
• the CPP is selected to promote the penetration of the mRNA into the target cells (e.g. according to the type of organ or cell line, for example muscle cells, or dermal cells).
• these compositions will be administered intramuscularly, intradermally, intraperitoneally or subcutaneously, by the respiratory route, or topically.
• These compositions are preferably intended to be injected into mammalian tissues, even more preferably in humans.
• These compositions are preferably intended to be injected intramuscularly, intravenously, intradermally, intraperitoneally or subcutaneously, according to methods known to those skilled in the art.
• the pharmaceutical composition of the invention may be administered repeatedly, over time. Its mode of administration, its dosage and its optimal dosage form can be determined according to the criteria generally taken into account in the establishment of a treatment adapted to a patient such as the age or the body weight of the patient, the gravity general condition, tolerance to treatment and side effects noted.
• Parenteral dosage forms include aqueous suspensions, isotonic saline solutions or sterile and injectable solutions which may contain pharmacologically compatible dispersing agents and / or wetting agents.
• the forms administered by the respiratory route include aerosols.
• Topically administrable forms include patches, gels, creams, ointments, lotions, sprays, eye drops.
• compositions suitable for administration will comprise a sufficient amount of mRNA molecules to be therapeutically effective.
• the effective dose of a compound of the invention varies according to many parameters such as, for example, the chosen route of administration, weight, age, sex, the progress of the pathology to treat and the sensitivity of the individual to treat.
• the subject of the invention is said composition for its use in gene therapy.
• the composition of the present invention can be used in the treatment or modulation of a variety of diseases, for example: cancer, genetic diseases (such as hemophilia, thalassemia, adenosine deaminase deficiency). , alpha-1 antitrypsin deficiency), diabetes, brain disorders such as Alzheimer's and Parkinson's disease, allergies, autoimmune diseases, and cardiovascular disease.
• diseases for example: cancer, genetic diseases (such as hemophilia, thalassemia, adenosine deaminase deficiency). , alpha-1 antitrypsin deficiency), diabetes, brain disorders such as Alzheimer's and Parkinson's disease, allergies, autoimmune diseases, and cardiovascular disease.
• the subject of the invention is said composition for its use in genetic vaccination.
• the composition of the present invention can be used in vaccination against cancer and influenza, as well as other viral and bacterial pathogens. Vaccination may involve humans as well as pets and livestock.
• the inventors have in particular shown that the mRNA of the present invention is stable in vivo. Indeed, the mRNA of the invention induces the synthesis of an amount of protein in vivo at least as high as that of a capped mRNA, indicating that its resistance to Xrn1 is at least as effective as that of mRNA. cap. In addition, the inventors have in particular shown that the mRNA of the present invention persists in long-term cells. Such an mRNA molecule is also very advantageous because its production cost is much lower than that of capped mRNA molecules.
• mRNA comprising an aptamer RNA penetrating directly into the cells (eg aptamer C RNA) or via a CPP peptide (eg aptamer A RNA), transfected the cells more efficiently, which advantageously allows , to improve the production of one or more proteins of interest.
• Figure 1 Diagram of messenger RNAs.
• the capped MB5-luc2 mRNA corresponds to a conventional luciferase-encoding messenger RNA, having a capping analogue at its 5 'end and a 3'-UTR region comprising a poly (A) tail.
• B) MB5-luc2 mRNA is identical to (A) except that it lacks a cap analog.
• C MB7-luc2 mRNA lacks a cap and has at its 5 'end a stem-loop and internal ribosome entry sequence (IRES) of the EMCV virus.
• MB8-luc2 mRNA lacks a cap and has both a loop-stem, two Xrn1-resistant sequences (xrRNA1 and xrRNA2) from West Nile Virus (WNV), and viruses TIRES. EMCV in the 5'UTR region.
• E MB9-luc2 mRNA is similar to (D) except that the 3'UTR and poly (A) thereof were replaced by Kunjin virus 3'UTR (KUN).
• Uncapped MB11-luc2 mRNA is similar to (D) except that aptamer A was added 5 'upstream of both Xrn1-resistant sequences (xrRNA1 and xrRNA2) from West Nile virus (WNV) and TIRES of the EMCV virus and that it does not have a stem-loop.
• MB13-luc2 (G) and MB14-luc2 (H) mRNAs have this same configuration, but include a 5 'loop stem followed by aptamer B and C RNAs, respectively.
• the capped MB15-luc2 mRNA is similar to (A) except that aptamer A was added 5 'after capping.
• (J) MB17-luc2 mRNA is similar to (D) except that aptamer A was added 5 'between the loop stem and xrRNA sequences.
• (K) MB18-luc2 mRNA is similar to (J) except that aptamer A is placed in the 5 'region downstream of the xrRNA sequences and upstream of the IRES sequence.
• Figure 2 Four-day luciferase expression kinetics in Caco-2 cells. Confluent Caco-2 cells were transfected with the capped MB5-luc2 (rhombic) and cap-free (circle) mRNAs, as well as the MB7-luc2 (triangle) and MB8-Iuc2 (square) mRNAs. The kinetics of expression was followed for four days.
• Figure 3 Ten-day luciferase expression kinetics in Caco-2 cells.
• Mesenchymal stem cells were transfected with 2 ⁇ g MB8-Iuc2 mRNA complexed to the pepMBI peptide at a positive charge ratio of the pepMBI peptide / mRNA negative charge of approximately 2.2 / 1 per well in platelets. 48 wells for 1 hour. Luciferase activity was then monitored for about 48 days.
• FIG. 1 Transfection of the muscle and dermis of mice.
• C2C12 cells were transfected with MB8-luc2 mRNA alone or MB8-Iuc2 mRNA mixed with MB8-2Apro mRNA at a ratio of 465/1 or 9/1, to evaluate the cytotoxic effect of the expression of the protein 2Apro. Cytotoxicity was determined by measuring the lactate dehydrogenase (LDH) released in the extracellular medium. The LDH activity is determined by measuring the absorbance at 490 nm. Negative Control: Cells untransfected or transfected with MB8-luc2 mRNA alone without lysis. Positive control: Untransfected cells lysed with Triton X-100.
• Figure 7 Effect of 2Apro protein on luciferase expression as a function of the MB8-luc2 mRNA / MB8-2Apro mRNA molar ratio.
• C2C12 cells were transfected with MB8-luc2 mRNA in the presence of an increasing amount of MB8-2Apro mRNA.
• the molar ratio MB8-luc2 mRNA / MB8-2Apro mRNA is 540/1 to 240/1.
• the total amount of transfected mRNA was 750 ng per well. Luciferase activity was measured 18 hours after transfection.
• Figure 8 Kinetics of luciferase expression over seven days in the presence or absence of MB8-2Apro mRNA.
• Confluent C2C12 cells were transfected with MB8-luc2 mRNAs alone (black line) or in combination with MB8-2Apro mRNA (dashed gray line).
• the molar ratio MB8-luc2 mRNA / MB8-2Apro mRNA is 465/1.
• the kinetics of expression, measured by the level of luciferase activity, was monitored for seven days.
• Figure 9 Effect of the 2Apro protein on the luciferase expression of different messenger RNAs.
• the effect of 2Apro protein on luciferase expression from different messenger RNAs was evaluated.
• the mRNA encoding luciferase was transfected alone (-) or co-transfected with a second mRNA encoding the 2Apro (+) protein and having the same characteristics at the optimal molar ratio of 465 / 1.
• Figure 10 Effect of different aptamers on the efficiency of transfection in the muscle of a mRNA molecule without a cap.
• the muscle of male BALB / cByJ mice was transfected with: 5 ⁇ g MB8-luc2 mRNA, MB13-luc2 mRNA, MB14-luc2 mRNA, MB11-luc2 mRNA complexed with M12-H6 peptide, or of MB11-luc2 mRNA complexed with M12-H6 peptide in the presence of 5 ⁇ g of chloroquine. Luciferase activity was measured 16 hours after the injections.
• Figure 11 Efficacy of transfection in the dermis of the mRNA according to the molar ratio of the CPP3-H6 peptide / MB11-luc1 mRNA.
• the dermis of male OF1 mice was transfected with 5.6 ⁇ g of MB11-luc2 mRNA complexed to the CPP3-H6 peptide at increasing molar ratios (molar ratios between 1/8 and 1, 125/1 of CPP3-H6 / MB11-luc2 mRNA). Luciferase activity was measured 18 hours after the injections. The molar ratio for optimal transfection was 1 CPP3-H6 / 4 MB11-luc2 mRNA.
• Figure 12 Efficacy of transfection in the dermis of the mRNA according to the molar ratio of the CPP1-H6 peptide / MB11-luc1 mRNA.
• the dermis of male OF1 mice was transfected with 5.6 ⁇ g of MB11-luc2 mRNA complexed to CPP1-H6 peptide at increasing molar ratios (molar ratios between 1/8 and 3/1 of CPP1-H6 / mRNA MB11-luc2). Luciferase activity was measured 18 hours after the injections. The molar ratio allowing optimal transfection was 1 CPP1-H6 / 4 mRNA MB1 1-luc2.
• Figure 13 Efficacy of transfection in the dermis of the mRNA according to the molar ratio of the CPP2-H6 peptide / MB11-luc1 mRNA.
• the dermis of male OF1 mice was transfected with 5.6 ⁇ g of MB11-luc2 mRNA complexed to the CPP2-H6 peptide at increasing molar ratios (molar ratios between 1/4 and 2.75 / 1 of CPP2-H6 / MB11-luc2 mRNA). Luciferase activity was measured 18 hours after the injections. The molar ratio allowing optimal transfection was 2 CPP2-H6 / 1 MB11-luc2 mRNA.
• Figure 14 Effect of aptamer A on the efficiency of transfection in the dermis of a capped mRNA molecule.
• a aptamer A RNA was inserted into the 5'UTR of the capped MB5-luc2 mRNA to generate the capped MB15-luc2 mRNA.
• the dermis of male OF1 mice was transfected with 5.6 ⁇ g of the capped MB15-luc2 mRNA complexed or not complexed with the CPP2-H6 peptide at the molar ratio 2 CPP2-H6 / 1 MB15-luc2 mRNA, in view of the results obtained previously. (see Fig. 12). Transfection was not improved with this construct, in the presence or absence of aptamer A as well as peptide CPP2-H6.
• Figure 15 Effect of 5'-SL on the efficiency of transfection in the dermis of an mRNA molecule.
• the effect of a stem-loop (here "5'-SL” having the sequence according to SEQ ID NO: 87) on the transfection efficiency when the stem-loop is followed by the sequence xrRNAI, 5 nucleotides downstream (MB8-luc2 mRNA and MB18-luc2), compared to stem-loop-free mRNA (MB11-luc2 mRNA) and mRNA whose stem-loop is more than 70 nucleotides upstream of xrRNAI (MB17-mRNA). luc2).
• DNA sequences corresponding to 5 'non-coding sequences of SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51 were chemically synthesized, integrated into a DNA vector and sequenced by ProteoGenix. The DNA fragments were excised by restriction enzymes. Plasmids of the pMBx-luc2 series were digested with the same restriction enzymes and the DNA fragments were integrated into these plasmids by the action of T4 DNA ligase. These plasmids contain the gene coding for the enzyme luciferase (SEQ ID NO: 62), inserted downstream of the bacteriophage T7 promoter.
• SEQ ID NO: 62 the gene coding for the enzyme luciferase
• the different non-coding 5'-UTR sequences separate the promoter from the luciferase gene. Plasmids thus constructed were amplified, verified and linearized downstream of the transcriptional polyadenylation sequence by a restriction enzyme.
• Sspl restriction site is located immediately downstream of the poly (A) of each plasmid (SEQ ID NO: 54).
• Ten micrograms of plasmid were digested with twenty units of the Sspl-HF restriction enzyme (New England Biolabs) in 1X CutSmart buffer for four hours at 37 ° C.
• MB5-luc2 mRNA is a classical mRNA encoding luciferase. It is endowed with a 5'-non-coding sequence (UTR) according to SEQ ID NO: 57, which has been selected for an optimal initiation of the translation as well as a 3'UTR region comprising a poly (A) tail according to SEQ ID No. NO: 60. This mRNA was synthesized with or without a cap (see Figure 1 (A) and (B)).
• the mRNA MB7-luc2 without cap has at its 5 'end, instead of 5'-UTR MB5-luc2 mRNA, a stem-loop at the 5' end, followed by the IRES sequence of EMCV virus. The latter would allow him to effectively recruit ribosomes, despite the absence of cap. On the other hand, this mRNA is sensitive to the enzyme Xrn1 (see Figure 1 (C)).
• the 5'-UTR region of MB7-luc2 mRNA corresponds to SEQ ID NO: 58 and the 3'-UTR region to SEQ ID NO: 60.
• the MB8-luc2 mRNA without a cap has a stem-loop at its 5 'end, followed, in the 5'UTR region, by two successive sequences resistant to Xrn1, resulting from the 3'-UTR Flavivirus WNV, called xrRNAI and xrRNA2 (Kieft et al., 2015). These sequences are followed by that of TIRES of the EMCV virus, which is therefore protected by the two Xrn1-resistant sequences (see Figure 1 (D)).
• the 5'-UTR region of the MB8-luc2 mRNA corresponds to SEQ ID NO: 59 and the 3'-UTR region to SEQ ID NO: 60.
• the cost of producing the MB8-luc2 mRNA without a cap is approximately 30 times less than the cost of producing a capped mRNA.
• Uncapped MB9-luc2 mRNA differs from the captive MB8-luc2 mRNA only at its 3 'end. Indeed, the 3'UTR and the poly (A) sequence of MB8-luc2 were replaced by the 3'UTR region of the Kunjin virus. This region does not have a poly (A) sequence (see Figure 1 (E)).
• the 3'-UTR region of the MB9-luc2 mRNA corresponds to SEQ ID NO: 61.
• the unlabeled MB11-luc2 mRNA differs from the cap-less MB8-luc2 mRNA only by the absence of a 5'-end-loop stem, and the presence of an aptamer in the 5 'upstream region.
• two successive sequences resistant to Xrn1 see Figure 1 (F)).
• MB11-luc2 mRNA comprises aptamer A of SEQ ID NO: 64; the 5'-UTR region of the MB11-luc2 mRNA thus corresponds to SEQ ID NO: 67.
• the MB13-luc2 mRNA, and unlabeled MB14-luc2 mRNA differs from the cap-free MB8-luc2 mRNA only by the presence of an aptamer in the 5 'region upstream of the two successive Xrn1-resistant sequences (see FIG. G, H)).
• MB13-luc2 mRNA comprises aptamer B of SEQ ID NO: 65; the 5'-UTR region of the MB13-luc2 mRNA corresponds therefore at SEQ ID NO: 68.
• MB14-luc2 mRNA comprises aptamer C of SEQ ID NO: 66; the 5'-UTR region of the MB14-luc2 mRNA therefore corresponds to SEQ ID NO: 69.
• MB15-luc2 mRNA corresponds to the capped MB5-luc2 mRNA, in which aptamer A (SEQ ID NO: 64) was inserted into the 5'-UTR region (see Figure 1 (I)); the 5'-UTR region of the MB15-luc2 mRNA therefore corresponds to SEQ ID NO: 70.
• the MB17-luc2 mRNA differs from the cap-less MB11-luc2 mRNA only by the presence of a stem-loop at the 5 'end upstream of the aptamer A (see Figure 1 (J)).
• MB17-luc2 mRNA comprises aptamer A of SEQ ID NO: 64; the 5'-UTR region of the MB17-luc2 mRNA thus corresponds to SEQ ID NO: 83.
• the uncapped MB18-luc2 mRNA differs from the unlabeled MB8-luc2 mRNA only by the presence of an aptamer. region 5 'between the two successive sequences resistant to Xrn1 and the sequence IRES (see Figure 1 (K)).
• MB18-luc2 mRNA comprises aptamer A of SEQ ID NO: 64; the 5'-UTR region of MB18-luc2 mRNA therefore corresponds to SEQ ID NO: 84.
• the purification of the different luciferase mRNAs was carried out using the MegaClear kit (Ambion). Seventy-nine ⁇ l of Elution Solution, 350 ⁇ l of Binding Solution Concentrate and 250 ⁇ l of 100% ethanol were added to the 21 ⁇ l of the previous mixture. These 700 mI were deposited on a Filter Cartridge and centrifuged at 10000 g for one minute. The filter retained the messenger RNA. Two washes were performed with 500 ml Wash Solution, centrifuging at 10,000 g for one minute. The RNA was then eluted from the filter by twice adding 50 ml of Elution Solution and heating at 70 ° C for ten minutes in a dry bath.
• Peptide peptide pepMBI was synthesized, purified and lyophilized by ProteoGenix. Its amino acid sequence is: CRRRRRRRRC. The lyophilizate was resuspended in sterile demineralized water. Five micrograms of luciferase mRNA was mixed with five micrograms of pepMBI at a final RNA concentration of 20 ⁇ g / ml. The mixtures were incubated at room temperature (20-25 ° C) for 15 minutes before being frozen at -80 ° C. The mRNA / pepMBI complexes were then lyophilized for about 20 hours.
• the Caco-2 cell line (ECACC) was cultured in DMEM (Gibco) supplemented with non-essential amino acids, a mixture of antibiotics and antimycotic, and fetal calf serum (15% final). The culture was carried out at 37 ° C in flasks of 75 cm 2 (Corning).
• C2C12 cells can be stored at confluence in the wells of a 48-well plate for a dozen days after seeding. Beyond this period, these cells differentiate into intestinal epithelium, which affects the translation of the mRNA. In contrast, human mesenchymal stem cells can be kept in confluent culture for more than 7 weeks.
• the mesenchymal stem cells (Millipore, Human Mesenchymal Stem Cell (Bone Marrow)) were therefore cultured in 48-well plates (Corning) in a ready-to-use medium (Millipore, Mesenchymal Stem Cell Expansion Medium) up to 48. days. For cells that will be lysed more than five days after transfection, the culture medium has been changed three times a week. b) Transfection of Caco-2 or C2C12 cells
• transfection by each mRNA was performed in five different wells. Lyophilisates of mRNA / pepMBI complexes (Proteogenix) were resuspended in 750 ⁇ l of transfection buffer (20 mM Hepes, 40 mM KCl and 100 mM trifluoroacetate).
• transfection with MB8 mRNA is carried out as indicated below.
• the MB8-luc2 / pepMBI mRNA complexes (Proteogenix) are assembled by incubating the mRNA in the presence of the peptides at a positive charge ratio of the peptide / mRNA negative charge of about 2.2 for 30 min at room temperature.
• the solution is then diluted with DMEM 3X to obtain final DMEM 1 X.
• RNA / pepMBI complex corresponding to 1 ⁇ g of mRNA per well for the Caco-2 cells and to 2 ⁇ g per well for C2C12 cells.
• Cells were incubated for 30 minutes (Caco-2 cells) or 1 hour (C2C12 cells) at 37 ° C in a CO 2 incubator.
• the mRNA / pepMBI complex solution was then aspirated and replaced with 250 ⁇ l of culture medium.
• the cells were then incubated for 6 hours to 48 days depending on cell type, at 37 ° C, in a CO 2 incubator.
• the cells were lysed to achieve expression kinetics of the luciferase protein.
• the culture medium was aspirated and replaced with 250 ⁇ l of lysis buffer (Luciferase Assay System, Promega). 20 ⁇ l of each cell lysate was introduced into a tube adapted to the luminometer (Berthold Technologies). 100 ⁇ l of luciferase substrate (Promega) was added to the cell lysate by the luminometer. The latter then measured the amount of light emitted by the enzymatic reaction catalyzed by luciferase. The results are expressed in relative light units (RLU).
• RLU relative light units
• Uncapped MB7-luc2 mRNA gives a stronger and more durable expression of the luciferase protein in Caco-2 cells than that of unlabeled MB5-luc2 mRNA.
• the IRES region recruits ribosomes, but does not provide significant resistance against Xrn1 (see Figure 2).
• Uncapped MB8-luc2 mRNA induces a luciferase expression kinetics similar to that obtained with capped MB5-luc2 mRNA (see Figures 2 and 3). This means that adding the two Xrn1-resistant sequences of the WNV virus provides the mRNA resistance to Xrn1 similar to that of the MB5-luc2 mRNA cap.
• MB7-luc2 mRNA which lacks a cap and Xrn1-resistant sequences, induces an intermediate luciferase expression between that of the uncapped MB8-luc2 mRNA and that of the uncapped MB5-luc2 mRNA (see FIG. 2).
• MB9-luc2 mRNA is differentiated from MB8-luc2 mRNA by its 3'UTR end lacking poly (A).
• the luciferase expression it induces in Caco-2 cells is significantly lower and less durable than that induced by MB8-luc2 mRNA (see Figure 3).
• the MB8-luc2 mRNA surprisingly and advantageously induces a luciferase expression that persists in the very long term. Indeed, even if the expression decreases over time, it is still detectable even 48 days after transfection (see Figure 4).
• mice For muscle, male BALB / cByJ mice, 8 weeks old, were housed in cages open to five animals per cage. The day / night cycles were managed by an automaton (12h of day / 12h of night). They were fed and had access to water filtered ad libitum. For the skin, male OF1 mice, 6 weeks old, were housed in cages open to four animals per cage. b) Extemporaneous preparation of the mRNA samples:
• mice Anesthesia of the mice was performed by the use of isoflurane.
• analgesia was performed by injection of buprenorphine.
• the back skin was mowed three to four days previously (see Example 10 for more details).
• each cell lysate 20 ⁇ l of each cell lysate was used for measurement of luciferase expression in each biopsy.
• a tube luminometer added 100 mI of luciferase substrate (Promega) in each sample and measured the amount of light emitted for 10 seconds. The results are expressed in relative units of light or RLU.
• the cell lysates were then diluted eight to twenty times for the protein assay using the 660 nm Protein Assay Kit (Pierce). One hundred microliters of diluted cell lysate was mixed with 1.5 ml of reagent for 6 minutes and the absorbance measured at 660 nm. A calibration range was performed with bovine serum albumin from 0 to 750 ⁇ g / ml.
• the results are illustrated in Figure 5.
• the cap-free MB8-luc2 mRNA induces luciferase expression, in skeletal muscle (A) and in skin (B), superior to the capped MB5-luc2 mRNA, 16 hours. (for the muscle) or 18 hours (for the dermis) after their injections.
• These results indicate that the presence of the two xrRNA sequences (here of the WNV virus) and the TIRES (here of EMCV) gives the mRNA greater resistance to Xrn1 and translation efficiency than those of the cap of the MB5 mRNA. lucu in vivo.
• the transfection of 10 ⁇ g of MB8-luc2 mRNA into the muscle tissue in the mouse results in a 2.6-fold higher luciferase expression than that obtained with the same dose of MB5-mRNA. luc2 with a cap (see Figure 5A).
• transfection of 5 ⁇ g of MB8-luc2 mRNA into the skin results in a luciferase expression 9.3 times higher than that obtained with the same dose of MB5-luc2 mRNA with a cap (see FIG. 5B).
• MB8-luc2 mRNA can therefore fully replace the capped MB5-luc2 mRNA, and would be even more advantageous.
• Example 7 Cellular Toxicity of MB8-2Apro mRNA Materials and Methods: Expression of 2Apro protein in a mammalian cell can induce toxicity to the point of causing apoptotic or necrotic cell death (Goldstaub et al. , 2000). During these processes, the cells release lactate dehydrogenase (LDH) into the extracellular medium. This LDH activity can be measured using a commercial kit, CytoTox96 Non-Radioactive Cytotoxicity Assay, Promega.
• LDH lactate dehydrogenase
• MB8-2Apro mRNA (SEQ ID NO: 80) is an mRNA encoding the non-structural protein 2A (2Apro, having the sequence of SEQ ID NO: 81) derived from the genome of human rhinovirus 2 (HRV2). It has a 5 'non-coding sequence (UTR) according to SEQ ID NO: 57, which is identical to that of the MB8-luc2 mRNA, as well as a 3'UTR region comprising a poly (A) tail according to SEQ ID NO: 60.
• UTR 5 'non-coding sequence
• co-transfection of MB8-luc2 and MB8-2Apro mRNAs increases luciferase expression of MB8-luc2 mRNA by at least 2.5-fold in C2C12 cells at all ratios tested.
• the best MB8-luc2 / MB8-2Apro mRNA mole ratio is 465/1, and the luciferase expression is increased 3.4-fold.
• Example 9 Effect of aptamer RNA on the efficiency of mRNA transfection in muscle
• Aptamers penetrating C2C12 cells were selected.
• double-stranded DNA was generated by hybridization of a 5 'primer followed by single-stranded DNA extension of a single-stranded DNA library.
• the double-stranded DNA thus obtained was then precipitated and purified according to methods well known to those skilled in the art.
• RNA library was then obtained by transcription of the purified fragments using the DuraScribe T7 transcription kit (20 mI / run) followed by purification of the RNA using a ssDNA and RNA purification kit. Finally, the solution is treated with DNAse I in order to eliminate the contaminating DNA. Aptamers are dissolved in 1X DMEM + ITS at 8 mM RNA (1288 mg / 5 ml). In order to select the aptamers, 5 ml of the DMEM / ITS / RNA solution is added to the cells previously washed twice with DMEM without antibiotics or serum. The cells are incubated at 37 ° C for 1 hour, briefly shaking the flask containing the mixture every 15 minutes.
• the flask containing the cells is then placed on ice and the cells are washed 5 times with 15 ml of cold 1x PBS in order to remove the aptamer RNAs that have not penetrated into the cells.
• the cells are then lysed with TRIzol (Invitrogen) and the total RNAs extracted by the "phenol-chloroform" method.
• TRIzol Invitrogen
• the Endogenous RNAs are digested with RNAse A, and the remaining RNAs hybridized with 3 'primers and retrotranscribed by the enzyme Superscript III (ThermoFisher), prior to amplification by PCR in the presence of 5' primer (100 mM), 3 'primer.
• RNAs B and C Two aptamer RNAs (B and C) entering C2C12 cells were selected and sequenced (SEQ ID NO: 65 and 66, respectively). Aptamer RNAs B and C were then inserted into the 5'UTR of MB8-luc2 mRNA, upstream of xrRNAI, to generate MB13-luc2 and MB14-luc2 mRNAs, respectively.
• a second strategy aimed at improving the internalization of mRNA consisted in inserting in the 5'UTR MB8-luc2 mRNA, upstream of xrRNAI, another aptamer RNA (aptamer A) capable of strongly binding the mRNA.
• aptamer A another aptamer RNA
• MRNA with aptamer A RNA was named MB11-luc2.
• Peptide penetrating the mouse muscle fibers, M12 was linked via a spacer (here including the glycine and serine amino acids) to the hexahistidine motif.
• SEQ ID NO: 75 RRQPPRSISSHPGGGGSGGGGSGGGGSGGGGSGGHHHHHH.
• SEQ ID NO: 76 PQRDTVGGRTT PPSWGPAKAGGGGSGGGGSGGGGHHHHHH.
• SEQ ID NO: 77 GPFHFYQFLFPPVGGGGSGGGGSGGG GSGGGGSGHHHHHH
• SEQ ID NO: 78 GSPWGLQHHPPRTGGGGSGGGGSGGGGSGGGGSGHH HHHH; spacer sequence underlined.
• the peptide thus formed was named M12-H6 (SEQ ID NO: 75).
• the MB11-luc2 mRNA was incubated for 30 minutes at room temperature with the M12-H6 peptide, before being injected into the mouse biceps femoris.
• the biceps femoris muscle of male BALB / cByJ mice was transfected with 5 ⁇ g MB8-luc2 mRNA, MB13-luc2 mRNA, MB14-luc2 mRNA, or MB11-luc2 mRNA complexed with M12-H6 peptide. in the presence or absence of 5 ⁇ g of chloroquine. Luciferase activity was measured 16 hours after the injections. Results:
• MB13-luc2 mRNA transfected muscle as well as MB8-luc2 mRNA ( Figure 10).
• the MB14-luc2 mRNA comprising aptamer C transfected the biceps femoris 2.1 times more efficiently than the MB8-luc2 mRNA.
• aptamer C RNA improved internalization in the muscle fibers of the mRNA molecule into which it was inserted.
• a 60 ⁇ l solution containing 20 ⁇ g of messenger RNA was prepared for each mouse. For this, demineralised water, 50 mM Hepes (1/8 Hepes, 7/8 sodium Hepes), NaCl (160 mM final), MgCl 2 , mRNA and optionally peptide were mixed. A 30 minute incubation at room temperature allowed the peptide to bind to the RNA. There was no incubation when the mRNA was not complexed to a peptide. The mRNA solutions were frozen at -80 ° C, to keep them until they were injected. b-Intradermal injection and biopsy samples
• mice Male OF1 mice 6 weeks old were used (Charles River). They were shaved three to four days before the intradermal injection. Anesthesia was performed using a mask. Induction of anesthesia was achieved by the use of 4% isoflurane (Piramal Heathcare). Maintenance of anesthesia was performed at a 2% isoflurane percentage. Prior to injection, the previously mowed back skin was cleaned with an alcohol soaked wipe. The mRNA solutions were slowly thawed at room temperature to fill the 0.3 mm x 8 mm U-100 (30G) insulin syringes (Becton-Dickinson). Three injections of approximately 17 ⁇ l of RNA solution were made into the skin of the mowed back of each mouse.
• Papules formed before resorbing. These were delineated with an indelible marker, to allow the identification of the skin area to be biopsied the next day. Tagging was also done with a marker to differentiate animals from the same cage. 18 hours after the injections, skin biopsies were performed at the injected areas. For this, the mice were anesthetized and then euthanized by cervical dislocation. The biopsies were cut into small pieces using a pair of scissors, to facilitate lysis of the cells, and introduced into tubes containing 500 ⁇ l 1X lysis buffer (Luciferase Cell Culture Lysis 5X (Promega), diluted in water). Each tube was frozen at -20 ° C until the next step. c-Lysis of cells, luciferase assays and proteins
• the lysis of the collected tissues was obtained by carrying out three cycles of freezing / thawing: -80 ° C for 10 minutes, 3 minutes in a water bath at room temperature and mixing with a vortex-stirrer for a few seconds .
• the tubes were then centrifuged at 5000 g for 5 minutes at 20 ° C to sediment tissue debris. The supernatant was transferred to another tube.
• the luciferase assay was performed using the Luciferase Assay System Kit (Promega). 20 ml of each sample were introduced into tubes dedicated to the luminometer (Berthold AutoLumat Plus LB 953). The apparatus injected 100 mI of substrate and measured the amount of light emitted (RLU).
• the protein assay was performed using the Pierce 660nm Protein Assay Kit (Thermo Scientific). A calibration range was previously performed with Bovine Albumin Serum (Thermo Scientific) and lysis buffer 1X as diluent. It has covered a range of 100 to 500 ⁇ g of protein per milliliter. White was obtained in using 100 mI 1X lysis buffer. The lysates were in some cases diluted with 1X lysis buffer. 100 ml of each sample were used for the protein assay. 1.5 ml of reagent was added to the blank and samples. After precisely 5 minutes of incubation at room temperature and in the dark, the absorbance at 660 nm of each sample was measured using a spectrophotometer.
• the strategy to improve the internalization of naked mRNA by binding of a cell-penetrating peptide (CPP) to aptamer A RNA is not restricted to muscle, as described in Example 9 herein. -above. This strategy has been applied here to the skin of mice using different CPP.
• CPP cell-penetrating peptide
• CPP1-H6, CPP2-H6 and CPP3-H6 Three CPPs were used here: CPP1, CPP2 and CPP3 (see Kamada et al., 2007 and Lee et al., 2012). They were connected to hexahistidine by a spacer consisting of glycines and serines to form the peptides CPP1-H6, CPP2-H6 and CPP3-H6 having the sequences of SEQ ID NO: 76, 77, and 78, respectively.
• MB11-luc2 mRNA was incubated with increasing amounts of CPP3-H6 peptide, CPP1-H6, or CPP2-H6 for 30 minutes in pre-optimized buffer comprising 160 mM NaCl, 0.7 mM MgCl 2 and 5 mM Hepes.
• pre-optimized buffer comprising 160 mM NaCl, 0.7 mM MgCl 2 and 5 mM Hepes.
• An intradermal injection of 5.6 ⁇ g MB8-luc2 mRNA or MB11-luc2 was carried out in the OF1 mouse according to the protocol detailed in Example 10.
• the mouse skin was also injected with a mixture of mRNA.
• MB11-luc2 and MB8-luc2 mRNA at a ratio of 1: 1, and the same amount of CPP3-H6 as the molar ratio of 0.5 to determine if the CPP3-H6 peptide can dissociate from aptamer RNA A, present in MB11-luc2 mRNA after intradermal injection.
• Luciferase activity was measured 18 hours after the injections.
• MB11-luc2 mRNA was also incubated with the CPP1-H6 peptide at different molar ratios. The best molar ratio was 1 CPP1-H6 peptide for 4 MB1 1-luc2 mRNA ( Figure 12). The transfection efficiency increased 5.4-fold relative to MB11-luc2 mRNA alone.
• CPP1-H6 gave a worse result than CPP3-H6. This can be explained by the number of copies of the receptor (s) of each CPP present in the plasma membrane of the skin cells and the affinity of each CPP for its receptor (s) .
• MB1 1-luc2 mRNA was also incubated with CPP2-H6 at various molar ratios.
• the best molar ratio was 2 CPP2-H6 peptides for 1 MB11-luc2 mRNA ( Figure 13).
• Transfection efficiency increased 10.6 fold over MB11-Iuc2 mRNA alone, and is also greater than that obtained with CPP3-H6 peptide.
• the expression luciferase is thus 22.8 times higher than that obtained with the conventional MB5-luc2 mRNA.
• aptamer A was inserted into the 5'UTR of the conventional MB5-luc2 mRNA capped to generate capped MB15-luc2 mRNA having the sequence of SEQ ID NO: 70.
• mRNA MB15-luc2 was incubated with CPP2-H6 for 30 minutes in buffer comprising 0.7 mM MgCl 2 and 5 mM Hepes, as described above in Example 11.
• the luciferase expression obtained with the capped MB15-Iuc2 mRNA is 1.68-fold lower than that obtained with the capped MB5-luc2 mRNA.
• the transfection efficiency remains much lower than that observed for the MB8-luc2 and MB11-luc2 mRNAs. It is therefore very advantageous to use the mRNA molecules of the invention rather than capped mRNA molecules.
• mRNAs MB8-luc2, MB11-luc2, MB17-luc2, MB18-luc2, and capped MB5-luc2 were injected into the skin and the luciferase activity measured 18 hours after injection, according to the protocol detailed in Example 10 above.
• the mRNA according to the invention comprising at least one xrRNA sequence and an IRES sequence (MB11-luc2) increases the luciferase expression in the skin by approximately 2-fold with respect to the capped mRNA ( MB 5-luc2).
• a stem-loop here the 5'-SL having the sequence of SEQ ID NO: 87
• the stem-loop when the stem-loop is located about 70 nucleotides upstream of the xrRNA sequence, the stem-loop does not improve the efficiency beyond what is already observed in the absence of the stem-loop. Indeed, the efficiency of the translation is similar for MB11-luc2 and MB17-luc2 mRNAs.
• the placement of aptamer A between the xrRNA sequences and the IRES sequence (MB18-luc2) increases the efficiency of the translation even more than the MB11-luc2 and MB17-luc2 mRNAs very surprisingly.
• the MB8-luc2 and MB18-luc2 mRNAs thus have a similar translation efficiency.
• An mRNA molecule according to the invention has a reduced synthesis cost of approximately 30 times, relative to a capped mRNA molecule, while having an increased yield of protein expression of at least 2 times, preferably about 10 times.
• the mRNA of the invention is at least as stable as capped mRNA, and its production by in vitro transcription is simplified by the absence of a capping molecule or an analogue thereof.
• An mRNA of the invention encoding HRV2 protease 2A makes it possible to increase the translation of an mRNA coding for a protein of interest when these two molecules are co-transfected.
• the efficiency of transfection into a tissue can be improved, for example, by insertion into the 5'-UTR region of the mRNA molecule according to the invention of an RNA aptamer penetrating directly into cells, a CPP (attached to an aptamer RNA as described above), and / or by adding a rod-loop at the 5 'end of region 5 -UTR upstream of the xrRNA sequence (s).

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• 2018
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