Two RNA sequences introduced at the 5' end of the messenger RNA replace the messenger RNA cap.

Abstract

This invention relates to messenger ribonucleic acid (mRNA) molecules lacking a cap molecule, which significantly reduce the cost of in vitro transcriptional synthesis, and which contain from the 5' to 3' ends: at least one copy of GUCAGRYC(N) resistant to Xrn1 exonuclease. 7‑19 )GCCA(N 2‑19 The UGCNRYCUG sequence includes a single copy of the internal ribosome entry site (IRES) RNA sequence and an open reading phase (une phase).

Description

Technical Field

[0001] This invention relates to the field of ribonucleic acid (RNA), and more specifically to the in vitro synthesis of messenger RNA (ARNm), its stability, and its translation into polypeptides, particularly in transfected cells. Background Technology

[0002] ARNm is an important molecule for the industrial-scale production of peptides. Its stability, as well as transcription and translation efficiency, strongly influences the downstream yield of peptides and the resulting cost.

[0003] ARNm is also a molecule of choice in pharmaceutical compositions used in gene therapy and genetic inoculation. In fact, unlike ADN molecules, ARNm molecules have never been shown to integrate into the genome of transfected cells. However, due to their sensitivity to ribonuclease degradation, they exhibit poor stability in solution. Therefore, the synthesis of stable ARN molecules both in vitro and in vivo is crucial for reducing the amount of ARNm required for optimal therapeutic efficacy and thus lowering costs. Furthermore, using smaller, more stable ARNm amounts reduces the risk of treatment-related side effects.

[0004] In vivo synthesis occurs in cultured cells (such as yeast or bacteria). However, this method has many drawbacks. In particular, purifying intact target ARNs is expensive and complex due to degradation issues and the presence of other cellular ARNs. It typically yields lower yields compared to those obtained through in vitro transcription.

[0005] As a result, the commonly used method for large-scale synthesis of ARNm is a cell-free in vitro transcription system. This method uses only a few purified compounds (i.e., reaction buffer, ADN molecules carrying the gene, recombinant ARN polymerase, and four ribonucleotide triphosphates), and the only ARN to be produced is the desired ARNm. At the end of the reaction, there are no ARNm degradation products because there is no ARNase in the reaction mixture. There are also no other ARN species, which is a major advantage over synthesizing ARNm using cultured cells. Therefore, the purification of ARNm is greatly simplified.

[0006] To ensure the stability of ARNm in eukaryotic cells, it possesses a cap molecule at its 5′ end, protecting it from exonucleases. The cap is a complex molecule composed of two guanosines, with their 5′ carbons linked by a chain of three phosphate groups. The terminal guanosine is methylated at the 7-position of the guanine. ARNm is stabilized by its resistance to the progressive enzymatic degradation performed by Xrn1 exonuclease from 5′ to 3′. In addition to its role in stabilizing ARNm, the cap also has other functions, including ribosome recruitment (Cowling, 2010). For this purpose, the cap initiates the translation initiation process by recruiting translation initiation factors, which in turn recruit ribosomes. The ribosomes then translate the ARNm into a protein.

[0007] On an industrial scale, to improve the stability of ARNm and achieve efficient translation of ARNm into peptides in transfected cells, a capping molecule should be incorporated at the 5′ end. In in vitro transcription, a capping molecule can be added in a post-transcriptional step using a capping enzyme (e.g., 2′-O-methyltransferase from vaccinia virus) (Martin et al., 1975). However, this additional step increases the complexity of synthesis, requires purification of the capping enzyme, and is not very efficient (Contreas et al., 1982). Furthermore, this enzyme requires S-adenosyl-L-methionine, which is unstable in aqueous solution.

[0008] To simplify in vitro transcription, cap analogs have been developed in existing technologies, but they are not satisfactory. For example, P... 1 -(5′-7-methyl-guanosyl)P 3 -(5′-(guanosine))triphosphate) analogues or P 1 -(5′-2,2,7-trimethyl-guanosine)P 3 -(5′-(guanosine)triphosphate) analogs. These analogs allow capping via phage-mediated co-transcriptionalization of ARNs, thus avoiding the additional steps of cap synthesis and improving the stability of ARNm. However, depending on the type of analog, up to 50% of the incorporating molecules have the opposite orientation, where the 7-methylguanosine nucleotide is adjacent to the ARN molecule rather than at the terminal position, thus reducing the stability and efficiency of ARNm translation (Pasquinelli et al., 1995). “Reverse-reverse” cap analogs (ARCA) have been developed, such as those described in US 7074596. These analogs prevent reverse incorporation of molecules. However, they remain unsatisfactory because their use causes a significant reduction in the yield of in vitro ARNm synthesis. Furthermore, the synthesis of cap molecules and their many chemically modified analogs is expensive due to the complexity of these compounds. Including one of these molecules in the mixture of in vitro transcription reactions on an industrial scale would significantly increase the cost of ARNm synthesis.

[0009] In practice, to ensure high capping efficiency during in vitro transcription, it is necessary to provide an excessive amount of cap analogues, resulting in high raw material costs. It is recommended to use a ratio of 4 cap analogue molecules per GTP molecule to maximize the chance of each ARNm molecule being capped. Nevertheless, it is estimated that only 80% of synthesized ARNm will be capped. Furthermore, the GTP concentration is reduced by 5-fold compared to the other three ribonucleoside triphosphates, resulting in a 5-fold reduction in transcription yield.

[0010] One approach to reducing the high cost of in vitro transcription is to reduce the production cost of the ADN and / or ARN polymerases used in the method. However, the cost reductions achieved remain relatively small. Alternatively, it is possible to synthesize circular, capless ARNm via in vitro transcription in a cell-free system (WO 2014 / 186334 A1). However, the authors demonstrated that circular ARNm exhibits lower translation efficiency in transfected cells compared to capped linear ARNm (see, for example, Figure 2 and Figure 3 And paragraphs

[00121] and

[00131] of WO 2014 / 186334A1.

[0011] As a result, there is still a need for ARNm molecules with high stability and high translation efficiency, preferably ARNm molecules with at least the same stability and translation efficiency as capped ARNm molecules. There is also a need for an ARN molecule with a simple production method and significantly reduced manufacturing costs. Summary of the Invention

[0012] This invention relates to a stable, cap-free ARNm molecule that can be efficiently translated. This ARNm is particularly advantageous because its production cost is significantly lower than that of conventional ARNm containing a capped molecule or its analogue. The invention also relates to an ARNm molecule that exhibits increased in vitro transcription yield compared to conventional ARNm containing a capped molecule or its analogue. Indeed, the ARNm of this invention is also advantageous because, despite its significantly reduced production cost and / or increased synthetic yield, it is at least as effective as conventional capped ARNm when transfected into cultured cells and tissues. In fact, the inventors have surprisingly demonstrated that the expression levels and duration obtained after in vivo transfection are at least as high as those of capped ARNm. Specifically, the expression kinetics of the reporter protein in Caco-2 cells are identical regardless of whether transfection is with the ARNm of this invention or a control capped ARNm. Figure 2 and Figure 3 Similarly, the inventors were quite surprised to demonstrate that when the ARNm of the present invention was transfected in vivo in the dermis or muscle of mice, the expression of the reporter protein was 2 to approximately 10 times higher than that of the control ARNm transfected with capped ARNm. Figure 5 and Figure 15 However, since the additional capping step is unnecessary, the reaction is simplified. Furthermore, the presence of cap-like molecules in the reaction mixture is also unnecessary during in vitro transcription. Therefore, compared to prior art ARNm, the ARNm molecules of this invention offer significant advantages in terms of cost reduction and production acceleration for at least the same level of expression.

[0013] For the purposes of this application, the term "ARNm molecule" refers to any linear chain of ribonucleotides. In this application, these sequences are expressed in a 5′ to 3′ orientation, starting with a 5′-UTR region.

[0014] "Ribosonucleotide" refers to any natural ribonucleotide (e.g., guanine, cytidine, uridine, adenosine), as well as analogs of these nucleotides and nucleotides with chemically or biologically modified bases (e.g., by methylation, alkylation, acylation, thiolation, etc.), inserted bases, modified ribose groups, and / or modified phosphate groups.

[0015] The inventors have unexpectedly demonstrated that the 5′ cap of the ARNm molecule can be replaced by at least one copy of a sequence resistant to Xrn1 exonuclease (xrRNA), said resistant sequence being derived from the 3′-UTR region of a Flavivirosis virus, preferably with an internal ribosome entry site (IRES) within the open reading frame. Significantly, compared to capped ARNm molecules, the production cost of such ARNm molecules via in vitro transcription is reduced by approximately 30-fold, and the yield is increased. Furthermore, the ARNm molecules of this invention are particularly stable in transfected cells and can be efficiently translated even without the cap.

[0016] "Cap" refers to 7-methylguanosine (N7-methylguanosine or m7G) nucleotide and any mutants, variants, analogs or fragments thereof, which can be linked to the first nucleotide transcribed from ARNm via a 5′-5′ triphosphate bond. Cap analogs include, without limitation, unmethylated analogs (e.g., P...). 1 -(guanosine)P 3- (5′-(guanosine))triphosphate), monomethylated analogs (e.g., P) 1 -(5′-7-methyl-guanosine)P 3 -(5′-(guanosine)triphosphate), trimethylated analogs (e.g., P... 1 -(5′-2,2,7-trimethyl-guanosine)P 3 -(5′-(guanosine))triphosphate), or with m replaced by 3′-O-methyl 7 Analogs of the 3′-OH group of the guanine moiety (e.g., ARCA P) 1 -(5′-(3′-O-methyl)-7-methyl-guanosine)P 3 -(5′-(guanosine)triphosphate)).

[0017] Therefore, according to a first aspect, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising:

[0018] • 5′-UTR region, which contains at least one copy of GUCAGRYC(N7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0019] • At least one copy of the internal ribosome entry site (IRES) ARN sequence; and

[0020] • At least one open reading box.

[0021] Preferably, the IRES ARN sequence is located upstream of each open reading frame.

[0022] According to a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising, from 5′ to 3′:

[0023] • 5′-UTR region, which contains at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0024] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0025] • Open reading box.

[0026] Preferably, the ARNm molecule also contains a 3′-UTR region.

[0027] Preferably, the ARNm molecule further comprises at least one ARN aptamer, which facilitates the penetration of the ARNm molecule into cells, preferably muscle cells. The ARN aptamer can significantly promote penetration, directly or indirectly, through peptides.

[0028] Preferably, the ARNm molecule also includes a stem-loop at the 5′ end.

[0029] In a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising, from 5′ to 3′, the following:

[0030] • 5′-UTR region, containing at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0031] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0032] • Open reading box.

[0033] In existing technology, the 3′-UTR untranslated region of flaviviruses has been shown to block Xrn1, thereby protecting downstream sequences (Chapman et al., 2014). This region of the ARN consists of at least one sequence that spontaneously folds to form a complex three-dimensional structure that inhibits the Xrn1 process, thus suppressing the degradation of the viral subgenomic ARN. However, such sequences have never been inserted into the 5′ of the ARNm before. Indeed, there are concerns that these sequences may need to function within the context of the 3′-UTR of the flavivirus genome. In particular, these three-dimensional structures may not fold correctly when no longer surrounded by sequences from the flavivirus 3′-UTR region.

[0034] Although existing technologies indicate that xrRNA sequences inhibit translation, the inventors have unexpectedly demonstrated that the protective function of the cap and the translation initiation function can be successfully replaced by at least one copy of the xrRNA sequence and at least one copy of the IRES sequence (preferably from EMCV virus) without affecting translation efficiency. Surprisingly, the use of xrRNA and IRES sequences can even significantly improve translation efficiency.

[0035] The term "flavivirus" refers to any virus in the genus Flavivirus, including yellow fever, dengue fever, West Nile virus (WNV), Zika virus, Japanese encephalitis virus, Rocio virus, Murray Valley virus, Bagza virus, Kokobera virus, Ntaya virus, Kedougou virus, Sepik virus, St. Louis virus, Usutu virus, Alfuy virus, Wesselbron virus, Ilheus virus, Bussuquara virus, Tembusu virus, Chaoyang virus, Yokose virus, Donngang virus, and any other virus in the genus Flavivirus.

[0036] Therefore, the present invention relates to a stable ARNm molecule containing at least one copy of an xrRNA sequence in its 5′ region. The ARNm is efficiently translated into protein at a level and duration similar to that of a capped molecule. According to a preferred embodiment of the invention, the ARNm molecule contains two copies of xrRNA.

[0037] The term "anti-Xrn1 ARN sequence" or "xrRNA" refers to any polynucleotide sequence that may reduce, slow down, or prevent the degradation of ARNm by the Xrn1 exonuclease. Preferably, the xrRNA sequence contains a common sequence.

[0038] "Common sequence" refers to any sequence that contains at least the following sequence: 5′-GUCAGRYC(N 7-19 )GCCA(N 12-19 )UGCNRYCUG-3′, where each N can independently represent a nucleotide selected from A, C, T, G, and U or their analogues. Each R represents a purine, and each Y represents a pyrimidine. As shown, the number of N nucleotides between conserved bases can vary in the shared sequence, from 5′ to 3′, from 7 to 19 bases, and from 12 to 19 bases. The sequence forms a stem-loop three-dimensional structure.

[0039] Preferably, the ARNm molecule contains at least one copy of an xrRNA sequence selected from the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 44. When the ARNm molecule contains more than one copy of the sequence, those copies may be the same or different. Therefore, more preferably, the ARNm molecule contains at least one copy of one of the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 26. Even more preferably, the ARNm molecule contains one copy of the sequence shown in SEQ ID NO: 11 and one copy of the sequence shown in SEQ ID NO: 26.

[0040] Preferably, when there are two xrRNA sequences in the 5′-UTR region, they are separated by a spacer sequence. Similarly, a spacer sequence may exist between the xrRNA sequence and the IRES sequence. Preferably, the spacer sequence between the two xrRNA sequences corresponds to SEQ ID NO: 47. Preferably, the spacer sequence between the xrRNA sequence and the IRES sequence corresponds to SEQ ID NO: 48. When the ARNm molecule contains at least two open reading frames, the spacer sequence may also exist between the two open reading frames or between the open reading frame and the IRES sequence.

[0041] "Spacer subsequence" refers to any non-coding polynucleotide sequence that makes it possible to physically separate the upstream and downstream sequences of the spacer subsequence. The ARNm molecule according to the invention may significantly contain one or more spacer subsequences.

[0042] In some embodiments, the length of the spacer sequence can be 2 to 300 nucleotides. Preferably, it is between 2 and 10 nucleotides, even more preferably between 2 and 5 nucleotides. Alternatively, it can be between 10 and 150 nucleotides, even more preferably between 15 and 40 nucleotides. Preferably, the spacer sequence does not generate secondary structures.

[0043] The ARNm of the present invention also includes at least one internal ribosome entry site (IRES) ARN sequence.

[0044] "Internal ribosome entry site" or "IRES" refers to any polynucleotide sequence that allows the initiation of translation of an ARNm molecule without relying on the cap. Such a sequence interacts directly with the translation initiation factor, which then recruits ribosomes at the translation start codon. Many IRES sequences are known (Mokrejs et al., 2010). Therefore, those skilled in the art will be able to identify sequences that function as IRES' and select those suitable for carrying out the invention. Thus, the IRES sequence according to the invention can be a eukaryotic or viral sequence, for example, from the genus Picornavirus. Preferably, it is derived from encephalomyocarditis virus (EMCV) (Borman et al., 1995) or from human eIF4G ARNm. Even more preferably, the ADN sequence of the IRES corresponds to SEQ ID NO: 45.

[0045] IRES sequences are typically located in the 5′-UTR region. They can also be located between two open reading frames, which allows bicistronic or polycistronic translation to be initiated from a single ARNm. In a preferred embodiment, the ARNm of the present invention contains a single copy of the IRES sequence in the 5′ region of the ARNm. According to a preferred embodiment, a second copy of the IRES sequence is located between two open reading frames. According to yet another preferred embodiment, the ARNm of the present invention contains a copy of the IRES sequence in the 5′ region and a copy of the IRES sequence between the two open reading frames. Advantageously, the IRES sequence is located downstream of the xrRNA sequence. This arrangement advantageously makes it possible to protect these sequences from Xrn1 exonuclease. When the ARNm molecule contains at least two IRES sequences, the sequences can be the same or different. Specifically, one or more IRES sequences can be selected based on their efficiency in initiating translation. It is particularly advantageous to select two different IRES sequences when the ARNm molecule contains at least two different open reading frames and the translation efficiency required for the first open reading frame is different from that required for the second open reading frame.

[0046] The IRES element also possesses a complex three-dimensional structure. Therefore, by forming pairings between the ARN sequences in each of the two regions, mutual interference can occur between the xrRNA and IRES sequences. Such interference can potentially prevent the correct structural formation of both the xrRNA and IRES sequences, respectively. Furthermore, the xrRNA structure can significantly inhibit the recruitment of translation initiation factors and ribosomes via the IRES. Even more surprisingly, the inventors have demonstrated that the presence of both xrRNA and IRES sequences makes it possible to obtain protein expression yields in transfected cells that are at least similar to those obtained with capped ARNm molecules. Therefore, the xrRNA and IRES sequences together can replace a cap or cap-like molecule. They are essential for ensuring translation of the open reading frames contained in the ARNm of this invention and for the stability of the ARNm.

[0047] Therefore, in transfected cells, the ARNm of the present invention is at least as stable as the capped ARNm, and is also at least as efficiently translated. Furthermore, its cost for in vitro transcription synthesis is significantly lower than that of the capped ARNm.

[0048] According to a specific implementation, the ARNm of the present invention is based on the common sequence GUCAGRYC(N) 7-19 )GCCA(N 12-19 The 5′-UTR region upstream of UGCNRYCUG (xrRNA) further includes a stem-loop, preferably at the 5′ end of the ARNm molecule.

[0049] A "stem-loop" refers to any polynucleotide sequence forming a double helix structure, in which the 5' end of one strand is physically connected to the 3' end of the other strand via an unpaired loop. Therefore, a stem-loop consists of a double-stranded stem and an unpaired single-stranded loop. The physical bond can be covalent or non-covalent. Preferably, the physical bond is covalent. The size of the ARN loop can be, for example, between 3 and 30 nucleotides. The loop size is preferably at least 3 nucleotides, more preferably at least 4 nucleotides. The length of the double-stranded stem can be, for example, between 5 and 50 nucleotides. The stem length is preferably 5 to 50, 5 to 40, 5 to 30, 5 to 25, or more preferably 5 to 10 nucleotides. Even more preferably, the stem length is 6, 7, or 8 nucleotides.

[0050] In the context of this invention, the stem-loop is preferably formed at the 5′ end of the ARNm molecule. According to a preferred embodiment, the stem-loop has the sequence of SEQ ID NO: 87. The stem-loop is preferably separated from the xrRNA sequence by a spacer sequence. Preferably, the spacer sequence is equal to or less than 5 nucleotides in length (i.e., 5, 4, 3, or 2 nucleotides). Indeed, the inventors have surprisingly demonstrated that adding a stem-loop structure (also referred to herein as 5′-SL, since the stem-loop is at the 5′ end) to the 5′ end of the ARNm molecule, when close to the xrRNA sequence (e.g., 5 nucleotides or less), may further improve in vivo translation. In fact, the inventors did not observe any beneficial effect when the stem-loop at the 5′ end was separated from the xrRNA sequence by a spacer sequence of approximately 70 nucleotides in length (see [link to invention]). Figure 15 ).

[0051] Unbound by theory and surprisingly, at least when the sequence is in stem-loop form and very close together, it can be assumed that the xrRNA sequence masks the 5′ end to protect it from phosphatases and Xrn1.

[0052] Therefore, in a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5′ to 3′:

[0053] • 5′-UTR region, containing at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0054] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0055] • Open reading box

[0056] The 5′ end also contains a stem-loop.

[0057] The ARNm molecule may further include a second IRES sequence, followed by a second open reading frame. The ARNm molecule may further include an ARN aptamer as defined herein, located between the stem-loop and the xrRNA sequence, or preferably, between the xrRNA sequence and the IRES sequence. The ARNm molecule may further include a 3′-UTR region as defined herein.

[0058] In a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5′ to 3′:

[0059] • A 5′-UTR region containing a stem-loop at the 5′ end, followed by at least one copy of GUCAGRYC(N) 7-19)GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0060] • Choose any location, ARN fitter;

[0061] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0062] • Open reading box.

[0063] In a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising, from 5′ to 3′, the following:

[0064] The 5′-UTR region contains a stem-loop at the 5′ end, followed by at least one copy of GUCAGRYC(N) 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0065] • Choose any location, ARN fitter;

[0066] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0067] • Open reading box; and

[0068] ·3′-UTR region.

[0069] "Aptamer" refers to any nucleic acid possessing recognition properties and specificity associated with the ability of a nucleic acid molecule to adopt a specific three-dimensional structure (particularly similar to a monoclonal antibody) (see, for example, Dunn et al., 2017). Aptamers may consist of ADNs, ARNs, and / or modified ARNs, preferably ARNs. The aptamer may consist of 6 to 50 nucleotides, such as ribonucleotides as defined above. As a non-limiting example, aptamers can be isolated according to various techniques known to those skilled in the art, for example by one or more in vitro selection cycles, or by an iterative selection method from a large library of compounds of random sequences ("SELEX" technique). Identifying aptamers in vitro by selection advantageously allows for the acquisition of aptamers with precise effects or functions without knowing the target against which the aptamer is targeted. The manufacture or selection of aptamers is described, for example, in European patent application EP0533838. Advantageously, according to the scope of the invention, ARN aptamers have been identified based on their ability to penetrate target cells (more specifically tissue cells, preferably muscle cells (e.g., myofibroblasts)). Advantageously, the aptamers according to the invention are capable of crossing cell membranes, more preferably the plasma membrane and / or endosomal membrane of mammalian cells.

[0070] The aptamers according to the invention preferably comprise 6 to 50 nucleotides, more preferably 10 to 45 nucleotides, 20 to 40 nucleotides, and even more preferably 30 to 40 nucleotides. Preferably, the aptamers are composed of ribonucleotides, such as those defined above. Preferably, the aptamers according to the invention share at least 70% identity with aptamer A having the sequence SEQ ID NO: 64, with aptamer B having the sequence SEQ ID NO: 65, or with aptamer C having the sequence SEQ ID NO: 66, more preferably at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and even more preferably at least 99% identity.

[0071] In the context of this invention, the percentage of identity referenced is determined based on the overall alignment of the sequences to be compared; that is, the sequences are aligned to their full length using any algorithm known to those skilled in the art (e.g., the Needleman and Wunsch algorithm, 1970). This sequence comparison can be performed using any software known to those skilled in the art, such as Needle software using a "Gap open" parameter of 10.0, a "Gap extend" parameter of 0.5, and a Blosum 62 matrix.

[0072] Preferably, the aptamer according to the invention is selected from aptamer A having the sequence SEQ ID NO: 64, aptamer B having the sequence SEQ ID NO: 65, and aptamer C having the sequence SEQ ID NO: 66, and even more preferably, from aptamer A having the sequence SEQ ID NO: 64 and aptamer C having the sequence SEQ ID NO: 66.

[0073] Preferably, the ARNm molecule according to the invention comprises at least one copy of a membrane-crossing aptamer, preferably a mammalian plasma membrane and / or endosome membrane. Therefore, advantageously, when the ARN molecule according to the invention comprises an aptamer, the aptamer facilitates its penetration into cells, preferably muscle or skin cells. Preferably, the ARNm molecule according to the invention comprises at least one copy of aptamer A having the sequence SEQ ID NO: 64, aptamer B having the sequence SEQ ID NO: 65, and / or aptamer C having the sequence SEQ ID NO: 66.

[0074] Aptamers that facilitate the entry of ARNm molecules into target cells do not necessarily need to be protected from exonuclease degradation, which primarily occurs in the cytoplasm of the cell. Therefore, depending on the specific implementation scheme, ARNm molecules containing aptamers are placed in one or more GUCAGRYC(N) nucleotides. 7-19 )GCCA(N12-19 The aptamer is located upstream of the 5′-UTR region of the GUCAGRYCUG (xrRNA) concordant sequence. Depending on the alternative implementation, the aptamer is placed in one or more GUCAGRYC (N) aptamers. 7-19 )GCCA(N 12-19 The UGCNRYCUG (xrRNA) sequence is downstream of the 5′-UTR region of the shared sequence, but upstream of the IRES sequence and the open reading frame. (See the diagram for representations of these two possibilities.) Figure 1 J and Figure 1 K). Therefore, according to a preferred embodiment, the present invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5′ to 3′:

[0075] • The 5′-UTR region contains at least one aptamer capable of crossing the cell membrane, preferably the plasma membrane and / or endosome membrane, preferably crossing the plasma membrane and / or endosome membrane of mammalian cells, preferably at least one aptamer selected from aptamers A, B and C described herein, and at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0076] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0077] • Open reading box.

[0078] Preferably, ARNm molecules are in GUCAGRYC(N 7-19 )GCCA(N 12-19 The upstream 5′-UTR region of the UGCNRYCUG (xrRNA) shared sequence contains the aptamer (see, for example, Figure 1 F). However, ARN molecules can also be found in GUCAGRYC(N). 7-19 )GCCA(N 12-19 The downstream of the UGCNRYCUG (xrRNA) common sequence contains an aptamer, for example, between the xrRNA common sequence and the IRES sequence.

[0079] Therefore, according to a preferred embodiment, the present invention relates to an ARNm molecule lacking a cap molecule, comprising from 5′ to 3′:

[0080] • A 5′-UTR region containing a stem-loop at the 5′ end, followed by at least one copy of GUCAGRYC(N) 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0081] • At least one aptamer capable of crossing the cell membrane;

[0082] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0083] • Open reading box.

[0084] While aptamers B and C were chosen for their ability to penetrate myofibroblasts, aptamer A corresponds to the “shot47” aptamer, identified by Tsuji et al. in 2013. Aptamer A advantageously binds to multihistidine-type peptide motifs with high affinity. Aptamer A can therefore bind to any molecule (e.g., peptide or protein) containing a multihistidine tag (e.g., an HHHHHH motif). As a non-limiting example, the ARNm molecule according to the invention can be linked via aptamer A to a molecule that allows it to better penetrate cells, such as a “cell-penetrating peptide” or “CPP”, which contains a multihistidine tag.

[0085] "Cell-penetrating peptide" or "CPP" refers to any peptide, polypeptide, or protein capable of crossing the cell membrane of mammalian cells, more preferably the plasma membrane and nuclear endosome membrane. Advantageously, when a CPP is attached to another molecule (particularly an ARNm molecule), the CPP retains this property, thereby allowing said other molecule to cross the membrane. In the context of this invention, any possible mechanisms for membrane crossing are considered, including, for example, energy-dependent transport mechanisms (i.e., active, e.g., endocytosis) and energy-independent transport mechanisms (e.g., diffusion). Typically, CPPs are cationic peptides (Poillot and De Waard, 2011). As a non-limiting example, ARNm molecules can non-covalently bind to CPPs due to their negative charge, utilizing electrostatic interactions and / or hydrophobicity. Alternatively, ARNm molecules can covalently bind to CPPs. CPPs can form oligomers consisting of at least two identical or different peptide molecules. In the context of this invention, CPPs are preferably non-covalently bound to ARN aptamers. In fact, given the simplicity of this bond, its low cost through simple mixing of CPP and ARNm molecules, and the fact that these molecules are fully biodegradable, this bond is advantageous. In fact, no non-natural and non-biodegradable chemical groups are required.

[0086] The length of the CPP according to the invention is preferably from about 8 amino acid residues to about 60 amino acid residues. More preferably, the length is from 8 to 40 amino acid residues, even more preferably from 8 to 30 amino acid residues, and even more preferably from 10 to 25 amino acid residues (e.g., 13 or 20 amino acid residues). However, those skilled in the art will recognize that the length of the CPP is not necessarily limited to those described above. For example, based on common knowledge in the art, CPP derivatives of different lengths described herein can be specifically created.

[0087] As a non-limiting example, the CPP of the present invention may be (as described by Gao X. et al., 2014) "M12" CPP, (as described by Kamada et al., 2007) "CPP2" or "CPP3" CPP, or (as described by Lee et al., 2012) CPP "CPP1", and any variants or derivatives thereof. According to a preferred embodiment, the CPP comprises a multi-histidine motif (e.g., hexahistidine), which is preferably linked to the CPP via a spacer. Preferably, the spacer is a hydrophilic spacer, advantageously unstructured and uncharged, and even more advantageously composed of glycine and serine. Preferably, the spacer is about 21 amino acids long, more preferably 21 amino acids.

[0088] Preferably, the CPP according to the invention has at least 70% identity with the following peptides, more preferably at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, and even more preferably at least 99% identity: M12-H6 peptide having the sequence SEQ ID NO: 75, CPP1-H6 peptide having the sequence SEQ ID NO: 76, CPP2-H6 peptide having the sequence SEQ ID NO: 77, and CPP3-H6 peptide having the sequence SEQ ID NO: 78. According to a particularly preferred embodiment, the CPP according to the invention is selected from: M12-H6 having the sequence SEQ ID NO: 75; CPP1-H6 having the sequence SEQ ID NO: 76; CPP2-H6 peptide having the sequence SEQ ID NO: 77; or CPP3-H6 having the sequence SEQ ID NO: 78; and even more preferably, selected from: CPP1-H6 having the sequence SEQ ID NO: 76; CPP2-H6 peptide having the sequence SEQ ID NO: 77; and CPP3-H6 having the sequence SEQ ID NO: 78. Advantageously, the CPP is non-covalently or covalently linked to the ARNm molecule, preferably non-covalently. Advantageously, the CPP is linked to an ARN aptamer contained in the ARNm molecule (i.e., aptamer A when the CPP contains a multihistidine tag).

[0089] Preferably, the CPPs of the present invention do not exhibit significant cytotoxic and / or immunogenic effects on their target cells after crossing the plasma membrane, i.e., they do not interfere with cell viability, cell transfection, and / or penetration. The term "non-significantly" as used herein means that after the ARNm molecule with the CPP attached crosses the plasma membrane and is internalized by the cell, less than 50%, preferably less than 40% or 30%, preferably less than 20% or 10%, and particularly less than 5% of the target cells are killed. Those skilled in the art are familiar with methods for determining the cytotoxicity of a given compound and / or the viability of target cells to which the compound is administered (see, for example, Ausubel et al., 2001). Corresponding analytical kits are commercially available from various suppliers. In specific embodiments, the potential inherent cytotoxic and / or immunogenic effects of the CPPs of the present invention can be "masked" by introducing one or more modifications into the peptide (e.g., by chemical synthesis or recombinant ADN). Such modifications may include, for example, the addition, removal, or substitution of functional groups or alteration of the position of these functional groups. Those skilled in the art are fully aware of how such "masking" can be accomplished for a given peptide.

[0090] Therefore, according to a preferred embodiment, the present invention relates to an ARNm molecule lacking a cap molecule, comprising from 5′ to 3′:

[0091] The 5′-UTR region contains at least the aptamer A ARN of SEQ ID NO:64 and at least one copy of GUCAGRYC(N) 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0092] • A copy of the internal ribosome entry site (IRES) ARN sequence, and

[0093] • Open reading box

[0094] The ARNm molecule is linked to a cell-penetrating peptide (CPP) fused with a multi-histidine tag, preferably selected from: M12-H6 having the sequence SEQ ID NO: 75; CPP1-H6 having the sequence SEQ ID NO: 76; CPP2-H6 having the sequence SEQ ID NO: 77; and CPP3-H6 having the sequence SEQ ID NO: 78. Preferably, the CPP is non-covalently linked to an aptamer.

[0095] An "open reading frame" refers to any polynucleotide sequence that can be translated into a target polypeptide. An open reading frame is a block of three consecutive nucleotides (called a codon), with each codon representing one amino acid. During translation, the polypeptide is synthesized by translating the codons of the open reading frame using ribosomes.

[0096] The first amino acid of a polypeptide is typically indicated on the ARNm molecule by the AUG codon, thus signifying the start of an open reading frame. Other known start codons include AUN or NUG, where N corresponds to A, C, U, or G. The terminator of a polypeptide is shown on the ARNm molecule as a stop codon, such as UAA, UGA, or UAG. The stop codon indicates the end of the open reading frame on the ARNm molecule.

[0097] The open reading frame according to the invention is more specifically an open reading frame that, when translated in transfected cells, produces a product for therapeutic or vaccine purposes in human or veterinary medicine. Preferably, the product for therapeutic purposes is a protein.

[0098] Among proteins targeted for therapeutic purposes, one more specific category might be enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 92 03120), growth factors, neurotransmitters or their precursors or synthases, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc., apolipoproteins: ApoAI, ApoAIV, ApoE, etc. (FR 9305125), dystrophin or small dystrophin (FR 91 11947), tumor suppressor proteins: p53, Rb, Rap1A, DCC, k-rev, etc. (FR 93 04745), factors involved in coagulation: factors VII, VIII, IX, etc., pro-apoptotic proteins: thymidine kinase, cytosine deaminase, etc., or even all or part of natural or artificial immunoglobulins (Fab, ScFv, etc., see, for example, WO). 2011 / 089527), ARN ligand (WO 91 / 19813), etc.

[0099] The target protein encoded by ARNm can also be an antigen capable of evoking an immune response in humans or animals to achieve vaccination. Specifically, these can be antigens specific to Epstein-Barr virus, HIV, hepatitis B virus (EP 185573), pseudorabies virus, or even tumors (EP 259 212). Finally, the target protein can be an adjuvant protein, which can stimulate an immune response to enhance vaccine efficacy.

[0100] The target protein encoded by the ARNm can be a protein that has a beneficial effect on uncapped ARNm molecules or proteins expressed by said molecules. This beneficial effect may arise from different mechanisms. As a non-limiting example, a protein encoded by an uncapped ARNm can increase the stability of the uncapped ARNm molecule by binding to said molecule or by degrading at least one protein with ARNase activity. As a non-limiting example, a protein encoded by an uncapped ARNm can increase the translation of said molecule by promoting the recruitment of initiation factors or by binding to capped cellular ARNm to inhibit its translation. As a non-limiting example, the target protein encoded by the ARNm is the 2Apro protein from a picornavirus, such as human rhinovirus type 2 (HRV2). Without being bound by theory, it can be considered that due to the protease activity of the 2Apro protein, cleaving the N-terminus of the eIF4G initiation factor, the initiation factor is prevented from recognizing the capped ARNm, and the 2Apro protein increases the expression of uncapped ARNm. This cleavage can reduce the competition between the ARNm of the present invention and capped ARNm in vivo. In fact, the inventors unexpectedly demonstrated that co-transfection of cells with a first ARNm encoding 2Apro according to the invention and a second ARNm encoding a reporter protein according to the invention resulted in increased reporter protein expression. Therefore, the presence of the 2Apro protein is particularly advantageous because it allows for a specific increase in the expression of the protein encoded by the capless ARNm of the invention. Figure 7 ).

[0101] According to a preferred embodiment, the ARNm of the present invention encodes the 2Apro protein, preferably a 2Apro protein derived from a piconese virus, and even more preferably from HRV2 virus. According to a specific embodiment, the 2Apro protein has the sequence SEQ ID NO: 81. According to a specific embodiment, the 2Apro protein is encoded by an ARNm having the sequence SEQ ID NO: 80.

[0102] The open reading frame can also encode therapeutic ARNm. This could be, for example, an antisense sequence whose expression in target cells allows for control over the transcription or translation of cellular ARNm. According to the technology described in patent EP 140 308, such a sequence could, for example, be transcribed in target cells into an ARN complementary to the intracellular ARNm, thereby preventing its translation into a protein.

[0103] Preferably, the ARNm of the present invention, in addition to containing the xrRNA sequence and the IRES sequence, also contains an open reading frame encoding the target polypeptide. Preferably, the open reading frame is located downstream of the xrRNA sequence. Those skilled in the art will readily understand that an ARNm can include several open reading frames. Therefore, an ARNm can be monocistronic, bicistronic, or polycistronic. When an ARNm has only one open reading frame, it is monocistronic. When it contains two open reading frames, it is bicistronic; when it contains at least two open reading frames, it is polycistronic.

[0104] The ARNm of the present invention may also include one or more non-coding regions. These non-coding regions may, in particular, be regions between two open reading frames. In this case, the IRES sequence advantageously exists in these non-coding regions located between the two open reading frames.

[0105] According to a specific implementation scheme, messenger RNA (ARNm) molecules lacking a cap or cap analogue molecule, from 5′ to 3′, contain:

[0106] • 5′UTR region, which contains at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The UGCNRYCUG (xrRNA) shared sequence is followed by a single copy of the internal ribosome entry site (IRES) ARN sequence.

[0107] • Open reading box; and

[0108] • 3′-UTR region containing poly(A) sequence.

[0109] Advantageously, the ARNm molecule further comprises at least one ARN aptamer that facilitates the penetration of the molecule into target cells, preferably muscle cells. Advantageously, the ARNm molecule comprises at least one aptamer selected from aptamer A having the sequence SEQ ID NO: 64, aptamer B having the sequence SEQ ID NO: 65, and aptamer C having the sequence SEQ ID NO: 66.

[0110] According to another specific implementation, the messenger ribonucleic acid (ARNm) molecule lacking a cap or cap analogue molecule, from 5′ to 3′, consists of the following:

[0111] • 5′-UTR region, which contains at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The UGCNRYCUG (xrRNA) shared sequence is followed by a single copy of the internal ribosome entry site (IRES) ARN sequence.

[0112] • Open reading box; and

[0113] • 3′-UTR region containing poly(A) sequence.

[0114] The “5′-UTR region” refers to any nucleic acid region located upstream of the translation start codon. This region is non-coding but may contain elements that regulate downstream ARNm expression. In addition to xrRNA and IRES elements, this region may also contain other elements such as riboswitch and / or T-box. In some embodiments, the 5′-UTR region can be 10 to 2000 nucleotides in length. Preferably, it is contained between 50 and 1500 nucleotides, more preferably between 200 and 1000 nucleotides.

[0115] The “3′-UTR region” refers to any nucleic acid region located downstream of the translation stop codon. This region may affect the expression and / or stability of the ARNm, its location in the cell, or contain a binding site for a protein or small interfering ARN or a tiny ARN. This region may include, for example, elements such as a poly(A) tail, a histone stem-loop structure, and / or a pyrimidine- or purine-rich region. It may contain coding or non-coding sequences. In some embodiments, the 3′-UTR region may be 50 to 500 nucleotides in length. Preferably, it is contained between 50 and 200 nucleotides, more preferably 50 to 100 nucleotides. In a preferred embodiment, the ARNm contains a poly(A) tail containing a nucleotide sequence of adenine or its analogues or variants thereof, which is 10 to 300 nucleotides, preferably 50 to 100 nucleotides.

[0116] According to a second aspect, the present invention relates to a deoxyribonucleic acid (ADN) molecule comprising a polynucleotide that can be transcribed into the ARNm molecule of the present invention. Preferably, the ADN molecule comprises a 5′-UTR region of SEQ ID NO: 50, 71, 72, 73, 85, or 86.

[0117] Preferably, the ADN molecule is contained in an expression cassette.

[0118] In this document, “expression cassette” refers to an ADN fragment containing a target polynucleotide, such as a polynucleotide that can be transcribed into the ARNm molecule of the present invention, which is operatively linked to one or more regulatory elements that control the expression of a gene sequence, such as, for example, promoter sequences and enhancer sequences.

[0119] Polynucleotides are "operably linked" to regulatory elements, meaning that when these different nucleic acid sequences are combined in this way on a single nucleic acid fragment, the function of one sequence is influenced by the others. For example, a regulatory ADN sequence is "operably linked" to an ADN sequence encoding an ARN or protein, such that if the two sequences are positioned in such a way that the regulatory ADN sequence influences the expression of the ADN-coding sequence (in other words, the ADN-coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in both the sense and antisense directions. Preferably, the coding sequence of the present invention is operably linked to the regulatory sequence in the sense direction.

[0120] In this paper, "regulatory sequences" or "regulatory elements" refer to polynucleotide sequences essential to the expression and maturation of the coding sequences they are linked to. Such regulatory sequences include, in particular, transcription initiation and termination sequences, promoter sequences and enhancer sequences; signals for efficient ARN maturation, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic ARNm; sequences that enhance translation efficiency (e.g., Kozak sequences); sequences that increase protein stability; and, where necessary, sequences that increase protein secretion.

[0121] Preferably, the regulatory sequence of the present invention includes a promoter sequence, i.e., the gene encoding the ARNm of the present invention is preferably operatively linked to a promoter that allows the expression of the corresponding ARNm. Preferably, when the gene encoding the ARNm of the present invention is located downstream of a promoter (i.e., promoter 3′), it is operatively linked to that promoter, thereby forming an expression cassette.

[0122] As used herein, the term "promoter" refers to a nucleotide sequence, most commonly located upstream (5′) of a coding sequence, that is recognized by ARN polymerase and other factors essential for transcription, thereby controlling the expression of said coding sequence. As used herein, "promoter" specifically includes minimal promoters, namely short ADN sequences consisting of a TATA box and other sequences that allow transcription start site specificity. Within the sense of this invention, "promoter" also includes nucleotide sequences comprising minimal promoters and regulatory elements capable of controlling the expression of the coding sequence. For example, the promoter sequences of this invention may contain regulatory sequences, such as enhancer sequences that can affect gene expression levels.

[0123] Advantageously, the promoters according to the invention are those that function in conjunction with ARN polymerases used in cell-free transcription systems. For example, promoters recognized by ARN polymerases of SP6 and T7 phages are well known to those skilled in the art. Therefore, the pMBx-luc2 vector (Rogé and Betton, 2005) containing a promoter recognized by T7 ARN polymerase was used in the experimental section below. Furthermore, vectors containing such promoters are commercially available.

[0124] In one specific embodiment, the present invention comprises an ADN molecule encoding the ARNm of the present invention, operably linked to at least one regulatory element. Preferably, the ADN molecule encoding the ARNm of the present invention is operably linked to an upstream promoter sequence to form an expression cassette. In another embodiment, the present invention comprises an ADN molecule encoding the ARNm of the present invention, operably linked to an upstream promoter sequence to form an expression cassette.

[0125] Even more preferably, the ADN molecule of the present invention comprises:

[0126] • A promoter recognized by T7 ARN polymerase, the promoter comprising the sequence shown in SEQ ID NO: 46;

[0127] • 5′-UTR region, which contains the sequence represented by SEQ ID NO: 50, 71, 72, 73, 85 or 86;

[0128] • Open reading box; and

[0129] • 3′-UTR region, which contains sequences selected from those shown in SEQ ID NO: 53, 54, 55 and 56.

[0130] Advantageously, the regulatory sequence of the present invention includes a transcription terminator sequence, that is, the gene encoding the ARNm of the present invention is preferably operatively linked to a transcription terminator. The term "transcription terminator" herein refers to a genomic sequence that marks the end of transcription of a gene or operon and becomes a messenger ARN. The mechanisms of transcription termination differ between prokaryotes and eukaryotes. Those skilled in the art know the signals used depending on the cell type. For example, if they wish to express the ARNm of the present invention in bacteria, they will use either a Rho-independent terminator (an inverted repeat sequence followed by a series of Ts (uracil in the transcribed ARN) or a Rho-dependent terminator (composed of a common sequence recognized by the Rho protein). When the gene encoding the ARNm of the present invention is located upstream of the terminator (i.e., at the 5′ end of the terminator), it is preferably operatively linked to the terminator to form an expression cassette.

[0131] Advantageously, the terminators according to the invention are those that function in conjunction with ARN polymerases used in cell-free transcription systems. For example, terminators recognized by ARN polymerases of SP6 and T7 phages are well known to those skilled in the art. Vectors containing such terminators are commercially available.

[0132] In one specific embodiment, the present invention includes an ADN molecule encoding the ARNm of the present invention, which is operatively linked to at least one regulatory element and at least one transcription terminator.

[0133] In a third aspect, the present invention also relates to a carrier comprising at least an ADN or ARNm molecule according to the present invention.

[0134] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid already linked to it. One type of vector is the "plasmid," which is a circular double loop of a single-stranded ADN to which other ADN fragments can be linked. Another type of vector is the viral vector, in which additional ADN fragments can be linked to the viral genome. Alternatively, viral vectors may contain ARNm (e.g., retroviruses or ARN viruses) within the viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., integrative mammalian vectors) can integrate into the host cell's genome upon introduction and thus replicate along with the host genome.

[0135] Those skilled in the art will know that, in order to introduce and maintain a target nucleic acid molecule in a variety of vectors, the target nucleic acid molecule can be inserted into a variety of vectors. The selection of a suitable vector depends on the intended use of the vector (e.g., replicating the target sequence, expressing the sequence, maintaining the sequence in an extrachromosomal form, or even integrating it into the host's chromosomal material) and the nature of the host cell (e.g., plasmids are preferably introduced into bacterial cells, while YACs are preferably used in yeast). These expression vectors can be plasmids, YACs, granules, retroviruses, episomes derived from EBV, and any vector that those skilled in the art deem suitable for expressing the sequence. In a preferred embodiment of the invention, the vector used to encode the ARNm of the invention is a vector capable of propagating in bacteria. More preferably, the plasmid contains a promoter recognized by an ARN polymerase used in cell-free transcription systems, such as the promoters of SP6 and T7 bacteriophages. Even more preferably, the promoter carried by the plasmid is capable of directing the expression of the ARNm of the invention in the presence of at least the aforementioned ARN polymerase.

[0136] Preferably, the vector of the present invention contains a replication origin to allow the vector to multiply in a host cell. The term "replication origin" (also called ori) is a unique ADN sequence that allows replication to begin. Unidirectional or bidirectional replication begins at this sequence. Those skilled in the art know that the structure of the replication origin differs from one species to another; therefore, although they all share certain characteristics, it is specific. A protein complex forms on this sequence, allowing the ADN to open and replication to begin.

[0137] Advantageously, the vector containing the expression cassette of the present invention also includes a selection marker to facilitate the identification of cells containing the vector, particularly after transformation. The "selection marker" according to the invention is a polynucleotide sequence carried by the vector, which allows for the identification and selection of cells possessing the vector. Selection markers are well known to those skilled in the art. Preferably, it is a gene encoding a protein confers antibiotic resistance.

[0138] The vector of the present invention containing the nucleic acid of the present invention is prepared using methods commonly used by those skilled in the art. To introduce the polynucleotide into a host cell, the resulting clone can be introduced into a suitable host using standard methods known to those skilled in the art. Such methods may include dextran conversion, calcium phosphate precipitation, polypropylene transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, biolistique injection, and direct microinjection of the ADN into the cell nucleus. It is also possible to combine the ADN or ARNm sequence (isolated or inserted into a plasmid or viral vector) with a substance that allows it to cross the host cell membrane (e.g., a transport protein (e.g., a nanotransporter) or a liposome formulation, or a cationic polymer). Furthermore, these methods can be advantageously combined, for example, by using liposome-associated electroporation.

[0139] According to a preferred embodiment of the present invention, the vector of the present invention comprises an ADN molecule encoding the ARNm of the present invention. Preferably, the vector of the present invention is a plasmid. Even more preferably, the vector of the present invention comprises an expression cassette, a transcription terminator, a replication origin, and a selection marker. In another preferred embodiment, the vector consists of an expression cassette.

[0140] According to another preferred embodiment of the invention, the vector comprises the ARNm molecule of the invention. Preferably, the vector of the invention is a virus or a synthetic ARN vector.

[0141] According to another aspect, the present invention relates to host cells comprising the vector. As used herein, the term "host cell" refers to a cell into which a recombinant expression vector for expressing the ARNm of the present invention has been introduced. This term should be understood to include not only the specific host cell but also its progeny. It should be understood that certain modifications may exist over multiple generations due to mutations or environmental influences. As a result, progeny cells may not be entirely identical to parent cells, but are still included in the term "host cell" as used herein.

[0142] The aforementioned ADN and / or carrier are particularly useful for generating large quantities of the ARNm of this invention.

[0143] According to another aspect, the present invention relates to a method for producing the ARNm of the present invention using the aforementioned ADN or carrier. Therefore, ARNm can be produced by any method known to those skilled in the art. This can be accomplished, for example, by chemical synthesis, by in vivo expression, or by in vitro expression.

[0144] Preferably, ARNm is expressed in vitro using a cell-free ARNm expression system. In the context of this invention, "cell-free ARNm expression system" refers to a biochemical system that allows the synthesis of the ARNm of this invention in the absence of cells. Cell-free systems are based on the production of specific ARNm from exogenous genetic information using the organism's transcriptional mechanisms. Therefore, in the context of this invention, a cell-free ARNm expression system contains all the elements required for the production of ARNm in the absence of cells. Many and diverse organisms from which this mechanism is derived, including both prokaryotes and eukaryotes, exist.

[0145] Specifically, the system particularly includes a cellular transcription mechanism. More specifically, the system contains an ARN polymerase capable of recognizing the aforementioned expression cassette promoter. Thus, in the presence of suitable nucleotides and under suitable ionic conditions, this ARN polymerase can direct the transcription of the ARNm gene encoding the present invention. Such systems have been well known to those skilled in the art for decades (see, for example, Beckert and Masquida, 2011, as a review). In cell-free systems, there are numerous methods for transcribing ADN into ARN. Kits from many companies are also available: New England Biolabs, Sigma Aldrich, Thermo Scientific, Promega, Roche Diagnostics, Ambion, Invitrogen, etc.

[0146] In addition to ARN polymerase, the cell-free in vitro transcription system also includes a reaction buffer. Advantageously, the system contains each of four ribonucleoside triphosphates. These four ribonucleoside triphosphates are very advantageously present in the same concentration. In particular, the concentration of GTP is the same as the concentration of the other three ribonucleoside triphosphates. Therefore, the in vitro transcription yield of the ARNm of the present invention is much higher than that of capped ARNm produced by in vitro reactions, which greatly reduces the cost of ARNm synthesis.

[0147] Preferably, the production method includes the step of purifying the ARNm.

[0148] In a preferred embodiment, in a cell-free in vitro transcription system, a plasmid ADN (containing a promoter recognized by a phage ARN polymerase, followed by an ADN sequence encoding the target ARNm) is contacted with a phage ARN polymerase. The ARNm synthesized by said method can then be purified. Preferably, the plasmid ADN in the cell-free in vitro transcription system is linearized before contact with the ARN polymerase. More preferably, the ADN is linearized downstream of the 3′-UTR region by enzymatic digestion. Even more preferably, the ADN is linearized by enzymatic digestion with Ssp1 or Eco53kI.

[0149] Preferably, the ARN polymerase is bacteriophage T7 ARN polymerase.

[0150] ARNm molecules can direct the production of target peptides in the eukaryotic organisms in which they are introduced. Therefore, they are particularly suitable for gene therapy or genetic inoculation. Thus, the ARNm molecules of this invention can be used as drugs or vaccines.

[0151] According to another aspect, the present invention also relates to pharmaceutical compositions or vaccine compositions.

[0152] Therefore, more specifically, the present invention relates to pharmaceutical or vaccine compositions comprising the ARNm of the present invention. Cells are transfected using the ARNm contained in the composition, which can then translate the ARNm into proteins. Preferably, these proteins have preventive or therapeutic activity.

[0153] In fact, the inventors unexpectedly demonstrated that, during tissue transfection, the ARNm according to the present invention is more effective than conventional capped ARNm. In fact, the inventors unexpectedly demonstrated that the expression levels and duration obtained after in vivo transfection are at least as high as those of capped ARNm. Specifically, the ARNm of the present invention exhibits greater in vivo expression in mouse dermis or muscle than the control capped ARNm. Figure 5 , Figure 15 Furthermore, the inventors unexpectedly demonstrated that the ARNm according to the invention persists in cells for an extended period (i.e., more than 7 weeks). Finally, the inventors unexpectedly demonstrated that co-transfecting cells with a first ARNm encoding the 2Apro protein according to the invention and a second ARNm encoding the second protein according to the invention makes it possible to increase the translation of the second protein.

[0154] Therefore, according to another aspect of the invention, the pharmaceutical composition comprises a 2Apro protein or an ARNm encoding said protein. Preferably, the 2Apro protein has the sequence of SEQ ID NO: 81. Preferably, the ARNm encoding said protein has the sequence of SEQ ID NO: 80. According to a specific embodiment of the invention, the pharmaceutical composition comprises at least two different uncapped messenger ribonucleic acid (ARNm) molecules, which comprise from 5′ to 3′:

[0155] • 5′-UTR region, containing at least one copy of GUCAGRYC(N 7-19 )GCCA(N 12-19 The shared sequence of UGCNRYCUG (xrRNA)

[0156] • A copy of the internal ribosome entry site (IRES) ARN sequence; and

[0157] • Open reading box

[0158] At least one of the ARNm molecules contains an open reading frame encoding the 2Apro protein.

[0159] Preferably, the pharmaceutical composition comprises a first ARNm molecule containing a reading frame encoding a 2Apro protein, and at least a second ARNm molecule containing an open reading frame encoding a target second protein. More preferably, the second target protein is not a 2Apro protein. Even more preferably, the target second molecule is an antigen or therapeutic protein as defined above. According to a specific embodiment of the invention, the molar ratio between the first ARNm and the second ARNm is between 540:1 and 240:1, preferably between 540:1 and 315:1, and even more preferably 465:1.

[0160] Preferably, the composition is supplemented with excipients and / or pharmaceutically acceptable carriers. In this specification, the term "pharmaceutically acceptable carrier" refers to a compound or combination of compounds included in a pharmaceutical composition that does not cause side effects and allows for, for example, improved ease of administration of the active compound, increased lifespan and / or efficacy in vivo, increased solubility in solution, or even improved storage. Such pharmaceutically acceptable carriers are well known, and those skilled in the art will adapt them according to the nature of the selected active compound and the method of administration. In this specification, the term "pharmaceutically acceptable excipient" refers to a compound or combination of compounds included in a pharmaceutical composition that does not cause side effects and allows for, for example, improved ease of administration of the active compound, increased lifespan and / or efficacy in organism, increased solubility in solution, or even improved storage. The excipient may be added to the composition specifically just before administration, for example, if the ARN is stored in lyophilized form. Pharmaceutically acceptable excipients / carriers are well known, and those skilled in the art will adapt them according to the nature of the selected active compound and the method of administration. The Science and Practice of Pharmacy, by Remington, 22nd edition, Pharmaceutical Press, London, UK (2013), provides specific descriptions of various excipients. For example, pharmaceutically acceptable compositions contain sterile water and / or chloroquine as excipients. “Chloroquine” excipients also include any variants or analogs thereof, such as primaquine.

[0161] In fact, the inventors unexpectedly demonstrated that, under certain conditions, co-injection of chloroquine and ARNm could potentially improve transfection efficiency. Specifically, the transfection efficiency of ARNm complexed with "CPP" type peptides was improved (see...). Figure 10 ).

[0162] According to a preferred embodiment, the pharmaceutical composition further comprises chloroquine. As a non-limiting example, the pharmaceutical composition comprises ARNm:chloroquine in a weight ratio between 1:0.5 and 1:4, and more specifically, the weight ratio of ARNm:chloroquine is 1:1. As a non-limiting example, the pharmaceutical composition further comprises 5 μg ARN: 2.5-20 μg chloroquine.

[0163] According to a preferred embodiment, the pharmaceutical composition further comprises at least one CPP described herein, which is non-covalently linked to the ARNm molecule according to the invention. Advantageously, the CPP is selected to facilitate ARNm penetration into target cells (e.g., depending on the type of organ or cell line, such as muscle or dermal cells).

[0164] Preferably, these compositions are administered via intramuscular, intradermal, intraperitoneal, or subcutaneous routes, via respiratory routes, or via local routes. These compositions are preferably intended for injection into mammalian tissues, and even more preferably, humans. These compositions are preferably intended for injection via intramuscular, intravenous, intradermal, intraperitoneal, or subcutaneous routes according to methods known to those skilled in the art. The pharmaceutical compositions of the present invention can be administered several times over a period of time. The method of administration, dosage, and optimal galen formulation can be determined based on criteria typically considered in establishing a treatment suitable for a patient, such as the patient's age or weight, the severity of the patient's general condition, tolerance to treatment, and observed side effects.

[0165] Parenteral formulations include aqueous suspensions, isotonic saline solutions, or sterile and injectable solutions, which may contain pharmacologically compatible dispersants and / or wetting agents. Forms administered via the respiratory route include aerosols. Topical formulations include patches, gels, creams, ointments, lotions, sprays, and eye drops.

[0166] Methods for preparing compounds suitable for parenteral administration will be known or obvious to those skilled in the art, and are described in more detail in, for example, Pharmaceutical Sciences of Remington, 17th edition, Mack Publishing Company, Easton, Pa. (1985), and its 18th and 19th editions. The use of media and reagents for the active pharmaceutical ingredient is well known in the art. To obtain a pharmaceutically acceptable composition suitable for administration, the composition will contain a sufficient amount of ARNm molecules to have a therapeutic effect.

[0167] The effective dose of the compounds of the present invention varies with many parameters, such as the chosen route of administration, body weight, age, sex, the progression of the pathology to be treated, and the sensitivity of the individual to be treated.

[0168] According to one specific aspect, the present invention relates to the use of the compositions in gene therapy. The compositions of the present invention can be used to treat or regulate a variety of diseases, such as: cancer, genetic diseases (e.g., hemophilia, thalassemia, adenosine deaminase deficiency, alpha-1 antitrypsin deficiency), diabetes, brain diseases (e.g., Alzheimer's disease and Parkinson's disease), allergies, autoimmune diseases, and cardiovascular diseases.

[0169] According to another aspect, the present invention relates to the use of the said composition in genetic vaccines. For example, the compositions of the present invention can be used for vaccination against cancer and influenza, as well as other viral and bacterial pathogens. Vaccination may involve humans as well as pets and livestock.

[0170] The inventors specifically demonstrate that the ARNm of the present invention is stable in vivo. In fact, the ARNm of the present invention induces at least as high a level of protein synthesis in vivo as capped ARNm, indicating that its resistance to Xrn1 is at least as effective as that of capped ARNm. Furthermore, the inventors specifically demonstrate that the ARNm of the present invention persists in cells for a long period. Such ARNm molecules are also highly advantageous because their production cost is significantly lower than that of capped ARNm molecules. Finally, the inventors demonstrate that ARNm containing ARN aptamers that penetrate cells directly (e.g., ARN aptamer C) or ARN aptamers that penetrate cells via CPP peptides (e.g., ARN aptamer A) can more effectively transfect cells, which advantageously improves the production of one or more target proteins.

[0171] The present invention will be described in more detail by way of the following embodiments. Attached Figure Description

[0172] Figure 1 The structure of the messenger ARN.

[0173] (A) The capped MB5-luc2 ARNm corresponds to the conventional messenger ARN encoding luciferase, with a cap analog at its 5′ end and a 3′-UTR region containing a poly(A) tail. (B) MB5-luc2 ARNm is the same as (A), except it lacks the cap analog. (C) MB7-luc2 ARNm lacks the cap, has a stem-loop at its 5′ end, and an internal ribosome entry site (IRES) for EMCV virus. (D) MB8-luc2 ARNm lacks the cap and has a stem-loop, with two Xrn1 resistance sequences (xrRNA1 and xrRNA2) from West Nile virus (WNV) and an IRES from EMCV virus in its 5′ UTR region. (E) MB9-luc2 ARNm is similar to (D), except that the 3′-UTR and poly(A) of (D) have been replaced by a 3′-UTR from Kunkinovirus (KUN). (F) The uncapped MB11-luc2 ARNm is similar to (D) except that aptamer A is added to the 5′ region upstream of the two Xrn1 resistance sequences (xrRNA1 and xrRNA2) of West Nile virus (WNV) and the IRES of EMCV virus, and it lacks a stem-loop. MB13-luc2(G) and MB14-luc2(H) ARNm have this same conformation, but contain a 5′ stem-loop following ARN aptamers B and C, respectively. (I) The capped MB15-luc2 ARNm is similar to (A) except that aptamer A is added to the 5′ region after the cap. (J) The MB17-luc2 ARNm is similar to (D) except that aptamer A is added at the 5′ region between the stem-loop and the xrRNA sequence. (K) The MB18-luc2 ARNm is similar to (J) except that aptamer A is located in the 5′ region downstream of the xrRNA sequence and upstream of the IRES sequence.

[0174] Figure 2 : Expression kinetics of luciferase in Caco-2 cells over four days.

[0175] Caco-2 cells were transfected with capped (diamond-shaped) and uncapped (circle-shaped) ARNm, as well as MB7-luc2 ARNm (triangle-shaped) and MB8-Iuc2 ARNm (square-shaped). Expression kinetics were monitored for four days.

[0176] Figure 3 : Expression kinetics of luciferase in Caco-2 cells over ten days.

[0177] Caco-2 cells were transfected with capped MB5-luc2 ARNm (rhombus), MB8-luc2 ARNm (square), and MB9-luc2 ARNm (triangle). Expression kinetics were monitored for ten days.

[0178] Figure 4 Expression kinetics of long-term luciferase in human mesenchymal stem cells.

[0179] MB8-Iuc2 ARNm was compounded with pepMB1 peptide (at a ratio of approximately 2.2:1 pepMB1 peptide positive charge to ARNm negative charge), and mesenchymal stem cells were transfected with the above complex at 2 μg per well in a 48-well plate for 1 hour. Luciferase activity was then monitored for approximately 48 days.

[0180] Figure 5 : Transfect mouse muscle and dermis.

[0181] The muscle and dermis of male BALB / cByJ mice were transfected with 10 μg and 5 μg of uncapped MB8-luc2 ARNm or capped MB5-luc2 ARNm, respectively. Luciferase activity was measured in muscle and skin at 16 and 18 hours post-injection.

[0182] Figure 6 Toxicological studies of MB8-2Apro ARNm.

[0183] To assess the cytotoxic effects of 2Apro protein expression, C2C12 cells were transfected with MB8-luc2 ARNm alone, or with a mixture of MB8-luc2 ARNm and MB8-2Apro ARNm at a ratio of 465:1 or 9:1. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) released into the extracellular medium. LDH activity was determined by measuring absorbance at 490 nm. Negative control: untransfected cells without lysis or cells transfected with MB8-luc2 ARNm alone. Positive control: untransfected cells lysed with Triton X-100.

[0184] Figure 7 The effect of 2Apro protein on luciferase expression varied with the molar ratio of MB8-luc2 ARNm to MB8-2Apro ARNm.

[0185] C2C12 cells were transfected with MB8-luc2 ARNm when an increased amount of MB8-2Apro ARNm was available. The molar ratio of MB8-luc2 ARNm to MB8-2Apro ARNm was 540:1 to 240:1. The total amount of ARNm transfected per well was 750 ng. Luciferase activity was measured 18 hours after transfection.

[0186] Figure 8 : Expression kinetics of luciferase over seven days in the presence or absence of MB8-2Apro ARNm.

[0187] C2C12 fusion cells were transfected with either MB8-luc2 ARNm alone (black line) or a combination of MB8-luc2 ARNm and MB8-2Apro ARNm (gray dashed line). The molar ratio of MB8-luc2 ARNm to MB8-2Apro ARNm was 465:1. Expression kinetics were measured by luciferase activity levels over a period of 7 days.

[0188] Figure 9 Effects of 2Apro protein on the expression of different messenger ARN luciferases.

[0189] The effect of the 2Apro protein on the expression of luciferase from different messenger ARNs was evaluated. In each case, the ARNm encoding luciferase was transfected alone (-) or co-transfected with a second ARNm encoding the 2Apro protein (+), exhibiting the same characteristics at an optimal molar ratio of 465:1. (1) MB8-luc2ARNm alone; (2) MB8-luc2 ARNm co-transfected with MB8-2AproARNm; (3) MB5-luc2 ARNm alone with capped cells; (4) MB5-luc2 ARNm co-transfected with uncapped cells; (5) MB5-luc2 ARNm alone without capped analogues; (6) MB5-luc2 ARNm without capped analogues co-transfected with MB8-2Apro ARNm without capped analogues; (7) MB7-luc2 ARNm alone; (8) MB7-luc2 ARNm co-transfected with MB8-2Apro ARNm. Luciferase activity was measured 18 hours after transfection.

[0190] Figure 10 The effect of different aptamers on the transfection efficiency of capped ARNm molecules in muscle.

[0191] The muscle of male BALB / cByJ mice was transfected with the following: 5 μg of MB8-luc2 ARNm, MB13-luc2 ARNm, MB14-luc2 ARNm, MB11-luc2 ARNm conjugated with M12-H6 peptide, or MB11-luc2 ARNm conjugated with M12-H6 peptide in the presence of 5 μg chloroquine. Luciferase activity was measured 16 hours after injection.

[0192] Figure 11 Transfection efficiency of ARNm based on the molar ratio of CPP3-H6 peptide to MB11-luc2 ARNm in the dermis.

[0193] The dermis of male OF1 mice was transfected with 5.6 μg of MB11-luc2 ARNm conjugated with CPP3-H6 peptide at varying molar ratios (CPP3-H6:MB11-luc2 ARNm molar ratios between 1:8 and 1125:1). Luciferase activity was measured 18 hours post-injection. The optimal permissible transfection molar ratio was 1 CPP3-H6:4 MB11-luc2 ARNm.

[0194] Figure 12 Transfection efficiency of ARNm based on the molar ratio of CPP1-H6 peptide to MB11-luc2 ARNm in the dermis.

[0195] The dermis of male OF1 mice was transfected with 5.6 μg of MB11-luc2 ARNm conjugated with CPP1-H6 peptide at varying molar ratios (CPP1-H6:MB11-luc2 ARNm molar ratios between 1:8 and 3:1). Luciferase activity was measured 18 hours post-injection. The optimal permissible transfection molar ratio was 1 CPP1-H6:4 MB11-luc2 ARNm.

[0196] Figure 13 Transfection efficiency of ARNm based on the molar ratio of CPP2-H6 peptide to MB11-luc2 ARNm in the dermis.

[0197] The dermis of male OF1 mice was transfected with 5.6 μg of MB11-luc2 ARNm conjugated with CPP2-H6 peptide at varying molar ratios (CPP2-H6:MB11-luc2 ARNm molar ratios between 1:4 and 2.75:1). Luciferase activity was measured 18 hours post-injection. The optimal permissible molar ratio for transfection was 2 CPP2-H6:1 MB11-luc2 ARNm.

[0198] Figure 14 Effect of aptamer A on transfection efficiency of capped ARNm molecules in the dermis.

[0199] ARN aptamer A was inserted into the 5′-UTR of a conventional capped MB5-luc2 ARNm to produce a capped MB15-luc2ARNm. This was based on previously obtained results (see...). Figure 12 The dermis of male OF1 mice was transfected with 5.6 μg of capped MB15-luc2 ARNm in a molar ratio of 2CPP2-H6:1MB15-luc2 ARNm or a different molar ratio containing the CPP2-H6 peptide. This construct did not improve transfection in the presence or absence of aptamer A or the CPP2-H6 peptide.

[0200] Figure 15Effect of 5′-SL on the transfection efficiency of ARNm molecules in the dermis.

[0201] The effect of stem-loop (here, “5′-SL” with the xrRNA1 sequence 5 nucleotides downstream) on transfection efficiency was compared to that of a stem-loop-deficient ARNm (MB11-luc2 ARNm) and an ARNm with its stem-loop located more than 70 nucleotides upstream of xrRNA1 (MB17-luc2 ARNm). Conventional MB5-luc2-capped ARNm was used as a control for the efficacy of the two xrRNA sequences following 5′-SL. Dermal cells of male OF1 mice were transfected with 5.6 μg of each different ARNm, and luciferase activity was measured 18 hours post-injection. The aptamer A of MB11-luc2 is not linked to the peptide and therefore had no effect on the transfection efficiency of this ARNm. Detailed Implementation

[0202] The invention is illustrated by the following non-limiting embodiments. As those skilled in the art will understand, these teachings include alternatives, modifications, and equivalents.

[0203] Example 1: Plasmid Construction

[0204] ADN sequences corresponding to the 5′ non-coding sequences of SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51 were chemically synthesized, integrated into ADN vectors, and sequenced using ProteoGenix. ADN fragments were digested with restriction enzymes. pMBx-luc2 series plasmids were digested with the same restriction enzymes, and ADN fragments were integrated into these plasmids using T4 ADN ligase. These plasmids contained the gene encoding luciferase (SEQ ID NO: 62), inserted downstream of the phage T7 promoter. A short non-coding 3′-UTR sequence, followed by a transcriptional polyadenylation sequence according to SEQ ID NO: 53, was located downstream of the luciferase gene. Different non-coding 5′-UTR sequences separated the promoter from the luciferase gene. The plasmids constructed in this way were amplified, validated, and linearized downstream of the transcriptional polyadenylation sequence using restriction enzymes.

[0205] Example 2: Plasmid linearization and in vitro transcription of ARNm

[0206] The Ssp1 restriction enzyme site is located immediately downstream of poly(A) in each plasmid (see SEQ ID NO: 54). 10 μg of plasmid was digested with 20 units of Ssp1-HF restriction enzyme (New England Biolabs) in 1×CutSmart buffer at 37°C for 4 hours.

[0207] Then, 8 μl of Ssp1-HF linearized plasmid was mixed with 2 μl of 10×T7 ARN polymerase reaction buffer, 2 μl each of the four nucleoside triphosphates (ATP, GTP, CTP, and UTP), and 2 μl of T7 ARN polymerase solution (New England Biolab). This reaction mixture included a cap analog for the synthesis of capped ARNm. The synthesis of uncapped ARNm was omitted.

[0208] Transcription was performed over three to ten hours in a block heater at 37°C. Then, 1 μL of TURBODNase (Thermo Fisher) was added to degrade the plasmid, and the mixture was incubated at 37°C for 15 minutes.

[0209] MB5-luc2 ARNm is a conventional ARNm encoding luciferase. It possesses a 5′ non-coding sequence (UTR) selected according to SEQ ID NO: 57 for optimal translation initiation, and a 3′-UTR region containing the Poly(A) tail according to SEQ ID NO: 60. Capped or uncapped versions of this ARNm are synthesized (see [link to original text]). Figure 1 (A) and Figure 1 (B)).

[0210] The capless MB7-luc2 ARNm has a 5′ stem-loop at its 5′ end, replacing the 5′-UTR of the MB5-luc2 ARNm, followed by the IRES sequence of the EMCV virus. Therefore, although it lacks a cap, the IRES sequence will allow it to efficiently recruit ribosomes. However, this ARNm is sensitive to the Xrn1 enzyme (see [link to ARNm]). Figure 1 (C)). The 5′-UTR region of MB7-luc2 ARNm corresponds to SEQ ID NO: 58 and the 3′-UTR region corresponds to SEQ ID NO: 60.

[0211] The capless MB8-luc2 ARNm has a stem-loop at its 5′ end, followed by two consecutive anti-Xrn1 sequences from the flavivirus WNV 3′-UTR in the 5′-UTR region, called xrRNA1 and xrRNA2 (Kieft et al., 2015). These sequences are followed by the EMCV virus IRES sequence, thus it is protected by the two anti-Xrn1 sequences (see [link to original text]). Figure 1(D)). The 5′-UTR region of MB8-luc2ARNm corresponds to SEQ ID NO: 59 and the 3′-UTR region corresponds to SEQ ID NO: 60. The cost of producing uncapped MB8-luc2 ARNm is about 30 times lower than the cost of producing capped ARNm.

[0212] The uncapped MB9-luc2 ARNm differs from the uncapped MB8-luc2 ARNm only at the 3′ end. In fact, the 3′-UTR and poly(A) sequences of MB8-luc2 have been replaced by the 3′-UTR region of Kunjin virus. This region lacks the poly(A) sequence (see [link to original text]). Figure 1 (E)). The 3′-UTR region of MB9-luc2 ARNm corresponds to SEQ ID NO: 61.

[0213] The only differences between capless MB11-luc2 ARNm and capless MB8-luc2 ARNm are the absence of a stem-loop at the 5′ end and the presence of an aptamer in the upstream 5′ region of the two consecutive sequences resistant to Xrn1 (see [link]). Figure 1 (F)). MB11-luc2 ARNm contains aptamer A of SEQ ID NO: 64; the 5′-UTR region of MB11-luc2 ARNm therefore corresponds to SEQ ID NO: 67.

[0214] The capless MB13-luc2 and MB14-luc2 ARNm differ from the capless MB8-luc2 ARNm only in the presence of an aptamer in the upstream 5′ region of the two consecutive sequences resistant to Xrn1 (see [link to relevant documentation]). Figure 1 (G, H)). MB13-luc2 ARNm contains aptamer B of SEQ ID NO: 65; therefore, the 5′-UTR region of MB13-luc2 ARNm corresponds to SEQ ID NO: 68. MB14-luc2 ARNm contains aptamer C of SEQ ID NO: 66; therefore, the 5′-UTR region of MB14-luc2 ARNm corresponds to SEQ ID NO: 69.

[0215] MB15-luc2 ARNm corresponds to capped MB5-luc2 ARNm, wherein aptamer A (SEQ ID NO: 64) has been inserted into the 5′-UTR region (see [link]). Figure 1 (I)); Therefore, the 5′-UTR region of MB15-luc2 ARNm corresponds to SEQ ID NO: 70.

[0216] The only difference between MB17-luc2 ARNm and uncapped MB11-luc2 ARNm is the presence of a stem-loop at the 5′ end upstream of aptamer A (see [link]). Figure 1 (J)). MB17-luc2 ARNm contains aptamer A of SEQ ID NO: 64; therefore, the 5′-UTR region of MB17-luc2 ARNm corresponds to SEQ ID NO: 83.

[0217] The only difference between capless MB18-luc2 ARNm and capless MB8-luc2 ARNm is the presence of an aptamer in the 5′ region between the two consecutive Xrn1-resistant sequences and the IRES sequence (see [link]). Figure 1 (K)). MB18-luc2 ARNm contains aptamer A of SEQ ID NO: 64; therefore, the 5′-UTR region of MB18-luc2 ARNm corresponds to SEQ ID NO: 84.

[0218] Example 3: Purification of Messenger ARN

[0219] Different luciferase ARNm were purified using the MegaClear kit (Ambion). 79 μl of eluent, 350 μl of conjugation solution concentrate, and 250 μl of 100% ethanol were added to 21 μl of the previous mixture. This 700 μl was placed on a filter cartridge and centrifuged at 10,000 × g for 1 min. The filter retained the messenger ARN. The filter was washed twice with 500 μl of washing buffer and centrifuged at 10,000 × g for 1 min. The ARN was then eluted from the filter by adding 50 μl of eluent twice and heating to 70 °C for 10 min in a block heater. The eluent was obtained by centrifugation at 10,000 × g for 1 min.

[0220] The second purification step involved precipitation with lithium chloride. 60 μl of the LiCl precipitation solution was added to 100 μl of eluent. The mixture was cooled at -20 °C for 1 hour, then centrifuged at maximum speed at 4 °C for 15 minutes. The precipitate was washed with 500 μl of 70% ethanol and centrifuged again at maximum speed at 4 °C for 5 minutes. The messenger ARN precipitate was air-dried for several minutes and then resuspended in sterile deionized water. The concentration of the ARNm solution was determined by measuring the absorbance at 260 nm using a spectrophotometer.

[0221] Example 4: Assembly of the messenger ARN / pepMB1 complex

[0222] The cationic peptide pepMB1 was synthesized, purified, and lyophilized by ProteoGenix. Its amino acid sequence is as follows: CRRRRRRRC. The lyophilized product was resuspended in sterile deionized water.

[0223] 5 μg of luciferase ARNm was mixed with 5 μg of pepMB1 to achieve a final ARN concentration of 20 μg / ml. The mixture was incubated at room temperature (20–25 °C) for 15 minutes and then frozen at -80 °C. The ARNm / pepMB1 complex was then lyophilized for approximately 20 hours.

[0224] Example 5: Transfection of Caco-2 or C2C12 cells

[0225] Materials and methods:

[0226] a) Culture and inoculation of Caco-2 or C2C12 cell lines

[0227] All cell manipulations were performed under a laminar flow hood. The Caco-2 cell line (ECACC) was cultured in DMEM (Gibco) supplemented with a mixture of non-essential amino acids, antibiotics, and antifungal agents, as well as fetal bovine serum (final 15%). The culture was carried out at 75 cm⁻¹. 2 The culture was carried out in a Corning flask at 37°C.

[0228] When the required cell count for seeding in 48-well plates (Corning) is reached, cells are isolated from the bottom of the flask by treating with 3 ml of TryPLE Select 1X (Gibco) at 37°C for 5 minutes. 7 ml of DMEM is added to neutralize the TryPLE Select 1X. Cells are centrifuged at 100 x g for 10 minutes at room temperature. The cell pellet is then resuspended in 10 ml of culture medium. 250 μl of this cell suspension is introduced into each well of a 48-well plate, which is then placed in an incubator at 37°C containing 5% CO2.

[0229] Following inoculation, C2C12 cells can remain confluenced in the wells of a 48-well plate for approximately 12 days. During this period, these cells differentiate into intestinal epithelium, thereby influencing ARNm translation. In contrast, human mesenchymal stem cells (Millipore) can be preserved in confluenced cultures for more than 7 weeks. Therefore, mesenchymal stem cells (Millipore, human mesenchymal stem cells (bone marrow)) were cultured in ready-to-use media (Millipore, mesenchymal stem cell expansion media) in 48-well plates (Corning) for up to 48 days. For cells that would be lysed more than 5 days post-transfection, the culture medium was changed 3 times per week.

[0230] b) Transfect Caco-2 or C2C12 cells

[0231] For Caco-2 cells, transfection was performed in five different wells for each ARNm. The lyophilized ARNm / pepMB1 complex (Proteogenix) was resuspended in 750 μl of transfection buffer (20 mM Hepes, 40 mM KCl and 100 mM trifluoroacetic acid).

[0232] For C2C12 cells, MB8 ARNm transfection was performed as follows. The MB8-luc2 ARNm / pepMB1 complex (Proteogenix) was assembled by incubating ARNm at room temperature for 30 minutes in the presence of peptides at a ratio of approximately 2.2 of positively charged peptides to negatively charged ARNm. The solution was then diluted with 3× DMEM to obtain 1× final DMEM.

[0233] In both cases, the culture medium in the wells was emptied to introduce 150 μl of ARN / pepMB1 complex solution (1 μg ARNm per well for Caco-2 cells; and 2 μg per well for C2C12 cells). Cells were incubated in a CO2 incubator at 37°C for 30 minutes (Caco-2 cells) or 1 hour (C2C12 cells). The ARNm / pepMB1 complex solution was then aspirated and replaced with 250 μl of culture medium. Cells were then incubated in a CO2 incubator at 37°C for 6 hours to 48 days, depending on the cell type.

[0234] c) Lyse Caco-2 or C2C12 cells and measure luciferase activity from 6 hours to 48 days post-transfection to assess luciferase protein expression kinetics. Aspirate the culture medium and replace it with 250 μl of lysis buffer (Luciferase Assay System, Promega). Place 20 μl of each cell lysate into a suitable tube for a luminometer (Berthold Technologies). Add 100 μl of luciferase substrate (Promega) to the cell lysate using a photometer. Then, measure the amount of light emitted by the enzymatic reaction catalyzed by luciferase. Results are expressed in relative light units (RLU). The amount of luciferase protein produced by Caco-2 or C2C12 cells was normalized to total cellular protein using a 660 nm protein assay kit (Pierce) via luciferase ARNm. For this purpose, 100 μl of cell lysate was mixed with 1.5 ml of reagent, and absorbance was measured at 660 nm. The calibration range was performed using bovine serum albumin solution. Therefore, luciferase activity is expressed as RLU per milligram of protein.

[0235] result:

[0236] The results are shown in Figure 2 , Figure 3 and Figure 4 In Caco-2 cells, capless MB5-luc2 ARNm induces low and short expression of luciferase protein. Without the cap, ARNm is rarely translated into protein and is rapidly degraded by Xrnl (see [link to relevant documentation]). Figure 2 ).

[0237] Compared to capless MB5-luc2 ARNm, capless MB7-luc2 ARNm provided stronger and more sustained luciferase protein expression in Caco-2 cells. The IRES region recruits ribosomes but does not show significant resistance to Xrn1 (see [link to relevant documentation]). Figure 2 ).

[0238] The expression kinetics of luciferase induced by uncapped MB8-luc2 ARNm were similar to those induced by capped MB5-luc2ARNm (see [link]). Figure 2 and Figure 3 This means that, similar to the cap on MB5-luc2 ARNm, adding two sequences from WNV virus that are resistant to Xrn1 confers resistance to Xrn1 to ARNm. The expression of luciferase induced by MB7-luc2 ARNm lacking both the cap and the Xrn1-resistant sequences is intermediate between that of capless MB8-luc2 ARNm and capless MB5-luc2 ARNm (see [link to relevant documentation]). Figure 2 ).

[0239] The difference between MB9-luc2 ARNm and MB8-luc2 ARNm lies in the absence of poly(A) at the 3′-UTR terminus. It induces significantly lower and shorter-lasting luciferase expression in Caco-2 cells compared to MB8-luc2 ARNm (see [link to relevant documentation]). Figure 3 ).

[0240] In human mesenchymal stem cells, MB8-luc2 ARNm unexpectedly and favorably induced luciferase expression, which persisted for a long time. In fact, even though expression decreased over time, it was still detectable 48 days post-transfection (see [link to relevant documentation]). Figure 4 ).

[0241] Example 6: Transfection of mouse muscle and dermis

[0242] Materials and methods:

[0243] a) Animal husbandry:

[0244] For muscle, 8-week-old male BALB / cByJ mice were housed in open cages, with 5 animals per cage. The day / night cycle was managed automatically (12h day / 12h night). They were fed and allowed free access to filtered water. For skin, 6-week-old male OF1 mice were housed in open cages, with 4 animals per cage.

[0245] b) Ad-hoc preparation of ARNm samples:

[0246] For each intramuscular injection, prepare 100 μl of bare ARNm solution containing 230 mM NaCl.

[0247] For each intradermal injection, prepare 17 μl of bare ARNm solution containing 160 mM NaCl.

[0248] When using the CPP-H6 peptide, mix it with ARNm and incubate at room temperature for 30 minutes in the presence of 5 mM Hepes at pH 7.5 and 0.7 mM MgCl2.

[0249] c) Intradermal and intramuscular injections of ARNm:

[0250] Mice were anesthetized using isoflurane. For intramuscular injection, analgesia was administered via buprenorphine injection. For intradermal injection, the skin on the back was shaved three to four days prior (see Example 10 for details).

[0251] Inject 100 μl and 17 μl of ARNm solution into the biceps femoris muscle and skin, respectively. Return the animal to its cage until the following morning.

[0252] d) Skin and muscle samples:

[0253] For muscle injections, mice were euthanized painlessly with CO2 16 hours post-injection. For skin injections, mice were euthanized painlessly with isoflurane anesthesia and cervical dislocation 18 hours post-injection. Skin and muscle injection sites were obtained. These biopsies were washed with saline, cut into small pieces, and placed in tubes (Promega) containing lysis buffer. These tubes were immediately frozen in liquid nitrogen.

[0254] e) Cells obtained from lysed skin and muscle biopsies:

[0255] Each skin and muscle biopsy underwent three freeze / thaw cycles. Specifically, tubes containing biopsy and lysis buffer were frozen at -80°C for 10 minutes. They were then thawed in a water bath at room temperature for two minutes, briefly vortexed. The tubes were then centrifuged at 5000 x g for 5 minutes at 20°C to precipitate tissue debris and obtain a clear cell lysis supernatant.

[0256] f) Measurement of luciferase activity:

[0257] 20 μl of each cell lysate was used to measure luciferase expression in each biopsy. 100 μl of luciferase substrate (Promega) was added to each sample using a tube luminometer, and the luminescence intensity was measured for 10 seconds. Results are expressed in relative light units (RLU).

[0258] The cell lysate was then diluted 8 to 20 times using the Pierce 660nm Protein Analysis Kit for protein analysis. 100 μl of diluted cell lysate was mixed with 1.5 ml of reagent over 6 minutes, and absorbance was measured at 660 nm. The correction range for bovine serum albumin was 0 to 750 μg / ml.

[0259] result:

[0260] The results are as follows Figure 5 As shown. At 16 hours (muscle) or 18 hours (dermis) post-injection, capless MB8-luc2ARNm induced higher luciferase expression in skeletal muscle (A) and skin (B) than capped MB5-luc2ARNm. These results indicate that the presence of two xrRNA sequences (from WNV virus) and IRES (from EMCV) confers Xrn1 resistance in ARNm in vivo and results in superior translation efficiency compared to capped MB5-luc2ARNm. Surprisingly, transfection of mouse muscle tissue with 10 μg of MB8-luc2ARNm resulted in luciferase expression 2.6-fold higher than that obtained with the same dose of capped MB5-luc2ARNm (see [link to relevant documentation]). Figure 5 A). Similarly, luciferase expression generated by transfecting 5 μg MB8-luc2ARNm in the skin was 9.3 times higher than that obtained by the same dose of capped MB5-luc2 ARNm (see A). Figure 5 B).

[0261] Therefore, MB8-luc2 ARNm can completely replace capped MB5-luc2 ARNm, and may even have more advantages.

[0262] Example 7: Cytotoxicity of MB8-2Apro ARNm

[0263] Materials and methods:

[0264] Expression of the 2Apro protein in mammalian cells can induce toxicity, ultimately leading to cell death, through apoptosis or necrosis (Goldstaub et al., 2000). During these processes, cells release lactate dehydrogenase (LDH) into the extracellular environment. LDH activity can be measured using the commercially available Promega CytoTox96 Non-Radioactive Cytotoxicity Assay kit.

[0265] MB8-2Apro ARNm (SEQ ID NO: 80) is an ARNm encoding a 2A nonstructural protein (2Apro, having the sequence of SEQ ID NO: 81) from the genome of human rhinovirus 2 (HRV2). It has a 5′ uncoding sequence (UTR) according to SEQ ID NO: 57, which is identical to MB8-luc2 ARNm, and a 3′-UTR region containing a poly(A) tail according to SEQ ID NO: 60.

[0266] As described in Example 4, 750 ng of MB8-luc2 ARNm alone or a mixture of MB8-luc2 ARNm and MB8-2Apro ARNm was combined with the pepMB1 peptide.

[0267] MB8-luc2 ARNm was transfected into C2C12 cells, either alone or in a mixture of MB8-luc2 ARNm and MB8-2Apro ARNm at two different molar ratios. Specifically, C2C12 cells from the wells of a 48-well plate were incubated with the ARNm / pepMB1 complex for one hour. After 18 hours, luciferase activity was measured. Figure 6 Untransfected, unlyzed C2C12 cells were used as a negative control, while untransfected C2C12 cells lysed with Triton X-100 to release all LDH into the culture medium were used as a positive control.

[0268] result:

[0269] Even at the highest molar ratio (MB8-luc2 ARNm:MB8-2Apro ARNm ratio of 9:1), the ARNm mixture did not induce cytotoxicity. This indicates that expression of the viral protease does not induce cytotoxicity. Figure 9 ).

[0270] Example 8: Optimization of luciferase expression kinetics by co-transfection of MB8-luc2 and MB8-2Apro ARNm

[0271] Materials and methods:

[0272] Under non-toxic conditions, MB8-luc2 and MB8-2Apro ARNm were co-transfected into C2C12 cells at different ratios according to the above method. After 18 hours, luciferase activity was measured. Figure 7 Then, following the method described above, kinetics were measured 6 hours to 7 days post-transfection to determine whether an improvement in luciferase expression was observed only 18 hours post-transfection.

[0273] result:

[0274] Surprisingly, in all tested ratios, co-transfection of MB8-luc2 and MB8-2Apro ARNm increased luciferase expression of MB8-luc2 ARNm in C2C12 cells by at least 2.5-fold. The optimal molar ratio of MB8-luc2 ARNm to MB8-2Apro ARNm was 465:1, which increased luciferase expression by 3.4-fold.

[0275] Kinetic studies conducted 6 hours to 7 days post-transfection unexpectedly demonstrated improved luciferase expression lasting for at least a week. In fact, luciferase expression increased by an average of 2.4-fold. Figure 7 ).

[0276] Example 9: Effect of ARN aptamers on ARNm transfection efficiency in muscle

[0277] To improve the internalization of the ARNm molecule according to the present invention, different aptamers are incorporated into the ARN molecule, as detailed below. The effects of these aptamers are then evaluated in vivo to determine whether an improvement in luciferase expression can be observed.

[0278] Materials and methods:

[0279] Fit Selection

[0280] Aptamers that penetrate C2C12 cells were selected. First, hybridization with a 5′ primer was performed, followed by extension of single-stranded ADNs from a single-stranded ADN library to generate double-stranded ADNs. Then, the resulting double-stranded ADNs were precipitated and purified according to methods well known to those skilled in the art.

[0281] The fragments were then purified by transcription using the T7 DuraScribe Transcription Kit (20 μl / round), followed by ARN purification using the ssDNA and ARN Purification Kit to obtain an ARN aptamer library. Finally, the solution was treated with DNAseI to remove contaminating ADN. The aptamers were dissolved in 1×DMEM+ITS (1288 μg / 5 ml) with 8 μM ARN. To select aptamers, 5 ml of DMEM / ITS / ARN solution was added to cells that had been pre-washed twice with DMEM without antibiotics or serum. Cells were incubated at 37°C for 1 hour, with brief shaking of the flask containing the mixture every 15 minutes. The flask containing the cells was then placed on ice, and the cells were washed five times with 15 mL of cold 1×PBS to remove ARN aptamers that had not penetrated the cells. Cells were then lysed with TRIzol (Invitrogen), and total ARN was extracted using the phenol-chloroform method. Endogenous ARN was digested with RNase A, and the remaining ARN was hybridized with a 3′ primer. Reverse transcription was then performed using Superscript III enzyme (ThermoFisher) before PCR amplification in the presence of a 5′ primer (100 μM), a 3′ primer (100 μM), Q5 High Fidelity ADN polymerase (NEB), and 1× Q5 High-Fidelity Master Mix buffer. All these steps were repeated to obtain an aptamer ARN library. Thus, two rounds of selection of ARN aptamers that penetrated C2C12 cells were performed.

[0282] Two ARN aptamers (B and C) that penetrated C2C12 cells were selected and sequenced (SEQ ID NO: 65 and 66, respectively). ARN aptamers B and C were then inserted into the 5′-UTR of MB8-luc2 ARNm upstream of xrRNA1, generating MB13-luc2 and MB14-luc2 ARNm, respectively.

[0283] aptamers that are firmly bound to multihistidine peptide motifs

[0284] A second strategy aimed at improving ARNm internalization consists of inserting another ARN aptamer (aptamer A) into the 5′-UTR upstream of xrRNA1 of the MB8-luc2 ARNm, capable of strongly binding to a multihistidine peptide motif in the presence of magnesium. The ARNm incorporating ARN aptamer A is named MB11-luc2. The mouse myofiber penetration peptide M12 (see Gao et al., 2014) is linked to a six-histidine motif via a spacer (containing glycine and serine amino acids). Different spacers are illustrated by the following sequence example: SEQ ID NO: 75 (PRQPPRSISSHP) GGGGSGGGGSGGGGSGGGGSGGHHHHHH), SEQID NO: 76 (PQRDTVGGRTT PPSWGPAKA GGGGSGGGGSGGGG HHHHHH), SEQ ID NO: 77 (GPFHFYQFLFPPV GGGGSGGGGSGGG GSGGGGSG HHHHHH) or SEQ ID NO: 78 (GSPWGLQHHPPRT) GGGGS GGGGSGGGGSGGGGGSG HH HHHH (underlined is a spacer sequence). The resulting peptide was named M12-H6 (SEQ ID NO: 75). MB11-luc2 ARNm was incubated with the M12-H6 peptide at room temperature for 30 minutes before being injected into the biceps femoris muscle of mice.

[0285] In vivo transfection

[0286] With or without 5 μg of chloroquine, the biceps femoris muscles of male BALB / cByJ mice were transfected with 5 μg of MB8-luc2 ARNm, MB13-luc2 ARNm, MB14-luc2 ARNm, or MB11-luc2 ARNm conjugated with the M12-H6 peptide. Luciferase activity was measured 16 hours post-injection.

[0287] result:

[0288] MB13-luc2 ARNm transfects muscle just like MB8-luc2 ARNm. Figure 10 Advantageously, MB14-luc2 ARNm containing the C aptamer was 2.1 times more efficient at transfecting the biceps femoris muscle than MB8-luc2 ARNm. Therefore, the ARN aptamer C improves the internalization of ARNm molecules into the muscle fibers into which they are inserted.

[0289] MB11-luc2 ARNm has moderate transfection efficiency. Figure 10 However, surprisingly, compared to MB11-luc2 ARNm in the presence of the peptide but without chloroquine, co-injection of 5 μg of MB11-luc2 ARNm, M12-H6 peptide, and 5 μg of chloroquine increased transfection efficiency by 31-fold. Furthermore, compared to MB8-Iuc2 ARNm, MB11-luc2ARNm exhibited a significantly higher luciferase expression rate of 2.7-fold in the presence of M12-H6 and chloroquine.

[0290] Without being bound by theory, it can be hypothesized that the high transfection efficiency of MB11-luc2 ARN complexed with the M12-H6 peptide in the presence of chloroquine is due to the reduction of endosome acidification by chloroquine after ARNm penetrates the cell. Chloroquine can slow down the protonation of hexahistine at acidic pH, thus preventing instability of the complex between MB11-luc2 ARNm and the M12-H6 peptide. As a result, ARNm can cross the endosome membrane with the help of the still-complexed M12-H6, which can enhance ARNm escape from the endosome, enter the cytoplasm, and subsequently be translated therein.

[0291] Example 10: Intradermal injection protocol in mice

[0292] a-Solution preparation

[0293] Prepare a 60 μl solution containing 20 μg ARNm for each mouse. To do this, mix deionized water, 50 mM Hepes (1 / 8 Hepes, 7 / 8 sodium Hepes), NaCl (final 160 mM), MgCl2, ARNm, and an optional peptide. Incubate at room temperature for 30 minutes to allow the peptide to bind to the ARN. No incubation is required if the ARNm does not bind to the peptide. Store the ARNm solution frozen at -80°C until injection.

[0294] b-Intradermal injection and biopsy samples

[0295] Six-week-old male OF1 mice (Charles River) were used. They were shaved three to four days prior to intradermal injection. Anesthesia was administered using a mask. Anesthesia was induced with 4% isoflurane (Piramal Heathcare). Maintenance anesthesia was maintained with 2% isoflurane. The previously shaved dorsal skin was cleaned with alcohol before injection. The ARNm solution was slowly melted at room temperature using a 0.3 mm x 8 mm U-100 (30G) insulin syringe (Becton-Dickinson). Approximately 17 μl of the ARN solution was injected three times into the shaved dorsal skin of each mouse. Papules formed before resorbing. These were demarcated using permanent markers, allowing identification of the skin area to be biopsied the following day. The tails were also marked to distinguish animals within the same cage.

[0296] Eighteen hours post-injection, skin biopsies were obtained from the injection site. Mice were anesthetized and then euthanized painlessly via cervical dislocation. The biopsies were cut into small pieces with scissors to promote cell lysis and placed in tubes containing 500 μl of 1× lysis buffer (5× luciferase cell culture lysis buffer (Promega), diluted with water). Each tube was frozen at -20°C until the next step.

[0297] c-cell lysis, luciferase and protein assay

[0298] The lysed tissue was obtained by performing three freeze / thaw cycles: incubation at -80°C for 10 minutes, followed by incubation in a water bath at room temperature for 3 minutes, and then mixing using a vortex mixer for a few seconds. The tube was then centrifuged at 5000 x g for 5 minutes at 20°C to precipitate tissue fragments. The supernatant was transferred to another tube.

[0299] Luciferase analysis was performed using the Luciferase Assay System kit (Promega). 20 μl of each sample was placed in a dedicated photometer tube (Berthold AutoLumat Plus LB 953). 100 μl of substrate was injected into the device, and the amount of emitted light (RLU) was measured.

[0300] Protein analysis was performed using the Pierce 660nm Protein Assay Kit (Thermo Scientific). The range was calibrated using bovine serum albumin (Thermo Science) and 1× lysis buffer as diluents. The range covered 100 to 500 μg of protein per ml. A 100 μl solution of 1× lysis buffer was used as a blank. In some cases, the lysate was diluted with 1× lysis buffer. 100 μl of each sample was used for protein analysis. 1.5 ml of reagent was added to both the blank and the sample. After incubation at room temperature and in the dark for an exact 5 minutes, the absorbance of each sample was measured at 660 nm using a spectrophotometer.

[0301] Example 11: Effect of CPP on ARNm transfection efficiency in skin

[0302] As described in Example 9 above, the strategy of improving naked ARNm internalization by binding cell-penetrating peptides (CPPs) to ARN aptamer A is not limited to muscle. This strategy using different CPPs has been applied here to mouse skin.

[0303] Materials and methods:

[0304] Three CPPs were used here: CPP1, CPP2, and CPP3 (see Kamada et al., 2007 and Lee et al., 2012). They were linked to six histidine residues via spacers composed of glycine and serine to form peptides CPP1-H6, CPP2-H6, and CPP3-H6, respectively, with sequences SEQ ID NO: 76, 77, and 78.

[0305] MB11-luc2 ARNm was incubated for 30 minutes with increased amounts of CPP3-H6, CPP1-H6, or CPP2-H6 peptides in a pre-optimized buffer containing 160 mM NaCl, 0.7 mM MgCl2, and 5 mM Hepes. MB11-luc2 ARNm alone, and MB8-luc2 ARNm diluted in sterile ultrapure deionized water, supplemented with 160 mM NaCl, were also injected as controls.

[0306] Following the protocol detailed in Example 10, 5.6 μg of MB8-luc2 ARNm or 5.6 μg of MB11-luc2 was administered intradermally to OF1 mice. A 1:1 mixture of MB11-luc2 ARNm and MB8-luc2 ARNm, along with an equal amount of CPP3-H6 at a 0.5 molar ratio, was also injected into the skin of mice to determine whether the CPP3-H6 peptide could separate from the ARN aptamer A present in MB11-luc2 ARNm after intradermal injection. Luciferase activity was measured 18 hours post-injection.

[0307] result:

[0308] As expected, injection of MB8-luc2 ARNm resulted in effective skin transfection. Co-injection of CPP3-H6, 0.7 mM MgCl2, and 5 mM Hepes with MB8-luc2 ARNm did not significantly affect transfection efficiency. Figure 11 ).

[0309] Compared to MB11-luc2 ARNm alone, the optimal amount of CPP3-H6 peptide that advantageously increases transcription efficiency by 9.2-fold corresponds to a molar ratio of 1 CPP3-H6 peptide: 2 ARNm. Figure 11 Furthermore, it showed a significantly higher luciferase activity than that obtained with MB8-luc2 ARNm, by 2.1 times, and a significantly higher luciferase activity than that obtained with conventional MB5-luc2-capped ARNm.

[0310] When one of every two ARNm cells does not bind CPP3-H6, transfection rate decreases by 12.3-fold. Figure 11 (MB8-luc2 and MB11-luc2 ARNm, 2.8 μg each). This means that after intradermal injection, the CPP3-H6 peptide can separate from the ARN aptamer A present in MB11-luc2 ARNm. If half of the ARNm lacks ARN aptamer A (MB8-luc2 ARNm), CPP3-H6 cannot effectively improve transfection.

[0311] MB11-luc2 ARNm was also incubated with CPP1-H6 peptide at different molar ratios. The optimal molar ratio was 1 CPP1-H6 peptide : 4 MB11-luc2 ARNm. Figure 12 Transfection efficiency was increased by 5.4 times compared to MB11-luc2 ARNm alone.

[0312] Therefore, CPP1-H6 performed worse than CPP3-H6. This can be explained by the copy number of each CPP receptor present on the plasma membrane of skin cells and the affinity of each CPP for its receptor.

[0313] Finally, MB11-luc2 ARNm was also incubated with CPP2-H6 in various molar ratios. The optimal molar ratio was 2 CPP2-H6 peptides : 1 MB11-luc2 ARNm ( Figure 13 Compared to MB11-Iuc2 ARNm alone, the transfection efficiency was increased by 10.6 times, and it was also higher than the transfection efficiency obtained by CPP3-H6 peptide. Therefore, luciferase expression was 22.8 times higher than that of conventional MB5-luc2 ARNm.

[0314] Example 12: Effect of CPP-H6 on the transfection efficiency of capped ARNm in the skin

[0315] Materials and methods:

[0316] To evaluate the effect of CPP (here, CPP2-H6) on the transfection efficiency of capped ARNm, aptamer A was inserted into the 5′-UTR of conventional capped MB5-luc2 ARNm to generate capped MB15-luc2 ARNm with the sequence SEQ ID NO: 70. As described in Example 11 above, MB15-luc2 ARNm was incubated with CPP2-H6 for 30 minutes in a buffer containing 0.7 mM MgCl2 and 5 mM Hepes.

[0317] Following the protocol detailed in Example 10, 5.6 μg of MB15-luc2 ARNm was administered intradermally to OF1 mice. Luciferase activity was measured 18 hours post-injection.

[0318] result:

[0319] In the absence of the CPP2-H6 peptide, capped MB15-Iuc2 ARNm showed 1.68-fold lower luciferase expression than capped MB5-luc2 ARNm.

[0320] Compared to MB15-luc2 ARNm alone, co-injection of capped MB15-luc2 ARNm and CPP2-H6 peptide at a molar ratio of 2 peptides per ARNm only increased transfection efficiency by 1.48-fold. This indicates that inserting ARN aptamer A into the 5′-UTR of a conventionally capped ARNm and attaching the CPP-H6 peptide to that ARN aptamer does not improve transfection efficiency compared to what was observed with capped ARNm MB11-luc2 (10.6-fold). Regardless of whether the capped ARNm molecule (with or without aptamer, whether or not it is attached to CPP2-H6 CPP) is used, the transfection efficiency remains significantly lower than that observed with MB8-luc2 and MB11-luc2 ARNm. Therefore, using the ARNm molecule of the present invention instead of capped ARNm molecules is highly advantageous.

[0321] Example 13: Effect of 5′-SL on the expression of in vivo luciferase in the skin

[0322] Materials and methods:

[0323] The following ARNm: According to the scheme detailed in Example 10 above, MB8-luc2, MB11-luc2, MB17-luc2, MB18-luc2 and capped MB5-luc2 were injected into the skin, and luciferase activity was measured 18 hours after injection.

[0324] result:

[0325] like Figure 15 As shown, compared to capped ARNm (MB5-luc2), the ARNm according to the invention, containing at least one xrRNA sequence and one IRES sequence (MB11-luc2), increases luciferase expression in the skin by approximately 2-fold. Surprisingly, when the stem-loop is located five nucleotides upstream of the xrRNA1 sequence (MB8-luc2 and MB18-luc2), the addition of the stem-loop (here, 5′-SL has the sequence of SEQ ID NO: 87) results in approximately 4.3-fold increase in luciferase expression in the skin compared to xrRNA and IRES sequence alone (MB11-luc2).

[0326] However, when the stem-loop is located approximately 70 nucleotides upstream of the xrRNA sequence, it does not improve efficiency, except as observed in the absence of a stem-loop. In fact, the translation efficiencies of MB11-luc2 and MB17-luc2 ARNm are similar. Surprisingly, placing aptamer A between the xrRNA sequence and the IRES sequence (MB18-luc2) improves translation efficiency by even more than MB11-luc2 and MB17-luc2 ARNm. Therefore, MB8-luc2 and MB18-luc2 ARNm have similar translation efficiencies.

[0327] in conclusion:

[0328] Compared to capped ARNm molecules, the ARNm molecules of the present invention reduce the synthesis cost by approximately 30-fold while increasing protein expression yield by at least 2-fold, preferably approximately 10-fold. Furthermore, the ARNm of the present invention is at least as stable as capped ARNm and simplifies its production via in vitro transcription due to the absence of a cap molecule or its analogue. When the ARNm encoding the HRV2 2A protease of the present invention is co-transfected with the ARNm encoding the target protein, the ARNm encoding the HRV2 2A protease of the present invention may increase the translation of the ARNm encoding the target protein. Additionally, for example, transfection efficiency in tissues (e.g., muscle or skin) can be improved by inserting a cell-penetrating ARN aptamer (CPP (linked to the aforementioned ARN aptamer)) into the 5′-UTR region of the ARNm molecule according to the present invention, and / or by adding a stem-loop to the 5′ end of the 5′-UTR region upstream of the xrRNA sequence.

[0329] References

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[0349] Tsuji S.et al., 2013, PLoS ONE 8(12):e83108. sequence list <110> Messinger Biopharmaceuticals <120> Two RNA sequences introduced at the 5' end of the messenger RNA replace the messenger RNA cap. <130> 375816D38002 <150> FR 1854052 <151> 2018-05-15 <160> 89 <170> PatentIn version 3.5 <210> 1 <211> 45 <212> RNA <213> Dengue virus type 1 <400> 1 gucaggccgg auuaagccau agcacgguaa gagcuaugcu gccug 45 <210> 2 <211> 45 <212> RNA <213> Dengue virus type 2 <400> 2 gucaggccgg auuaagccau aguacggaaa aaacuaugcu accug 45 <210> 3 <211> 45 <212> RNA <213> Dengue virus type 3 <400> 3 gucaggccac cuuaagccac aguacggaag aagcugugcu gccug 45 <210> 4 <211> 43 <212> RNA <213> CocoBella virus <400> 4 gucaggccug aaaagccacc ugauccggug aaggugcugc cug 43 <210> 5 <211> 47 <212> RNA <213> Zika virus <400> 5 gucaggccug cuagucagcc acaguuuggg gaaagcugug cagccug 47 <210> 6 <211> 50 <212> RNA <213> St. Louis encephalitis virus <400> 6 gucaggccaa ucaguuuugc caccggaugu cagguaaacg gugcugucug 50 <210> 7 <211> 50 <212> RNA <213> Japanese encephalitis virus <400> 7 gucaggccag caaaagcugc caccggauac uggguagacg gugcugucug 50 <210> 8 <211> 50 <212> RNA <213> Entaia virus <400> 8 gucaggccag gcaaugccug ccaccggaag uuggaugacg gugcugucug 50 <210> 9 <211> 50 <212> RNA <213> Usutu virus <400> 9 gucaggccag ggcaaccugc caccggaagu ugaguagacg gugcugccug 50 <210> 10 <211> 52 <212> RNA <213> Murray Valley Encephalitis Virus <400> 10 gucaggccag ccgguuaggc ugccaccgaa gguugguaga cggugcugcc ug 52 <210> 11 <211> 51 <212> RNA <213> West Nile virus <400> 11 gucaggccag auuaaugcug ccaccggaag uugaguagac ggugcugccu g 51 <210> 12 <211> 45 <212> RNA <213> Iguazu virus <400> 12 gucaggccgg aaacgccacc ggauggucgu aaacggugcg gccug 45 <210> 13 <211> 52 <212> RNA <213> Bagza virus <400> 13 gucaggccgg acuacguguc cgccaccgga uguuggauga cggugcugcc ug 52 <210> 14 <211> 52 <212> RNA <213> Israel-Turkey meningoencephalitis virus <400> 14 gucaggccgg auuauauguc cgccaccgga uguuggauga cggugcugcc ug 52 <210> 15 <211> 42 <212> RNA <213> New Mapun virus <400> 15 gucaggccga auagccaucu gauccgguga agaugcugcc ug 42 <210> 16 <211> 41 <212> RNA <213> Dengue virus type 1 <400> 16 gucaggccga aagccacggu ucgagcaagc cgugcugccu g 41 <210> 17 <211> 46 <212> RNA <213> Dengue virus type 2 <400> 17 gucaggccau cauaaaugcc auagcuugag uaaacuaugc agccug 46 <210> 18 <211> 43 <212> RNA <213> Dengue virus type 4 <400> 18 gucaggccac uugugccacg guuugagcaa accgugcugc cug 43 <210> 19 <211> 37 <212> RNA <213> CocoBella virus <400> 19 gucagauccg aaaggccacc aguuuggugc agaacug 37 <210> 20 <211> 44 <212> RNA <213> Zika virus <400> 20 gucaggccga gaacgccaug gcacggaaga agccaugcug ccug 44 <210> twenty one <211> 46 <212> RNA <213> St. Louis encephalitis virus <400> twenty one gucagaccag aaaugccacc ugaaagcaug cuaaaggugc ugucug 46 <210> twenty two <211> 44 <212> RNA <213> Japanese encephalitis virus <400> twenty two gucaggccac aaauuuggc caccccgcua gggggugcgg ccug 44 <210> twenty three <211> 43 <212> RNA <213> Entaia virus <400> twenty three gucaggccgu agguuuuacg ccacuagcau gcagugcugc cug 43 <210> twenty four <211> 41 <212> RNA <213> Usutu virus <400> twenty four gucaggccgc aaagcgccac uucgccaagg agugcagccu g 41 <210> 25 <211> 42 <212> RNA <213> Murray Valley Encephalitis Virus <400> 25 gucagaucgc gaaagcgcca cuucgccgag gagugcaauc ug 42 <210> 26 <211> 45 <212> RNA <213> West Nile virus <400> 26 gucagaccac acuuuaaugu gccacucugc ggagagugca gucug 45 <210> 27 <211> 43 <212> RNA <213> Iguazu virus <400> 27 gucaggccga aagccaccac aagcgguaca guggugcugc cug 43 <210> 28 <211> 43 <212> RNA <213> Bagza virus <400> 28 gucaggccac agguuuugg ccacuagcau gcagugcugc cug 43 <210> 29 <211> 43 <212> RNA <213> Israel-Turkey meningoencephalitis virus <400> 29 gucaggccac agguuuugg ccacuagcau gcagugcugc cug 43 <210> 30 <211> 48 <212> RNA <213> New Mapun virus <400> 30 gucagaccac uuagugccac caguaugaug auaagcuggu gcugucug 48 <210> 31 <211> 50 <212> RNA <213> Wessel Brown virus <400> 31 gucagcccau cauugaugc cauggcuaag cugugaggcc augcuggcug 50 <210> 32 <211> 49 <212> RNA <213> Illeus virus <400> 32 gucaggccau ggaaacaugc cacccaaagc uuguagaggg ugcagccug 49 <210> 33 <211> 51 <212> RNA <213> Sepik virus <400> 33 gucagcccgu cauaaugacg ccauggcuaa gcugugaggc caugcuggcu g 51 <210> 34 <211> 45 <212> RNA <213> Busuqua virus <400> 34 gucaggccag aaaugccacc gguaaaaggu agacggugcu gccug 45 <210> 35 <211> 50 <212> RNA <213> Tembusu virus <400> 35 gucaggccag ggaaucccug ccaccggaug uuggaugacg gugcugucug 50 <210> 36 <211> 44 <212> RNA <213> Chaoyang virus <400> 36 gucaggccua aaugccaccg gaugauagua gacggugcug ccug 44 <210> 37 <211> 50 <212> RNA <213> Kedu virus <400> 37 gucaggccac ucgugagagu gccacaguac gguaaagacu gugcggccug 50 <210> 38 <211> 53 <212> RNA <213> Yokose virus <400> 38 gucaggccaa gauugagaaa aucuugccac agcuuggcag acuugcagc cug 53 <210> 39 <211> 51 <212> RNA <213> Donggang virus <400> 39 gucaggccuc acgaauguga gccaccggau gggacuagac ggugcugccu g 51 <210> 40 <211> 59 <212> RNA <213> Yellow fever virus <400> 40 gucagcccag aaccccacac gaguuuugcc acugcuaagc ugagaggcag ugcaggcug 59 <210> 41 <211> 48 <212> RNA <213> Rossio virus <400> 41 gucaggccgu ccuuggacgc cacccaaagc augggagggu gcugccug 48 <210> 42 <211> 49 <212> RNA <213> Alfredo <400> 42 gucaggccag ugaaaacugc caccggaugu ugguagacgg ugcugccug 49 <210> 43 <211> 43 <212> RNA <213> Stratford virus <400> 43 gucaggccug aaaagccauc ugauccggug aagaugcugc cug 43 <210> 44 <211> 50 <212> RNA <213> Duck Egg Drop Syndrome Virus <400> 44 gucaggccag ggaaucccug ccaccggaug uuggaugacg gugcugucug 50 <210> 45 <211> 547 <212> DNA <213> Encephalomyocarditis virus <400> 45 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac 60 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag 120 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa 180 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag 240 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga 300 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag 360 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc 420 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag 480 gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga 540 tgataat 547 <210> 46 <211> 17 <212> DNA <213> Bacteriophage T7 <400> 46 taatacgact cactata 17 <210> 47 <211> 83 <212> RNA <213> Artificial sequence <220> <223> Spacer 1 between xrRNA sequences from pMB8-luc2 and pMB9-luc2 RNA <400> 47 gaccaaagcu gcgaggugau ccacguaagc ccucagaacc gucucggaag gaggacccca 60 cgugcuuuag ccucaaagcc cag 83 <210> 48 <211> 17 <212> RNA <213> Artificial sequence <220> <223> Spacer 2 between xrRNA2 and IRES sequences in RNA from pMB8-luc2 and pMB9-luc2 <400> 48 uaacaaaggc aaaacau 17 <210> 49 <211> 63 <212> DNA <213> Artificial sequence <220> <223> 5'UTR pMB5-luc2 (without T7 promoter) <400> 49 gggaagctta agtgttcttt ttgcagaagc tcagaataaa cgctcaactt tggcagatct 60 acc 63 <210> 50 <211> 818 <212> DNA <213> Artificial sequence <220> <223> 5'UTR of pMB8-luc2 and pMB9-luc2 <400> 50 gggaagctta agcttcccaa aaaagtcagg ccagattaat gctgccaccg gaagttgagt 60 agacggtgct gcctgcggct caaccccagg aggactgggt gaccaaagct gcgaggtgat 120 ccacgtaagc cctcagaacc gtctcggaag gaggacccca cgtgctttag cctcaaagcc 180 cagtgtcaga ccacacttta atgtgccact ctgcggagag tgcagtctgc gatagtgccc 240 caggtggact gggttaacaa aggcaaaaca ttactggccg aagccgcttg gaataaggcc 300 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 360 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 420 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 480 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt 540 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 600 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 660 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 720 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca 780 A cggggacgtg gttttccttt gaaaaacacg atgataat 818 <210> 51 <211> 583 <212> DNA <213> Artificial Sequence <220> <223> 5' UTR of pMB7-luc2 (without T7 promoter) <400> 51 gggaagctta agcttccctt aacaaaggca aaacattact ggccgaagcc gcttggaata 60 aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 120 gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 180 cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 240 ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 300 caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 360 ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 420 attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatggggat ctgatctggg 480 gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg 540 aaccacgggg acgtggtttt cctttgaaaa acacgatgat aat 583 <210> 52 <211> 44 <212> DNA <213> Artificial sequence <220> <223> The 3'-UTR of pMB5-luc2, pMB7-luc2, and pMB8-luc2 lacks a poly A tail or restriction site. <400> 52 ttctagaatg tccgaatggt tgacacttga tctcggcaac gcat 44 <210> 53 <211> 109 <212> DNA <213> Artificial sequence <220> <223> The 3'-UTR of pMB5-luc2, pMB7-luc2, and pMB8-luc2 has no restriction sites. <400> 53 ttctagaatg tccgaatggt tgacacttga tctcggcaac gcataaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 109 <210> 54 <211> 113 <212> DNA <213> Artificial sequence <220> <223> Complete 3'-UTR of pMB5-luc2, pMB7-luc2 and pMB8-luc2 (with SspI restriction site) <400> 54 ttctagaatg tccgaatggt tgacacttga tctcggcaac gcataaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaat att 113 <210> 55 <211> 625 <212> DNA <213> Kunjin virus <400> 55 aatactttgt taattgtaaa taaatattgt tattatgtgt agaagtttag ctttataata 60 gtgtttagtg tgtttagagt tagaaaaatt ttagtgagga agtcaggccg gaaaattccc 120 gccaccggaa gttgagtaga cggtgctgcc tgcgactcaa ccccaggagg actgggtgaa 180 caaagctgcg aagtgatcca tgtaagccct cagaaccgtc tcggaaagag gaccccacat 240 gttgtagctt caaggcccaa tgtcagacca cgccatggcg tgccactctg cggagagtgc agtctgcgac agtgccccag gaggactggg tgaacaaagg cgaatcaacg tcccacgcgg ccctagctct ggcaatggtg ttaaccagag tgaaaggact agaggttaga ggagaccccg cgttctgaag tgcacggccc agcctggctg aagctgtagg tcaggggaag gactagaggt 480 cccgtgccgc cccgtgccgc aaaacaccac aacaacacag catattgaca cctgggatag actaggagat cttctgctct gcacaaccag ccacacggca cagtgcgccg acaatggtgg ctggtggtgc gagacacag gatct <210> 56 <211> 718 <212> DNA <213> The snowstorm <220> <223> 3'UTR (unlocked)‐Eco53kI(pMB9‐luc2) <400> 56 ttctgaata ctttgttaat tgtaaataaa tattgttatt atgtgtagaa gtttagcttt father ttagtgtgtt taggttag aaaattttag tgaggaagtc aggccggaa attcccgcca ccggaagttg agtagcggt gctgcctgcg actcaacccc aggaggactg 180 ggtgaacaaa gctgcgaagt gatccatgta agccctcaga accgtctcgg aaagaggacc 240 ccacatgttg tagcttcaag gcccaatgtc agaccacgcc atggcgtgcc actctgcgga 300 gagtgcagtc tgcgacagtg ccccaggagg actgggtgaa caaaggcgaa tcaacgtccc 360 acgcggccct agctctggca atggtgttaa ccagagtgaa aggactagag gttagaggag 420 accccgcgtt ctgaagtgca cggcccagcc tggctgaagc tgtaggtcag gggaaggact 480 agaggttagt ggagaccccg tgccgcaaaa caccacaaca acacagcata ttgacacctg 540 ggatagacta ggagatcttc tgctctgcac aaccagccac acggcacagt gcgccgacaa 600 tggtggctgg tggtgcgaga acacaggatc tgggtcggca tggcatctcc acctcctcgc 660 ggtccgacct gggcatccga aggaggacgc acgtccactc ggatggctaa gggagctc 718 <210> 57 <211> 63 <212> RNA <213> Artificial sequence <220> <223> 5'-UTR of RNA from MB5-luc2 <400> 57 gggaagcuua aguguucuuu uugcagaagc ucagaauaaa cgcucaacuu uggcagaucu 60 acc 63 <210> 58 <211> 583 <212> RNA <213> Artificial sequence <220> <223> 5'-UTR of RNA from MB7-luc2 <400> 58 gggaagcuua agcuucccuu aacaaaggca aaacauuacu ggccgaagcc gcuuggaaua 60 aggccggugu gcguuugucu auauguuauu uuccaccaua uugccgucuu uuggcaaugu 120 gagggcccgg aaaccuggcc cugucuucuu gacgagcauu ccuagggguc uuuccccucu 180 cgccaaagga augcaagguc uguugaaugu cgugaaggaa gcaguuccuc uggaagcuuc 240 uugaagacaa acaacgucug uagcgacccu uugcaggcag cggaaccccc caccuggcga 300 caggugccuc ugcggccaaa agccacgugu auaagauaca ccugcaaagg cggcacaacc 360 ccagugccac guugugaguu ggauaguugu ggaaagaguc aaauggcucu ccucaagcgu 420 auucaacaag gggcugaagg augcccagaa gguaccccau uguaugggau cugaucuggg 480 gccucggugc acaugcuuua cauguguuua gucgagguua aaaaacgucu aggccccccg 540 aaccacgggg acgugguuuu ccuuugaaaa acacgaugau aau 583 <210> 59 <211> 818 <212> RNA <213> Artificial sequence <220> <223> 5'UTR of pMB8-luc2 and pMB9-luc2 <400> 59 gggaagcuua agcuucccaa aaaagucagg ccagauuaau gcugccaccg gaaguugagu 60 agacggugcu gccugcggcu caaccccagg aggacugggu gaccaaagcu gcgaggugau 120 ccacguaagc ccucagaacc gucucggaag gaggacccca cgugcuuuag ccucaaagcc 180 cagugucaga ccacacuuua augugccacu cugcggagag ugcagucugc gauagugccc 240 cagguggacu ggguuaacaa aggcaaaaca uuacuggccg aagccgcuug gaauaaggcc 300 ggugugcguu ugucuauaug uuauuuucca ccauauugcc gucuuuuggc aaugugaggg 360 cccggaaacc uggcccuguc uucuugacga gcauuccuag gggucuuucc ccucucgcca 420 aaggaaugca aggucuguug aaugucguga aggaagcagu uccucuggaa gcuucuugaa 480 gacaaacaac gucuguagcg acccuuugca ggcagcggaa ccccccaccu ggcgacaggu 540 gccucugcgg ccaaaagcca cguguauaag auacaccugc aaaggcggca caaccccagu 600 gccacguugu gaguuggaua guuguggaaa gagucaaaug gcucuccuca agcguauuca 660 acaaggggcu gaaggaugcc cagaagguac cccauuguau gggaucugau cuggggccuc 720 ggugcacaug cuuuacaugu guuuagucga gguuaaaaaa cgucuaggcc ccccgaacca 780 cggggacgug guuuuccuuu gaaaaacacg augauaau 818 <210> 60 <211> 110 <212> RNA <213> Artificial sequence <220> <223> 3'-UTR of the RNA of MB5-luc2, MB7-luc2, MB8-luc2 <400> 60 uucuagaaug uccgaauggu ugacacuuga ucucggcaac gcauaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaau 110 <210> 61 <211> 715 <212> RNA <213> Artificial sequence <220> <223> 3'-UTR of the RNA of MB9-luc2 <400> 61 uucuagaaua cuuuguuaau uguaaauaaa uauuguuauu auguguagaa guuuagcuuu 60 auaauagugu uuaguguguu uagaguuaga aaaauuuuag ugaggaaguc aggccggaaa 120 auucccgcca ccggaaguug aguagacggu gcugccugcg acucaacccc aggaggacug 180 ggugaacaaa gcugcgaagu gauccaugua agcccucaga accgucucgg aaagaggacc 240 ccacauguug uagcuucaag gcccaauguc agaccacgcc auggcgugcc acucugcgga 300 gagugcaguc ugcgacagug ccccaggagg acugggugaa caaaggcgaa ucaacguccc 360 acgcggcccu agcucuggca augguguuaa ccagagugaa aggacuagag guuagaggag 420 accccgcguu cugaagugca cggcccagcc uggcugaagc uguaggucag gggaaggacu 480 agagguuagu ggagaccccg ugccgcaaaa caccacaaca acacagcaua uugacaccug 540 ggauagacua ggagaucuuc ugcucugcac aaccagccac acggcacagu gcgccgacaa 600 ugguggcugg uggugcgaga acacaggauc ugggucggca uggcaucucc accuccucgc 660 gguccgaccu gggcauccga aggaggacgc acguccacuc ggauggcuaa gggag 715 <210> 62 <211> 1656 <212> DNA <213> Artificial sequence <220> <223> luc2 <400> 62 atggaagatg ccaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120 gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180 gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240 tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420 aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540 ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600 agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660 catgcccgg accccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780 cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840 aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900 atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960 aaagggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020 ggcctgacag aaaaaccag cgccattctg atcaccccccg aaggggacga caagcctggc 1080 gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140 acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200 tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380 caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440 cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560 gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620 aaggccaaga agggcggcaa gatcgccgtg taataa 1656 <210> 63 <211> 1656 <212> RNA <213> Artificial Sequence <220> <223> luc2 <400> 63 auggaagaug ccaaaaacau uaagaagggc ccagcgccau ucuacccacu cgaagacggg 60 accgccggcg agcagcugca caaagccaug aagcgcuacg cccuggugcc cggcaccauc 120 gccuuuaccg acgcacauau cgagguggac auuaccuacg ccgaguacuu cgagaugagc 180 guucggcugg cagaagcuau gaagcgcuau gggcugaaua caaaccaucg gaucguggug 240 ugcagcgaga auagcuugca guucuucaug cccguguugg gugcccuguu caucggugug 300 gcuguggccc cagcuaacga caucuacaac gagcgcgagc ugcugaacag caugggcauc 360 agccagccca ccgucguauu cgugagcaag aaagggcugc aaaagauccu caacgugcaa 420 480 uuccaaagca ugacaaccuu cgugacuucc cauuugccac ccggcuucaa cgaguacgac 540 uucgugcccg agagcuucga ccgggacaaa accaucgccc ugaucaugaa caguaguggc 600 aguaccggau ugcccaaggg cguagcccua ccgcaccgca ccccuugugu ccgauucagu 660 caugcccgcg accccaucuu cggcaaccag aucaucccg acaccgcuau ccucagcgug 720 gugccauuuc accacggcuu cggcauguuc accacgcugg gcuacuugau cugcggcuuu 780 840 aagauucaau cugcccugcu ggugcccaca cuauuuagcu ucuucgcuaa gagcacucuc 900 aucgacaagu agaccuaag caacuugcac gagaucgcca gcggcggggc gccgcucagc 960 aaggagguag gugaggccgu ggccaaacgc uuccaccuac caggcauccg ccagggcuac 1020 ggccugacag aaacaaccag cgccauucug aucaccccg aaggggacga caagccuggc 1080 gcaguaggca agguggugcc cuucuucgag gcuaaggugg uggacuugga caccgguaag 1140 acacugggug ugaaccagcg cggcgagcug ugcguccgug gccccaugau caugagcggc 1200 uacguuaaca accccgaggc uacaaacgcu cucaucgaca aggacggcug gcugcacagc 1260 ggcgacaucg ccuacuggga cgaggacgag cacuucuuca ucguggaccg gcugaagagc 1320 cugaucaaau acaagggcua ccagguagcc ccagccgaac uggagagcau ccugcugcaa 1380 caccccaaca ucuucgacgc cggggucgcc ggccugcccg acgacgaugc cggcgagcug 1440<00​​​​​​​​​​​​​​​​​​​​​​​ <210> 65 <211> 32 <212> RNA <213> Artificial sequence <220> <223> Aptamer B <400> 65 gggaggacga ugcggacaga cgacucgccc ga 32 <210> 66 <211> 36 <212> RNA <213> Artificial sequence <220> <223> Aptamer C <400> 66 gggaggacga ugcggacgug cagacgacuc gcccga 36 <210> 67 <211> 869 <212> RNA <213> Artificial sequence <220> <223> 5'-UTR of RNA from MB11-luc2 <400> 67 gggaagcuua aguggugagg uauauuggcg ccuucgugga augucagugc cucaccagca 60 ugccuuccca aaaaagucag gccagauuaa ugcugccacc ggaaguugag uagacggugc 120 ugccugcggc ucaaccccag gaggacuggg ugaccaaagc ugcgagguga uccacguaag 180 cccucagaac cgucucggaa ggaggacccc acgugcuuua gccucaaagc ccagugucag 240 accacacuuu aaugugccac ucugcggaga gugcagucug cgauagugcc ccagguggac 300 uggguuaaca aaggcaaaac auuacuggcc gaagccgcuu ggaauaaggc cggugugcgu 360 uugucuauau guuauuuucc accauauugc cgucuuuugg caaugugagg gcccggaaac 420 cuggcccugu cuucuugacg agcauuccua ggggucuuuc cccucucgcc aaaggaaugc 480 aaggucuguu gaaugucgug aaggaagcag uuccucugga agcuucuuga agacaaacaa 540 cgucuguagc gacccuuugc aggcagcgga accccccacc uggcgacagg ugccucugcg 600 gccaaaagcc acguguauaa gauacaccug caaaggcggc acaaccccag ugccacguug 660 ugaguuggau aguuguggaa agagucaaau ggcucuccuc aagcguauuc aacaaggggc 720 ugaaggaugc ccagaaggua ccccauugua ugggaucuga ucuggggccu cggugcacau 780 gcuuuacaug uguuuagucg agguuaaaaa acgucuaggc cccccgaacc acggggacgu 840 gguuuuccuu ugaaaaacac gaugauaau 869 <210> 68 <211> 874 <212> RNA <213> Artificial Sequence <220> <223> 5'-UTR of RNA from MB13-luc2 <400> 68 gggaagcuua agcuucccaa aaaagggagg acgaugcgga cagacgacuc gcccgaaaaa 60 aaucuagagc augcaaaaaa gucaggccag auuaaugcug ccaccggaag uugaguagac 120 ggugcugccu gcggcucaac cccaggagga cugggugacc aaagcugcga ggugauccac 180 guaagcccuc agaaccgucu cggaagggagg accccacgug cuuuagccuc aaagcccagu 240 gucagaccac acuuuaaugu gccacucugc ggagagugca gucugcgaua gugccccagg 300 uggacugggu uaacaaaggc aaaacauuac uggccgaagc cgcuuggaau aaggccggug 360 ugcguuuguc uauauguuau uuuccaccau auugccgucu uuuggcaaug uggggcccg 420 gaaaccuggc ccugucuucu ugacgagcau uccuaggggu cuuuccccuc ucgccaaagg 480 aaugcaaggu cuguugaaug ucgugaagga agcaguuccu cuggaagcuu cuugaagaca 540 aacaacgucu guagcgaccc uuugcaggca gcggaacccc ccaccuggcg acaggugccu 600 cugcggccaa aagccacgug uauaagauac accugcaaag gcggcacaac cccagugcca 660 cguugugagu uggauaguug uggaaagagu caaauggcuc uccucaagcg uauucaacaa 720 ggggcugaag gaugcccaga agguacccca uuguauggga ucugaucugg ggccucggug 780 cacaugcuuu acauguguuu agucgagguu aaaaaacguc uaggcccccc gaaccacggg 840 gacgugguuu uccuuugaaa aacacgauga uaau 874 <210> 69 <211> 878 <212> RNA <213> Artificial sequence <220> <223> 5'-UTR of RNA from MB14-luc2 <400> 69 gggaagcuua agcuucccaa aaaagggagg acgaugcgga cgugcagacg acucgcccga 60 aaaaaaucua gagcaugcaa aaaagucagg ccagauuaau gcugccaccg gaaguugagu 120 agacggugcu gccugcggcu caaccccagg aggacugggu gaccaaagcu gcgaggugau 180 ccacguaagc ccucagaacc gucucggaag gaggacccca cgugcuuuag ccucaaagcc 240 cagugucaga ccacacuuua augugccacu cugcggagag ugcagucugc gauagugccc 300 cagguggacu ggguuaacaa aggcaaaaca uuacuggccg aagccgcuug gaauaaggcc 360 ggugugcguu ugucuauaug uuauuuucca ccauauugcc gucuuuuggc aaugugaggg 420 cccggaaacc uggcccuguc uucuugacga gcauuccuag gggucuuucc ccucucgcca 480 aaggaaugca aggucuguug aaugucguga aggaagcagu uccucuggaa gcuucuugaa 540 gacaaacaac gucuguagcg acccuuugca ggcagcggaa ccccccaccu ggcgacaggu 600 gccucugcgg ccaaaagcca cguguauaag auacaccugc aaaggcggca caaccccagu 660 gccacguugu gaguuggaua guuguggaaa gagucaaaug gcucuccuca agcguauuca 720 acaaggggcu gaaggaugcc cagaagguac cccauuguau gggaucugau cuggggccuc 780 ggugcacaug cuuuacaugu guuuagucga gguuaaaaaa cgucuaggcc ccccgaacca 840 cggggacgug guuuuccuuu gaaaaacacg augauaau 878 <210> 70 <211> 114 <212> RNA <213> Artificial Sequence <220> <223> 5'-UTR of Capped MB15-luc2 RNA <400> 70[[ID=二十九]]<00014八十四>gggaagcuua aguguucuuu uugcagaagc ucagaauugg ugagguauau uggcgccuuc 60 guggaauguc agugccucac cagcaugcaa acgcucaacu uuggcagauc uacc 114 <210> 71 <211> 869 <212> DNA <213> Artificial Sequence <220> <223> 5'-UTR of MB11-luc2 <400> 71 gggaagctta agtggtgagg tatattggcg ccttcgtgga atgtcagtgc ctcaccagca 60 tgccttccca aaaaagtcag gccagattaa tgctgccacc ggaagttgag tagacggtgc 120 tgcctgcggc tcaaccccag gaggactggg tgaccaaagc tgcgaggtga tccacgtaag 180 ccctcagaac cgtctcggaa ggaggacccc acgtgcttta gcctcaaagc ccagtgtcag 240 accacacttt aatgtgccac tctgcggaga gtgcagtctg cgatagtgcc ccaggtggac 300 tgggttaaca aaggcaaaac attactggcc gaagccgctt ggaataaggc cggtgtgcgt 360 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac 420 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc 480 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa 540 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg 600 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg 660 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc 720 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat 780 gctttacatg tgtttagtcg aggttaaaaa acgtctaggc cccccgaacc acggggacgt 840 ggttttcctt tgaaaaacac gatgataat 869 <210> 72 <211> 874 <212> DNA <213> Artificial sequence <220> <223> 5'-UTR of MB13-luc2 <400> 72 gggaagctta agcttcccaa aaaagggagg acgatgcgga cagacgactc gcccgaaaaa 60 aatctagagc atgcaaaaaa gtcaggccag attaatgctg ccaccggaag ttgagtagac 120 ggtgctgcct gcggctcaac cccaggagga ctgggtgacc aaagctgcga ggtgatccac 180 gtaagccctc agaaccgtct cggaaggagg accccacgtg ctttagcctc aaagcccagt 240 gtcagaccac actttaatgt gccactctgc ggagagtgca gtctgcgata gtgccccagg 300 tggactgggt taacaaaggc aaaacattac tggccgaagc cgcttggaat aaggccggtg 360 tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 420 gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 480 aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 540 aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 600 ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 660 cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 720 ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 780 cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc gaaccacggg 840 gacgtggttt tcctttgaaa aacacgatga taat 874 <210> 73 <211> 878 <212> DNA <213> Artificial sequence <220> <223> 5'-UTR of MB14-luc2 <400> 73 gggaagctta agcttcccaa aaaagggagg acgatgcgga cgtgcagacg actcgccccga 60 aaaaaatcta gagcatgcaa aaaagtcagg ccagattaat gctgccaccg gaagttgagt 120 agacggtgct gcctgcggct caaccccagg aggactgggt gaccaaagct gcgaggtgat 180 ccacgtaagc cctcagaacc gtctcggaag gaggaccca cgtgctttag cctcaaagcc 240 cagtgtcaga ccacacttta atgtgccact ctgcggagag tgcagtctgc gatagtgccc 300 caggtggact gggttaacaa aggcaaaaca ttactggccg aagccgcttg gaataaggcc 360 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg 420 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca 480 aaaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 540 gaaaaac gtctgtagcg accctttgca ggcagcgggaa ccccccacct ggcgacaggt 600 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt 660 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca 720 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc 780 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca 840 cggggacgtg gttttccttt gaaaaacacg atgataat 878 <210> 74 <211> 114 <212> DNA <213> Artificial sequence <220> <223> 5'-UTR of capped MB15-luc2 <400> 74 gggaagctta agtgttcttt ttgcagaagc tcagaattgg tgaggtatat tggcgccttc 60 gtggaatgtc agtgcctcac cagcatgcaa acgctcaact ttggcagatc tacc 114 <210> 75 <211> 40 <212> PRT <213> Artificial sequence <220> <223> M12-H6 <400> 75 Arg Arg Gln Pro Pro Arg Ser Ile Ser Ser His Pro Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 Gly Gly His His His His His His 35 40 <210> 76 <211> 40 <212> PRT <213> Artificial sequence <220> <223> CPP1-H6 <400> 76 Pro Gln Arg Asp Thr Val Gly Gly Arg Thr Thr Pro Pro Ser Trp Gly 1 5 10 15 Pro Ala Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly His His His His His His 35 40 <210> 77 <211> 40 <212> PRT <213> Artificial sequence <220> <223> CPP2-H6 <400> 77 Gly Pro Phe His Phe Tyr Gln Phe Leu Phe Pro Pro Val Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly His His His His His His 35 40 <210> 78 <211> 40 <212> PRT <213> Artificial sequence <220> <223> CPP3-H6 <400> 78 Gly Ser Pro Trp Gly Leu Gln His His Pro Pro Arg Thr Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30 Ser Gly His His His His His His 35 40 <210> 79 <211> 462 <212> RNA <213> Human rhinovirus 2 <400> 79 auggugacaa ggcccaucau cacaacagcc ggcccuuccg auauguacgu gcacgugggc 60 aaccugaucu acaggaaccu gcaccuguuc aacagcgaga ugcacgagag cauccuggug 120 agcuacagca gcgaucugau caucuacaga accaacacag ugggcgacga uuacaucccc 180 agcugugacu gcacacaggc caccuacuac ugcaagcaca agaacagaua cuucccuauc 240 acagugacaa gccacgauug guacgagauc caggagagcg aguacuaccc uaagcacauc 300 caguacaacc ugcugaucgg cgagggcccu ugcgagcccg gagacugugg cggaaagcug 360 cuguguaagc acggcgugau cggcaucgug accgccggcg gagauaacca cguggccuuc 420 aucgaccuga ggcacuucca cugcgccgag gagcaguaau aa 462 <210> 80 <211> 1390 <212> RNA <213> Artificial Sequence <220> <223> MB8-2Apro mRNA <400> 80 gggaagcuua agcuucccaa aaaagucagg ccagauuaau gcugccaccg gaaguugagu 60 agacggugcu gccugcggcu caaccccagg aggacugggu gaccaaagcu gcgaggugau 120 ccacguaagc ccucagaacc gucucggaag gaggacccca cgugcuuuag ccucaaagcc 180 cagugucaga ccacacuuua augugccacu cugcggagag ugcagucugc gauagugccc 240 cagguggacu ggguuaacaa aggcaaaaca uuacuggccg aagccgcuug gaauaaggcc 300 ggugugcguu ugucuauaug uuauuuucca ccauauugcc gucuuuuggc aaugugaggg 360 cccggaaacc uggcccuguc uucuugacga gcauuccuag gggucuuucc ccucucgcca 420 aaggaaugca aggucuguug aaugucguga aggaagcagu uccucuggaa gcuucuugaa 480 gacaaacaac gucuguagcg acccuuugca ggcagcggaa ccccccaccu ggcgacaggu 540 gccucugcgg ccaaaagcca cguguauaag auacaccugc aaaggcggca caaccccagu 600 gccacguugu gaguuggaua guuguggaaa gagucaaaug gcucuccuca agcguauuca 660 acaaggggcu gaaggaugcc cagaagguac cccauuguau gggaucugau cuggggccuc 720 ggugcacaug cuuacaugu guuuagucga gguuaaaaa cgucuaggcc ccccgaacca 780 cggggacgug guuuuccuuu gaaaaacacg augauaauau ggugacaagg cccaucauca 840 icacagccgg cccuuccgau auguacgugc acgugggca ccugaucuac aggaccugc 900 accuguaca cagcgagaug accugugag accugucag accucagc gaucugauca 960 ucuacagaac cacacagug ggcgacgauu acacccag cugugacugc acacaggcca 1020 cuacuacu cuckacacag cucuacuacu cucuacuc agugacaagc cucuauggu 1080 acgagaucca ggagagcgag uacuacccua agcacaucca guacaaccug cugaucggcg 1140 agggcccuug cgagcccgga gacuguggcg gaagcugcu guguaagcac ggcgugaucg 1200 gcaucgugac cgccggcgga gauaaccacg uggccuucau cgaccugagg cacuuccacu 1260 gcgccgagga gcaguaauaa ucuagaaug uccgaauggu ogacacuuga ucucggcaac 1320 gcauaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1380 aaaaaaaaau 1390 <210> 81 <211> 143 <212> PRT <213> Human rhinovirus 2 <400> 81 Met Gly Pro Ser Asp Met Tyr Val His Val Gly Asn Leu Ile Tyr Arg 1 5 10 15 Asn Leu His Leu Phe Asn Ser Glu Met His Glu Ser Ile Leu Val Ser 20 25 30 Tyr Ser Ser Asp Leu Ile Ile Tyr Arg Thr Asn Thr Val Gly Asp Asp 35 40 45 Tyr Ile Pro Ser Cys Asp Cys Thr Gln Ala Thr Tyr Tyr Cys Lys His 50 55 60 Lys Asn Arg Tyr Phe Pro Ile Thr Val Thr Ser His Asp Trp Tyr Glu 65 70 75 80 Ile Gln Glu Ser Glu Tyr Tyr Pro Lys His Ile Gln Tyr Asn Leu Leu 85 90 95 Ile Gly Glu Gly Pro Cys Glu Pro Gly Asp Cys Gly Gly Lys Leu Leu 100 105 110 Cys Lys His Gly Val Ile Gly Ile Val Thr Ala Gly Gly Asp Asn His[[ID=4l]] 115 120 125 Val Ala Phe Ile Asp Leu Arg His Phe His Cys Ala Glu Glu Gln 130 135 140 <210> 82 <211> 547 <212> RNA <213> encephalomyocarditis virus <400> 82 uacuggccga agccgcuugg aauaaggccg gugugcguuu gucuauaugu uauuuuccac 60 cauauugccg ucuuuuggca augugagggc ccggaaaccu ggcccugucu ucuugacgag 120 cauuccuagg ggucuuuccc cucucgccaa aggaaugcaa ggucuguuga augucgugaa 180 ggaagcaguu ccucuggaag cuucuugaag acaaacaacg ucuguagcga cccuuugcag 240 gcagcggaac cccccaccug gcgacaggug ccucugcggc caaaagccac guguauaaga 300 uacaccugca aaggcggcac aaccccagug ccacguugug aguuggauag uuguggaaag 360 agucaaaugg cucuccucaa gcguauucaa caaggggcug aaggaugccc agaagguacc 420 ccauuguaug ggaucugauc uggggccucg gugcacaugc uuuacaugug uuuagucgag 480 guuaaaaaac gucuaggccc cccgaaccac ggggacgugg uuuuccuuug aaaaacacga 540 ugauaau 547 <210> 83 <211> 885 <212> RNA <213> artificial sequence <220> <223> 5' UTR of MB17-luc2 RNA <400> 83 gggaagcuua agcuucccac aaaaaaucgg uauauuggcg ccuucgugga augucagugc 60 cucaacaucu agagcaugcc uucccaaaaa agucaggcca gauuaaugcu gccaccggaa 120 guugaguaga cggugcugcc ugcggcucaa ccccaggagg acugggugac caaagcugcg 180 aggugaucca cguaagcccu cagaaccguc ucggaaggag gaccccacgu gcuuuagccu 240 caaagcccag ugucagacca cacuuuaaug ugccacucug cggagagugc agucugcgau 300 agugccccag guggacuggg uuaacaaagg caaaacauua cuggccgaag ccgcuuggaa 360 uaaggccggu gugcguuugu cuauauguua uuuuccacca uauugccguc uuuuggcaau 420 gugagggccc ggaaaccugg cccugucuuc uugacgagca uuccuagggg ucuuuccccu 480 cucgccaaag gaaugcaagg ucuguugaau gucgugaagg aagcaguucc ucuggaagcu 540 ucuugaagac aaacaacguc uguagcgacc cuuugcaggc agcggaaccc cccaccuggc 600 gacaggugcc ucugcggcca aaagccacgu guauaagaua caccugcaaa ggcggcacaa 660 ccccagugcc acguugugag uuggauaguu guggaaagag ucaaauggcu cuccucaagc 720 guauucaaca aggggcugaa ggaugcccag aagguacccc auuguauggg aucugaucug 780 gggccucggu gcacaugcuu uacauguguu uagucgaggu uaaaaaacgu cuaggccccc 840 cgaaccacgg ggacgugguu uuccuuugaa aaacacgaug auaau 885 <210> 84 <211> 873 <212> RNA <213> Artificial sequence <220> <223> 5' UTR of MB18-luc2 RNA <400> 84 gggaagcuua agcuucccaa aaaagucagg ccagauuaau gcugccaccg gaaguugagu 60 agacggugcu gccugcggcu caaccccagg aggacugggu gaccaaagcu gcgaggugau 120 ccacguaagc ccucagaacc gucucggaag gaggacccca cgugcuuuag ccucaaagcc 180 cagugucaga ccacacuuua augugccacu cugcggagag ugcagucugc gauagugccc 240 cagguggacu ggguuaacaa aggcaaaaca uggugaggua uauuggcgcc uucguggaau 300 gucagugccu caccuaagca ugccguuacu ggccgaagcc gcuuggaaua aggccggugu 360 gcguuugucu auauguuauu uuccaccaua uugccgucuu uuggcaaugu gagggcccgg 420 aaaccuggcc cugucuucuu gacgagcauu ccuagggguc uuuccccucu cgccaaagga 480 augcaagguc uguugaaugu cgugaaggaa gcaguuccuc uggaagcuuc uugaagacaa 540 acaacgucug uagcgacccu uugcaggcag cggaaccccc caccuggcga caggugccuc 600 ugcggccaaa agccacgugu auaagauaca ccugcaaagg cggcacaacc ccagugccac 660 guugugaguu ggauaguugu ggaaagaguc aaauggcucu ccucaagcgu auucaacaag 720 gggcugaagg augcccagaa gguaccccau uguaugggau cugaucuggg gccucggugc 780 acaugcuuua cauguguuua gucgagguua aaaaacgucu aggccccccg aaccacgggg 840 acgugguuuu ccuuugaaaa acacgaugau aau 873 <210> 85 <211> 885 <212> DNA <213> Artificial sequence <220> <223> 5' UTR of MB17-luc2 <400> 85 gggaagctta agcttcccac aaaaaatcgg tatattggcg ccttcgtgga atgtcagtgc 60 ctcaacatct agagcatgcc ttcccaaaaa agtcaggcca gattaatgct gccaccggaa 120 gttgagtaga cggtgctgcc tgcggctcaa ccccaggagg actgggtgac caaagctgcg 180 aggtgatcca cgtaagccct cagaaccgtc tcggaaggag gaccccacgt gctttagcct 240 caaagcccag tgtcagacca cactttaatg tgccactctg cggagagtgc agtctgcgat 300 agtgccccag gtggactggg ttaacaaagg caaaacatta ctggccgaag ccgcttggaa 360 taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat 420 gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct 480 ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct 540 tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc 600 gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa 660 ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc 720 gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg 780 gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc 840 cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataat 885 <210> 86 <211> 873 <212> DNA <213> Artificial sequence <220> <223> 5' UTR of MB18-luc2 <400> 86 gggaagctta agcttcccaa aaaagtcagg ccagattaat gctgccaccg gaagttgagt 60 agacggtgct gcctgcggct caaccccagg aggactgggt gaccaaagct gcgaggtgat 120 ccacgtaagc cctcagaacc gtctcggaag gaggacccca cgtgctttag cctcaaagcc 180 cagtgtcaga ccacacttta atgtgccact ctgcggagag tgcagtctgc gatagtgccc 240 caggtggact gggttaacaa aggcaaaaca tggtgaggta tattggcgcc ttcgtggaat 300 gtcagtgcct cacctaagca tgccgttact ggccgaagcc gcttggaata aggccggtgt 360 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg 420 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga 480 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa 540 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc 600 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac 660 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag 720 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc 780 acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg aaccacgggg 840 acgtggtttt cctttgaaaa acacgatgat aat 873 <210> 87 <211> 18 <212> RNA <213> Artificial sequence <220> <223> 5'-SL <400> 87 gggaagcuua agcuuccc 18 <210> 88 <211> 10 <212> RNA <213> Artificial sequence <220> <223> pepMB1 <400> 88 crrrrrrrrc 10 <210> 89 <211> 23 <212> RNA <213> Artificial sequence <220> <223> A common sequence, where the "N" at position 9 corresponds to 7 to 19 nucleotides in any sequence, and The "N" at position 14 corresponds to 12 to 19 nucleotides in any sequence. <220> <221> misc_feature <222> (9)..(9) <223> n is a, c, g, or u <220> <221> misc_feature <222> (14)..(14) <223> n is a, c, g, or u <220> <221> misc_feature <222> (18)..(18) <223> n is a, c, g, or u <400> 89 gucagrycng ccanugcnry cug 23

Claims

1. An mRNA molecule lacking a cap molecule, comprising from 5' to 3': -5'-UTR region, containing at least one copy of GUCAGRYC(N 7-19 GCCA (N) 12-19 The xrRNA with the common sequence UGCNRYCUG; wherein N is A, U, G or C, R represents purine A or G, Y represents pyrimidine U or C; and at least one xrRNA sequence is selected from SEQ ID NO:1 to 44; - One copy of the internal ribosome entry site (IRES) RNA sequence; -Open reading box; and - Contains a 3'-UTR region containing the poly(A) sequence; in, The mRNA molecule contains a stem-loop sequence located at the 5' end of the 5'-UTR region, wherein the stem-loop sequence is shown in SEQ ID NO:87; and the stem-loop sequence is separated from the xrRNA sequence by a spacer sequence of 5 nucleotides in length.

2. The mRNA molecule lacking a cap molecule according to claim 1, comprising two copies of xrRNA.

3. The mRNA molecule lacking a cap molecule according to claim 1 or 2, comprising SEQ ID NO: 11 and SEQ ID NO:

26.

4. The mRNA molecule lacking a cap molecule according to claim 1, comprising the IRES sequence of encephalomyocarditis virus.

5. The mRNA molecule lacking a cap molecule according to claim 2, comprising the IRES sequence of encephalomyocarditis virus.

6. The mRNA molecule lacking a cap molecule according to claim 3, comprising the IRES sequence of encephalomyocarditis virus.

7. The mRNA molecule lacking a cap molecule according to claim 1, comprising at least one aptamer selected from aptamer A shown in SEQ ID NO: 64 and aptamer C shown in SEQ ID NO:

66.

8. The mRNA molecule lacking a cap molecule according to claim 2, comprising at least one aptamer selected from aptamer A shown in SEQ ID NO: 64 and aptamer C shown in SEQ ID NO:

66.

9. The mRNA molecule lacking a cap molecule according to claim 3, comprising at least one aptamer selected from aptamer A shown in SEQ ID NO: 64 and aptamer C shown in SEQ ID NO:

66.

10. The mRNA molecule lacking a cap molecule according to claim 7, wherein the mRNA molecule is further bound to a cell-penetrating peptide fused with a multihistidine tag.

11. The mRNA molecule lacking a cap molecule according to claim 8, wherein the mRNA molecule is further bound to a cell-penetrating peptide fused with a multihistidine tag.

12. The mRNA molecule lacking a cap molecule according to claim 11, wherein the cell-penetrating peptide is selected from M12-H6 shown in SEQ ID NO: 75, CPP1-H6 shown in SEQ ID NO: 76, CPP2-H6 shown in SEQ ID NO: 77, and CPP3-H6 shown in SEQ ID NO:

78.

13. The mRNA molecule lacking a cap molecule according to claim 1, wherein the open reading frame encodes the 2Apro protein of HRV2 virus.

14. A DNA molecule comprising: The sequence encoding the mRNA molecule lacking the cap molecule as described in any of the preceding claims, and A promoter recognized by T7 RNA polymerase, the promoter comprising the sequence shown in SEQ ID NO:

46.

15. The DNA molecule of claim 14, further comprising a 5'-UTR region, wherein the 5'-UTR region comprises the sequence shown by SEQ ID NO: 50, 73, 85 or 86.

16. The DNA molecule according to claim 14 or 15, further comprising a 3'-UTR region, wherein the 3'-UTR region comprises a sequence selected from those shown in SEQ ID NO: 53 and 54.

17. A vector comprising an mRNA molecule lacking a cap molecule as described in any one of claims 1 to 13 or a DNA molecule as described in claim 14.

18. An in vitro method for producing at least one cap-deficient mRNA molecule, comprising contacting the DNA molecule of claim 14 with at least one RNA polymerase.

19. The production method according to claim 18, further comprising the step of purifying the mRNA molecule lacking the cap molecule.

20. A pharmaceutically acceptable composition comprising an mRNA molecule lacking a cap molecule as described in any one of claims 1 to 13 and a physiologically acceptable excipient.

21. The composition of claim 20, further comprising a second mRNA molecule. The open reading frame of the mRNA molecule lacking the cap molecule encodes the 2Apro protein.

22. A pharmaceutically acceptable composition comprising an mRNA molecule lacking a cap molecule as described in any one of claims 1 to 13 and a physiologically acceptable adjuvant.

23. The composition of claim 22, further comprising a second mRNA molecule. The open reading frame of the mRNA molecule lacking the cap molecule encodes the 2Apro protein.

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