阅读说明：本技术 信使rna 5’端引入的两个rna序列取代信使rna帽 (Messenger RNA cap substituted by two RNA sequences introduced at the 5' end of messenger RNA ) 是由 J·勒穆瓦安 于 2019-05-15 设计创作，主要内容包括：本发明涉及缺少帽分子的信使核糖核酸(mRNA)分子,因其在体外转录合成的成本大大降低,其从5’至3’端包含：至少一个拷贝的对Xrn1外切核酸酶具有抗性的GUCAGRYC(N-(7-19))GCCA(N-(2-19))UGCNRYCUG共有序列、一个拷贝的内部核糖体进入位点(IRES)RNA序列、以及开放阅读相(une phase)。(The present invention relates to a messenger ribonucleic acid (mRNA) molecule lacking a cap molecule, comprising from the 5 'to the 3' end: at least one copy of GUCAGRYC (N) resistant to Xrn1 exonuclease 7‑19 )GCCA(N 2‑19 ) UGCNRYCUG consensus sequence, one copy of internal ribosome entry siteDot (IRES) RNA sequences, and open reading phase (une phase).)

1. A messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5 'to 3':

-5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) Ugcnrycag (xrna) consensus sequence;

-one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

-an open reading frame.

2. The ARNm molecule of claim 1, comprising two copies of xrna.

3. The ARNm molecule of claim 1 or 2, comprising at least one of SEQ ID NOs 1 to 44, preferably comprising SEQ ID NO: 11 and SEQ ID NO: 26.

4. the ARNm molecule of any of claims 1 to 3, comprising an IRES sequence of encephalomyocarditis virus (EMCV).

5. The ARNm molecule according to any one of claims 1 to 4, comprising a stem-loop, preferably according to SEQ ID NO:87, located 5 'of the 5' -UTR region.

6. The ARNm molecule of any of claims 1 to 5, comprising a sequence selected from those having SEQ ID NOs: aptamer a of sequence 64 and a nucleic acid molecule having the sequence of SEQ ID NO: 66 sequence of aptamer C.

7. An ARNm molecule comprising an aptamer a according to claim 6, wherein the ARNm molecule binds to a Cell Penetrating Peptide (CPP) fused to a polyhistidine tag, preferably selected from the group consisting of peptides having the amino acid sequence of SEQ ID NO: 75, M12-H6 having the sequence of SEQ ID NO: 76, CPP1-H6 having the sequence of SEQ ID NO: 77, and CPP2-H6 having the sequence of SEQ ID NO: 78 sequence CPP 3-H6.

8. The ARNm molecule of any of claims 1 to 7, wherein the open reading frame encodes the 2Apro protein of the HRV2 virus.

9. A deoxyribonucleic Acid (ADN) molecule comprising a sequence encoding an ARNm according to any of the preceding claims, preferably comprising:

-a promoter recognized by the T7 ARN polymerase, said promoter comprising the sequence shown by SEQ ID NO. 46;

-a 5' -UTR region comprising a sequence defined by SEQ ID NO: 50. 71, 72, 73, 85, or 86;

-an open reading frame; and

-a 3' -UTR region comprising a sequence selected from SEQ ID NO: 53. 54, 55 and 56, respectively.

10. A vector comprising the ARNm molecule of any of claims 1 to 5 or the ADN of claim 9.

11. An in vitro method for producing at least one ARNm comprising contacting an ADN molecule of claim 9 with at least one ARN polymerase.

12. The production process according to claim 11, comprising a step of purifying the ARNm.

13. A pharmaceutically acceptable composition comprising an ARNm according to any of claims 1 to 7 and a physiologically acceptable excipient and/or adjuvant.

14. Use of a composition according to claim 13 in gene therapy or genetic vaccination.

15. The composition of claim 13 or 14, comprising a second ARNm molecule of any of claims 1 to 7, wherein the open reading frame encodes a 2Apro protein.

Technical Field

The present invention relates to the field of ribonucleic acids, and more particularly to the in vitro synthesis of messenger ribonucleic acid (ARNm), its stability and its translation into polypeptides, particularly in transfected cells.

Background

ARNm is an important molecule for the production of polypeptides on an industrial scale. Its stability and transcription and translation efficiency strongly influence the downstream yield of the polypeptide, and thus the cost.

ARNm is also a molecule of choice for pharmaceutical compositions used in gene therapy and genetic vaccination. In fact, integration of the ARNm molecule into the genome of transfected cells has never been demonstrated, in contrast to ADN molecules. However, since it is sensitive to ribonuclease degradation, its stability in solution is poor. Therefore, stable ARN molecular synthesis in vitro and in vivo is crucial to reduce the amount of ARNm required for optimal therapeutic effect and thus to reduce its cost. Furthermore, the use of lower amounts of more stable ARNm may reduce the risk of side effects associated with the treatment.

In vivo synthesis occurs in cultured cells (e.g., yeast or bacteria). However, this approach has a number of disadvantages. In particular, due to degradation problems and the presence of other cellular ARNs, purification of the complete target ARN is expensive and complicated. It generally results in lower yields compared to yields obtained by in vitro transcription.

As a result, the synthetic approach that commonly uses large-scale ARNm is a cell-free in vitro transcription system. This method uses only a few purified compounds (i.e., reaction buffer, ADN molecule with gene, recombinant ARN polymerase and four ribonucleotide triphosphates), the only ARN to be produced is the ARNm one wishes to synthesize. At the end of the reaction, there were no ARNm degradation products due to the absence of ARN enzymes in the reaction mixture. There are also no other ARN species, which is a major advantage over the synthesis of ARNm using cultured cells. The purification of ARNm is thus greatly simplified.

To ensure stability of ARNm in eukaryotic cells, the 5 'end of the ARNm has a cap molecule that protects the 5' end from exoribonucleases. The cap is a complex molecule consisting of two guanosines, the 5' carbons of which are linked by a chain of three phosphate groups. The terminal guanosine is methylated at the 7-position of guanine. ARNm is stabilized by the cap being resistant to the progressive enzymatic degradation performed by Xrn1 exoribonuclease from 5 'to 3'. In addition to the effect of stabilizing ARNm, the cap also has other functions, including ribosome recruitment (Cowling, 2010). To this end, the cap begins with the recruitment of translation initiation factors, which in turn recruit ribosomes. The ribosome then translates the ARNm into protein.

On an industrial scale, to increase the stability of ARNm and to achieve efficient translation of ARNm into a polypeptide in transfected cells, a cap molecule should be incorporated at the 5' end. In the in vitro transcription process, a capping molecule can be added using a capping enzyme (e.g., 2' -O-methyltransferase from vaccinia (Viccia) virus) in a post-transcriptional step (Martin et al, 1975). However, this additional step adds complexity to the synthesis, requires purification of the capping enzyme, and is inefficient (Contreas et al, 1982). In addition, the enzyme requires S-adenosyl-L-methionine, which is an unstable molecule in aqueous solution.

To simplify in vitro transcription, cap analogs have been developed in the prior art, but are not satisfactory. For example, P may be referenced¹- (5' -7-methyl-guanosine) P³- (5' - (guanosine)) triphosphate) analogues or P¹- (5' -2,2, 7-trimethyl-guanosine) P³- (5' - (guanosine) triphosphate) analogs. These analogs allow capping by phage ARN polymerase mediated co-transcription, thereby avoiding the additional step of cap synthesis and also improving the stability of ARNm. However, depending on the type of analog, up to 50% of the incorporated molecules have the opposite orientation, with the 7-methylguanosine nucleotide adjacent to the ARN molecule rather than in the terminal position, thus reducing the stability and efficiency of ARNm translation (Pasquinelli et al, 1995). "inverted" cap analogs (ARCA) have been developed, for example, as described in US 7074596. These analogs prevent reverse incorporation of the molecule. However, they are still unsatisfactory because their use causes a significant reduction in the yield of ARNm synthesis in vitro. Furthermore, due to the complexity of these compounds, the synthesis of cap molecules and many of their chemically modified analogs is expensive. Inclusion of one of these molecules in a mixture of in vitro transcription reactions on an industrial scaleWhich greatly increases the cost of ARNm synthesis.

In fact, in order to ensure high capping efficiency during in vitro transcription, it is necessary to provide an excess of the cap analogue, resulting in high raw material costs. In fact, it is suggested to use 4 cap analogue molecules per GTP molecule in a ratio to maximize the chance of having a cap per ARNm molecule. Nevertheless, it is estimated that only 80% of the synthetic ARNm will be capped. Furthermore, the GTP concentration is reduced 5-fold compared to the other three ribonucleoside triphosphates, thus reducing the transcription yield 5-fold.

One way to reduce the high cost of in vitro transcription is to reduce the production cost of ADN and/or ARN polymerases used in the method. However, the cost reduction obtained is still relatively small. Alternatively, it is possible to synthesize circular cap-free ARNm by in vitro transcription in a cell-free system (WO 2014/186334 a 1). However, the authors demonstrated that circular ARNm translated less efficiently in transfected cells than does capped linear ARNm (see, e.g., fig. 2 and 3 and paragraphs [00121] and [00131] of WO 2014/186334 a 1).

As a result, there remains a need for ARNm molecules with high stability as well as high translation efficiency, preferably ARNm molecules with at least as high stability and translation efficiency as capped ARNm molecules. There is also a need for an ARN molecule that has a simple production process and whose manufacturing costs are significantly reduced.

Disclosure of Invention

The present invention relates to stable, cap-free ARNm molecules that can be translated efficiently. The ARNm is particularly advantageous because its production cost is much lower than that of a conventional ARNm containing a capping molecule or an analogue thereof. The invention also relates to ARNm molecules with increased in vitro transcription yield compared to conventional ARNm comprising a cap molecule or an analogue thereof. Indeed, the ARNm of the present invention is also advantageous in that it is at least as effective as a conventional capped ARNm when transfecting cultured cells and tissues, despite its significantly reduced production cost and/or its improved synthetic yield. Indeed, the inventors have very surprisingly demonstrated that the expression levels and durations obtained after in vivo transfection are at least as high as the capped ARNm. Specifically, the kinetics of reporter protein expression in Caco-2 cells were identical whether transfected with ARNm of the invention or control-capped ARNm (fig. 2 and 3). Also, the inventors have very surprisingly demonstrated that when ARNm of the invention is transfected in vivo in the dermis or muscle of mice, the expression of the reporter protein is 2-fold to about 10-fold higher than that of the control capped ARNm transfection (fig. 5 and 15). However, the reaction is simplified because an additional capping step is not necessary. It is also not necessary to include cap analog molecules in the reaction mixture during in vitro transcription. Thus, the ARNm molecules of the invention are clearly advantageous in terms of reduced cost and facilitated production for at least as high an expression as compared to ARNm of the prior art.

For the purposes of this application, the term "ARNm molecule" refers to any linear strand of ribonucleotides. In the present application, these sequences are expressed in the 5' to 3' direction starting with the 5' -UTR region.

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

The inventors herein surprisingly demonstrated that the cap at the 5 'end of the ARNm molecule can be replaced by at least one copy of a Xrn1 exoribonuclease (xrna) resistant sequence derived from the 3' -UTR region of a virus of the flavivirus genus, preferably with an Internal Ribosome Entry Site (IRES) in the open reading frame. Advantageously, the production cost of such ARNm molecules by in vitro transcription is reduced by about 30-fold and the yield is increased compared to capped ARNm molecules. In addition, the ARNm molecules of the invention are particularly stable in transfected cells and can be efficiently translated even without caps.

"Cap" refers to a 7-methylguanosine (N7-methylguanosine or m7G) nucleoside and any mutant, variant, analogue or fragment thereof which may be transcribed from the ARNm via a 5 '-5' triphosphate linkageThe first nucleotide of (a) is linked. Without limitation, cap analogs include unmethylated analogs (e.g., P)¹- (guanosine) P^3-(5' - (guanosine)) triphosphate), monomethylated analogs (e.g., P)¹- (5' -7-methyl-guanosine) P³- (5' - (guanosine) triphosphates), trimethylated analogs (e.g. P)¹- (5' -2,2, 7-trimethyl-guanosine) P³- (5 '- (guanosine)) triphosphate), or substitution of m with 3' -O-methyl⁷Analogs of the 3' -OH group of the guanine moiety (e.g., ARCA P¹- (5 '- (3' -O-methyl) -7-methyl-guanosine) P³- (5' - (guanosine) triphosphate)).

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

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

at least one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

at least one open reading frame.

Preferably, the IRES ARN sequences are located upstream of each open reading frame.

According to a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5 'to 3':

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

open reading frames.

Preferably, the ARNm molecule further comprises a 3' -UTR region.

Preferably, the ARNm molecule further comprises at least one ARN aptamer that facilitates penetration of the ARNm molecule into a cell, preferably a muscle cell. ARN aptamers can significantly facilitate penetration directly or indirectly through peptides.

Preferably, the ARNm molecule further comprises a stem-loop at the 5' end.

In a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, consisting of from 5 'to 3':

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

open reading frames.

The untranslated region of the 3' -UTR of flaviviruses (flaviviruses) has been shown in the prior art to have the property of blocking 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, thereby inhibiting degradation of the viral subgenomic ARN. However, such sequences have never been previously inserted 5' to ARNm. Indeed, there is a concern that these sequences may need to be in the context of the 3' -UTR of the flavivirus genome in order to function. In particular, these three-dimensional structures may not fold properly when no longer surrounded by the sequence of the flavivirus 3' -UTR region.

Although the prior art indicates that xrna sequences inhibit translation, the inventors herein surprisingly demonstrate that the protective function and translation initiation function of the cap can be successfully replaced by at least one copy of an xrna sequence and at least one copy of an IRES sequence (preferably from an EMCV virus) without affecting translation efficiency. Surprisingly, the use of xrna and IRES sequences can even greatly improve translation efficiency.

By "flavivirus" is meant any virus of the genus flavivirus, including yellow fever, dengue fever, West Nile (WNV), zika, japanese encephalitis, Rocio (Rocio), Murray (Murray) stream valley, Bagaza (Bagaza) virus, koku bera (kokokobera), enaya (Ntaya), kadu guo (kedou), seebeck (Sepik), saint louis, wusu picture (Usutu), alfy (Alfuy), wessel brown (Wesselbron), ilex (ileus), brusura (busuu), tembusuhua), tembususu (tenoyang), changogar (yong), yose (yose), hong kong (dogang) virus, and any other virus of the genus flavivirus.

Accordingly, the present invention relates to a stable ARNm molecule comprising at least one copy of an xrna sequence in the 5' region of the ARNm. The ARNm is efficiently translated into protein at a level and duration similar to that of the capping molecule. According to a preferred embodiment of the invention, the ARNm molecule comprises two copies of xrna.

By "ARN sequences resistant to Xrn 1" or "xrna" is meant any polynucleotide sequence that may reduce, slow or prevent Xrn1 exonuclease degradation of ARNm. Preferably, the xrna sequences comprise a consensus sequence.

"consensus sequence" refers to any sequence comprising at least the sequence shown below: 5' -GUCAGRYC (N)_7-19) GCCA(N_12-19) Ugcnryug-3', wherein each N may independently represent a nucleotide selected from A, C, T, G and U or an analog thereof. Each R represents a purine and each Y represents a pyrimidine. As shown, the number of N nucleotides between conserved bases may vary in the consensus sequence from 5 'to 3', from 7 to 19 bases and from 12 to 19 bases. The sequences form a three-dimensional structure of stem-loop type.

Preferably, the ARNm molecule comprises at least one copy of a sequence selected from SEQ ID NOs: 1 to SEQ ID NO: 44, or an xrna sequence of the sequence shown in figure 44. When an ARNm molecule contains more than one copy of the sequence, those copies may be the same or different. Thus, more preferably, the ARNm molecule comprises SEQ ID NO: 11 and SEQ ID NO: 26 at least one copy of one of the sequences shown. Even more preferably, the ARNm molecule comprises one copy of the sequence of SEQ ID NO: 11 and one copy of SEQ ID NO: 26, and (b) a sequence shown in 26.

Preferably, when there are two xrna sequences in the 5' -UTR region, they are separated by a spacer sequence. Likewise, a spacer sequence may be present between the xrna sequence and the IRES sequence. Preferably, the spacer sequence between the two xrna sequences corresponds to SEQ ID NO: 47. preferably, the spacer sequence between the xrna sequence and the IRES sequence corresponds to SEQ ID NO: 48. when the ARNm molecule comprises at least two open reading frames, a spacer sequence may also be present between the two open reading frames or between the open reading frames and the IRES sequence.

"spacer sequence" refers to any non-coding polynucleotide sequence that makes it possible to physically separate a sequence upstream from a sequence downstream of the spacer sequence. ARNm molecules according to the present invention may significantly comprise one or more spacer sequences.

In some embodiments, the spacer sequence may be 2 to 300 nucleotides in length. Preferably, it is between 2 and 10 nucleotides, even more preferably between 2 and 5 nucleotides. Alternatively, it may be between 10 and 150 nucleotides, even more preferably between 15 and 40 nucleotides. Preferably, the spacer sequence does not produce secondary structure.

The ARNm of the invention further comprises at least one Internal Ribosome Entry Site (IRES) ARN sequence.

An "internal ribosome entry site" or "IRES" refers to any polynucleotide sequence that allows cap-independent initiation of translation of an ARNm molecule. Such sequences interact directly with the translation initiation factor, which then recruits ribosomes at the translation initiation codon. A number of IRES sequences are known (Mokrejs et al, 2010). Thus, one skilled in the art will be able to identify sequences that function as IRES' and select those suitable for use in the practice of the present invention. Thus, the IRES sequences according to the invention may be eukaryotic or virally derived sequences, e.g. from the genus Picornavirus (picornaviridus). Preferably, it is derived from encephalomyocarditis virus (EMCV) (Borman et al, 1995) or from human eIF4G ARNm. Even more preferably, the ADN sequence of IRES corresponds to SEQ ID NO: 45.

IRES sequences are usually located in the 5' -UTR region. They may also be located between two open reading frames, which allows bi-cistronic or polycistronic translation to be initiated from a single ARNm. In a preferred embodiment, an ARNm of the invention comprises a single copy of an IRES sequence in the 5' region of the ARNm. According to a preferred embodiment, the second copy of the IRES sequence is located between two open reading frames. According to yet another preferred embodiment, the ARNm of the invention comprises one copy of an IRES sequence in the 5' region and one copy of the IRES sequence between two open reading frames. Advantageously, the IRES sequence is located downstream of the xrna sequence. This tissue advantageously makes it possible to protect these sequences from Xrn1 exoribonuclease. When the ARNm molecule comprises at least two IRES sequences, the sequences may be the same or different. In particular, one or more IRES sequences may be selected according to their efficiency in initiating translation. The choice of two different IRES sequences is particularly advantageous when the ARNm molecule comprises at least two different open reading frames and the required translational efficiency of the first open reading frame is different from the required translational efficiency of the second open reading frame.

IRES elements also have complex three-dimensional structures. Thus, by forming a pair between the ARN sequences in each of the two regions, it is possible to generate mutual interference between the xrna and IRES sequences. Such interference may prevent correct structural formation of xrna and IRES sequences, respectively. In addition, the xrna structure can significantly prevent recruitment of translation initiation factors and ribosomes by IRES. Even more surprisingly, the inventors have demonstrated that the presence of xrna and IRES sequences makes it possible to obtain protein expression yields in transfected cells that are at least similar to the yields obtained with capped ARNm molecules. Thus, the xrna and IRES sequences together can replace a cap or cap analog molecule. They are essential to ensure translation of the open reading frame contained in the ARNm of the invention and ARNm stability.

Thus, the ARNm of the invention is at least as stable as a capped ARNm in transfected cells, while also being at least efficiently translated. In addition, the cost of its synthesis by in vitro transcription is greatly reduced compared to capped ARNm.

According to a particular embodiment, the ARNm of the invention is in the consensus sequence GUCAGRYC (N)_7-19) GCCA(N_12-19) The 5'-UTR region upstream of ugcnrycag (xrna) further includes a stem-loop, preferably at the 5' end of the ARNm molecule.

"stem-loop" refers to any polynucleotide sequence that forms a double helix structure in which the 5 'end of one strand is physically linked to the 3' end of the other strand by an unpaired loop. Thus, the stem-loop consists of a double-stranded stem and an unpaired single-stranded loop. The physical bond may be covalent or non-covalent. Preferably, the physical bond is a covalent bond. The size of the ARN loop may be, for example, between 3 and 30 nucleotides. The loop size is preferably at least 3 nucleotides, preferably at least 4 nucleotides. The double-stranded stem may be, for example, between 5 and 50 nucleotides in length. The length of the stem 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 is 6, 7 or 8 nucleotides in length.

In the context of the present 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, in a sequence of seq id no. The stem-loop is preferably separated from the xrna sequence by a spacer sequence. Preferably, the spacer sequence is equal to or less than 5 nucleotides (i.e., 5, 4, 3, or 2 nucleotides) in length. Indeed, the inventors surprisingly demonstrated that the addition of a stem-loop structure (also referred to herein as 5' -SL, since the stem-loop is at the 5' end) at the 5' end of the ARNm molecule may further improve translation in vivo as it approaches xrna sequences (e.g., 5 nucleotides or less). In fact, the inventors have not observed a favorable effect when the stem-loop at the 5' end is separated from the xrna sequence by a spacer sequence of about 70 nucleotides in length (see fig. 15).

Without being bound by theory and unexpectedly, it is contemplated that the xrna sequence covers the 5' end from phosphatase and Xrn1, at least when the sequences are in stem-loop form and in close proximity.

Thus, in a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5 'to 3':

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

an Open Reading Frame (ORF),

also included at the 5' end is a stem-loop.

The ARNm molecule may further comprise a second IRES sequence followed by a second open reading frame. The ARNm molecule may further comprise an ARN aptamer as defined herein, located between the stem-loop and the xrna sequence, or preferably, between the xrna sequence and the IRES sequence. The ARNm molecule may further comprise a 3' -UTR region as defined herein.

In a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5 'to 3':

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) UGCNRYCUG (xrRNA) consensus sequence,

optionally, an ARN aptamer;

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

open reading frames.

In a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, consisting of from 5 'to 3':

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) UGCNRYCUG (xrRNA) consensus sequence,

optionally, an ARN aptamer;

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

an open reading frame; and

the 3' -UTR region.

An "aptamer" refers to any nucleic acid having recognition properties and specificity that are related to the ability of the nucleic acid molecule to adopt a particular three-dimensional structure (particularly similar to a monoclonal antibody) (see, e.g., Dunn et al, 2017). Aptamers may consist of ADN, ARN and/or modified ARN, preferably ARN. The aptamer may consist of 6 to 50 nucleotides, such as the ribonucleotides defined above. By way of non-limiting example, aptamers may be isolated according to various techniques well known to those skilled in the art, for example, by one or more in vitro selection cycles, or by iterative selection methods to identify aptamers in vitro from a combinatorial library of a large number of compounds of random sequence ("SELEX" technique). By identifying aptamers in vitro by selection, it is advantageously possible to obtain aptamers with precise action or function without the need to know the target to which the aptamer is directed. The production or selection of aptamers is described, for example, in european patent application EP 0533838. Advantageously, according to the scope of the present invention, ARN aptamers have been identified according to their ability to penetrate cells of interest, more particularly tissue cells, preferably muscle cells (e.g., myofibroblasts). Advantageously, the aptamer according to the invention is capable of crossing a cell membrane, more preferably the plasma membrane and/or the endosomal (endosomal) membrane of a mammalian cell.

The aptamer according to the invention preferably comprises 6 to 50 nucleotides, more preferably 10 to 45 nucleotides, 20 to 40 nucleotides, even more preferably 30 to 40 nucleotides. Preferably, the aptamer consists of ribonucleotides, such as those defined above. Preferably, the aptamer according to the invention binds to a polypeptide having the sequence of SEQ ID NO:64, and an aptamer having the sequence of SEQ ID NO: 65 or an aptamer of SEQ ID NO: aptamer C of sequence 66, having at least 70% identity, 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, even more preferably at least 99% identity.

The percentage of identity referred to in the context of the present description is determined based on the overall alignment of the sequences to be compared, that is, the sequences are aligned over their entirety using any algorithm known to those skilled in the art (e.g., the algorithms of Needleman and Wunsch, 1970). This sequence comparison can be performed using any software known to those skilled in the art, for example, Needle software using a "Gap open" parameter equal to 10.0, a "Gap extended" parameter equal to 0.5, and a Blosum 62 matrix.

Preferably, the aptamer according to the invention is selected from the group having SEQ ID NO: aptamer a of sequence 64, having the sequence of SEQ ID NO: 65, and an aptamer having the sequence of SEQ ID NO: 66, even more preferably, selected from the group consisting of aptamers having SEQ ID NOs: aptamer a of sequence 64 and a nucleic acid molecule having the sequence of SEQ ID NO: aptamer C of sequence 66.

Preferably, ARNm molecules according to the invention comprise at least one copy of a membrane-capable aptamer, preferably a mammalian plasma membrane and/or a nuclear endosomal membrane. Advantageously, therefore, when the ARN molecule according to the invention comprises an aptamer, said aptamer facilitates its penetration into a cell, preferably into a muscle or skin cell. Preferably, ARNm molecules according to the invention comprise at least one copy of a polypeptide having the sequence of SEQ ID NO: aptamer a of sequence 64, having the sequence of SEQ ID NO: 65, and/or an aptamer having the sequence of SEQ ID NO: aptamer C of sequence 66.

Aptamers that facilitate the passage of ARNm molecules into target cells do not necessarily need to be protected from exonuclease degradation, which occurs primarily in the cytoplasm of the cells. Thus, according to a specific embodiment, the ARNm molecule containing the aptamer is in one or more guacagrycs (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence upstream of the 5' -UTR region. According to an alternative embodiment, the aptamer is placed in one or more GUCAGRYCs (N)_7-19)GCCA(N_12-19) Ugcnrycag (xrna) consensus sequence is downstream of the 5' -UTR region, but upstream of the IRES sequence and open reading frame. (representative diagrams for these two possibilities, see FIGS. 1J and 1K). Thus, according to a preferred embodiment, the invention relates to a messenger ribonucleic acid (ARNm) molecule lacking a cap molecule, comprising from 5 'to 3':

a 5' -UTR region comprising at least one aptamer capable of crossing a cell membrane, preferably the plasma membrane and/or the endosomal membrane of a mammalian cell, preferably at least one aptamer selected from the group consisting of aptamers A, B and C as described herein, and at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

open reading frames.

Preferably, the ARNm molecule is in GUCAGRYC (N)_7-19)GCCA(N_12-19) The upstream 5' -UTR region of the ugcnrycag (xrna) consensus sequence comprises an aptamer (see, e.g., fig. 1F).However, the ARN molecule may also be in GUCAGRYC (N)_7-19)GCCA(N_12-19) Ugcnrycag (xrna) contains aptamers downstream of the consensus sequence, e.g., between the xrna consensus sequence and the IRES sequence.

Thus, according to a preferred embodiment, the present invention relates to ARNm molecules lacking a cap molecule comprising from 5 'to 3':

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) UGCNRYCUG (xrRNA) consensus sequence,

at least one aptamer capable of crossing the cell membrane;

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

open reading frames.

While aptamers B and C were chosen because of their ability to penetrate myofibroblasts, aptamer a corresponded to the "shot 47" aptamer, identified by Tsuji et al in 2013. Aptamer a advantageously binds with high affinity to a polyhistidine-type peptide motif. Aptamer a can thus bind to any molecule (e.g., peptide or protein) that comprises a polyhistidine tag (e.g., hhhhhhhh motif). As a non-limiting example, ARNm molecules according to the present invention may be linked by aptamer a to a molecule that allows it to better penetrate cells, such as a "cell penetrating peptide" or "CPP", which contains a poly-histidine tag.

"cell-penetrating peptide" or "CPP" refers to any peptide, polypeptide or protein that is capable of passing through the cell membrane of a mammalian cell, more preferably through the plasma membrane and the endosomal membrane. Advantageously, when a CPP is linked to another molecule (particularly an ARNm molecule), the CPP retains this property, allowing the other molecule to pass through the membrane. In the context of the present invention, any possible mechanism of transport across the membrane is contemplated, 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, due to its negative charge, ARNm molecules may bind non-covalently to CPPs using electrostatic interactions and/or hydrophobicity. Alternatively, an ARNm molecule may be covalently bound to a CPP. CPPs may form oligomers composed of at least two identical or different peptide molecules. In the context of the present invention, a CPP is preferably non-covalently bound to an ARN aptamer. Indeed, such a bond is advantageous in view of its simplicity, its low cost through simple mixing of CPP and ARNm molecules, and the fact that these molecules are fully biodegradable. In fact, chemical groups that are not natural and are not biodegradable are not required.

The length of a CPP according to the present 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, more preferably from 8 to 30 amino acid residues, 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 a CPP need not be limited to those described above. For example, CPP derivatives described herein may be specifically created with varying lengths, in accordance with common general knowledge of those skilled in the art.

As non-limiting examples, the CPP of the present invention may be an "M12" CPP (as described by Gao x et al, 2014), a "CPP 2" or a "CPP 3" CPP (as described by Kamada et al, 2007), or a CPP "CPP 1" (Lee et al, 2012), as well as any variants or derivatives of these. According to a preferred embodiment, said CPP comprises a poly-histidine motif (e.g. hexa-histidine), which is preferably linked to said CPP by a spacer. Preferably, the spacer is a hydrophilic spacer, advantageously unstructured and uncharged, even more advantageously consisting of glycine and serine. Preferably, the spacer is about 21 amino acids in length, preferably 21 amino acids.

Preferably, a CPP according to the present invention has at least 70% identity, 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, even more preferably at least 99% identity with the following peptides: has the sequence shown in SEQ ID NO: 75, M12-H6 peptide having the sequence of SEQ ID NO: 76 CPP1-H6 peptide having the sequence of SEQ ID NO: 77, and a CPP2-H6 peptide having the sequence of SEQ ID NO: 78 sequence CPP3-H6 peptide. According to a particularly preferred embodiment, the CPP according to the invention is selected from: has the sequence shown in SEQ ID NO: M12-H6 of sequence 75; has the sequence shown in SEQ ID NO: CPP1-H6 of sequence 76; has the sequence shown in SEQ ID NO: 77 sequence CPP2-H6 peptide; or has the sequence of SEQ ID NO: 78 sequence CPP 3-H6; even more preferably, selected from: has the sequence shown in SEQ ID NO: CPP1-H6 of sequence 76; has the sequence shown in SEQ ID NO: 77 sequence CPP2-H6 peptide; and a polypeptide having the sequence of SEQ ID NO: 78 sequence CPP 3-H6. 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 an ARNm molecule (i.e., aptamer a when the CPP comprises a polyhistidine tag).

Preferably, the CPPs of the present invention have no significant cytotoxic and/or immunogenic effect 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 less than 50%, preferably less than 40% or 30%, preferably less than 20% or 10% and especially less than 5% of the target cells are killed after the ARNm molecule to which the CPP is attached has been internalized by the cell across the plasma membrane. One skilled in the art is familiar with methods for determining the cytotoxicity of a given compound and/or the viability of target cells administered the compound (see, e.g., Ausubel et al, 2001). Corresponding assay kits are commercially available from various suppliers. In particular embodiments, the potentially inherent cytotoxic and/or immunogenic effects of a CPP of the invention may be "masked" (masquage) "by introducing one or more modifications in the peptide (e.g., by means of chemical synthesis or recombinant ADN). Such modifications may include, for example, the addition, removal, or substitution of functional groups or the changing of the position of such functional groups. The skilled person is well aware of how this "masking" can be done for a given peptide.

Thus, according to a preferred embodiment, the present invention relates to ARNm molecules lacking a cap molecule comprising from 5 'to 3':

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) UGCNRYCUG (xrRNA) consensus sequenceThe columns of the image data are,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence, and

an Open Reading Frame (ORF),

wherein the ARNm molecule is linked to a Cell Penetrating Peptide (CPP) fused to a polyhistidine tag, said CPP preferably being selected from: has the sequence shown in SEQ ID NO: M12-H6 of sequence 75; has the sequence shown in SEQ ID NO: CPP1-H6 of sequence 76; has the sequence shown in SEQ ID NO: 77 sequence CPP2-H6 peptide; and a polypeptide having the sequence of SEQ ID NO: 78 sequence CPP 3-H6. Preferably, the CPP is non-covalently linked to the aptamer.

By "open reading frame" is meant any polynucleotide sequence that can be translated into a polypeptide of interest. The open reading frame is read for a block of three consecutive nucleotides (called codons), each codon representing one amino acid. During translation, the polypeptides are synthesized by ribosome translation of the codons of the open reading frame.

The first amino acid of a polypeptide is generally indicated by the AUG codon on the ARNm molecule, thus indicating the beginning of the open reading frame. Other initiation codons are known, such as AUN, or NUG, where N corresponds to A, C, U or G. The ends of the polypeptide are shown on the ARNm molecule as UAA, UGA or UAG stop codons. Stop codons indicate the end of the open reading frame on the ARNm molecule.

The open reading frame according to the present invention is more particularly an open reading frame which is translated in transfected cells to produce a product of therapeutic or vaccine interest for use in human or veterinary medicine. Preferably, the product of therapeutic interest is a protein.

Among the proteins of therapeutic interest, one may more particularly refer to enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 9203120), growth factors, neurotransmitters or their precursors or synthetases, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, etc., apolipoprotein: ApoAI, ApoAIV, ApoE, etc. (FR 9305125), dystrophin or dystrophin small (FR 9111947), oncostatin: p53, Rb, Rap1A, DCC, k-rev, etc. (FR 9304745), are involved in the factors in coagulation: factors VII, VIII, IX, etc., pro-apoptotic proteins: thymidine kinase, cytosine deaminase, etc., or even all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc., see, e.g., WO 2011/089527), ARN ligand (WO 91/19813), etc.

The protein of interest encoded by ARNm may also be an antigen capable of generating an immune response in a human or animal to achieve vaccination. They may in particular be antigenic proteins specific for Epstein Barr virus, HIV virus, hepatitis B virus (EP 185573), pseudorabies virus, even specific for tumors (EP 259212). Finally, the protein of interest may be an adjuvant protein, which may stimulate an immune response to increase the efficacy of the vaccine.

The protein of interest encoded by the ARNm may be a protein that has a beneficial effect on the uncapped ARNm molecule or the protein expressed by the molecule. This advantageous effect may come from different mechanisms. As a non-limiting example, the protein encoded by the capped ARNm may increase the stability of the capped ARNm molecule by binding to the molecule or by degrading at least one protein having ARN enzyme activity. By way of non-limiting example, a protein encoded by a cap-free ARNm may increase translation of the molecule by promoting recruitment of initiation factors or by binding to a capped cellular ARNm to inhibit its translation. As a non-limiting example, the protein of interest encoded by ARNm is a 2Apro protein from a picornavirus, such as human rhinovirus type 2(HRV 2). Without being bound by theory, it is believed that cleavage of the N-terminus of the eIF4G initiation factor due to the protease activity of the 2Apro protein prevents the initiation factor from recognizing capped ARNm, and that the 2Apro protein increases the expression of uncapped ARNm. Such cleavage can reduce competition between ARNm of the invention and capped ARNm in vivo. Indeed, the inventors surprisingly demonstrated that cells co-transfected with a first ARNm according to the invention encoding a 2Apro, and a second ARNm according to the invention encoding a reporter protein, resulted in increased expression of the reporter protein. Thus, the presence of the 2Apro protein is particularly advantageous as it allows for an increased specific expression of the cap-free ARNm encoded protein of the invention (fig. 7).

According to a preferred embodiment, the ARNm of the invention encodes a 2Apro protein, preferably a 2Apro protein from a picornavirus, even more preferably from an HRV2 virus. According to a particular embodiment, the 2Apro protein has the amino acid sequence of SEQ ID NO: 81, in sequence. According to a specific embodiment, the 2Apro protein consists of a polypeptide having the sequence of SEQ ID NO: 80 sequence of ARNm coding.

The open reading frame may also encode a therapeutic ARNm. This may be, for example, an antisense sequence, the expression of which in the target cell allows the transcription or translation of the cellular ARNm to be controlled. Such sequences may be transcribed, for example, in target cells to ARN complementary to intracellular ARNm, thereby preventing its translation to protein, according to the techniques described in patent EP 140308.

Preferably, the ARNm of the invention comprises, in addition to an xrna sequence and an IRES sequence, an open reading frame encoding a polypeptide of interest. Preferably, the open reading frame is located downstream of the xrna sequence. Those skilled in the art will readily appreciate that ARNm may comprise several open reading frames. Thus, ARNm may be monocistronic, bicistronic, or polycistronic. When ARNm has only one open reading frame, it is a monocistron. When it comprises two open reading frames, it is a bicistronic; when it comprises at least two open reading frames, it is polycistronic.

ARNm of the invention may also comprise one or more non-coding regions. These non-coding regions may in particular be the regions between two open reading frames. In this case, the IRES sequence is advantageously present in these non-coding regions between two open reading frames.

According to a particular embodiment, a messenger ribonucleic acid (ARNm) molecule, lacking a cap or cap analogue molecule, comprises from 5 'to 3':

5' UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) Ugcnrycag (xrna) consensus sequence, a copy of which is followed by a single copy of an Internal Ribosome Entry Site (IRES) ARN sequence;

an open reading frame; and

the 3' -UTR region comprising the poly (A) sequence.

Advantageously, the ARNm molecule further comprises at least one ARN aptamer that facilitates penetration of the molecule into a target cell, preferably a muscle cell. Advantageously, the ARNm molecule comprises at least one aptamer selected from the group having SEQ ID NO: aptamer a of sequence 64, having the sequence of SEQ ID NO: 65, and an aptamer having the sequence of SEQ ID NO: aptamer C of sequence 66.

According to another specific embodiment, a messenger ribonucleic acid (ARNm) molecule, lacking a cap or cap analogue molecule, consists from 5 'to 3' of:

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) Ugcnrycag (xrna) consensus sequence, a copy of which is followed by a single copy of an Internal Ribosome Entry Site (IRES) ARN sequence;

an open reading frame; and

the 3' -UTR region comprising the poly (A) sequence.

"5' -UTR region" refers to any nucleic acid region located upstream of the translation initiation codon. This region is non-coding, but may contain elements that modulate downstream ARNm expression. In addition to the xrna and IRES elements, the region may also contain other elements, such as riboswitches and/or T-boxes. In some embodiments, the 5' -UTR region can be 10 to 2000 nucleotides in length. Preferably, it comprises between 50 and 1500 nucleotides, more preferably 200 to 1000 nucleotides.

"3' -UTR region" refers to any nucleic acid region located downstream of a translation stop codon. This region may affect the expression and/or stability of ARNm, its location in a cell, or a binding site comprising a protein or a small interfering ARN or mini ARN. The region may include, for example, elements such as a polyadenylated tail (poly (a)), a histone stem-loop structure, and/or a pyrimidine or purine rich region. It may comprise coding or non-coding sequences. In some embodiments, the 3' -UTR region can be 50 to 500 nucleotides in length. Preferably, it comprises between 50 and 200 nucleotides, more preferably 50 to 100 nucleotides. In a preferred embodiment, the ARNm comprises a polyadenylated tail (poly (a)) comprising a nucleotide sequence of adenine or an analogue or variant thereof which is from 10 to 300 nucleotides, preferably from 50 to 100 nucleotides.

According to a second aspect, the present invention relates to a deoxyribonucleic Acid (ADN) molecule comprising a polynucleotide which can be transcribed into an ARNm molecule of the invention. Preferably, the ADN molecule comprises SEQ ID NO: 50. 71, 72, 73, 85 or 86.

Preferably, the ADN molecule is comprised in an expression cassette.

By "expression cassette" is meant herein an ADN fragment comprising a polynucleotide of interest, e.g., a polynucleotide that can be transcribed into an ARNm molecule of the invention, operably linked to one or more regulatory elements that control the expression of gene sequences, such as, for example, promoter sequences and enhancer sequences.

Polynucleotides are "operably linked" to regulatory elements, wherein the function of one sequence is affected by the other when these different nucleic acid sequences are combined in this manner on a single nucleic acid fragment. For example, a regulatory ADN sequence is "operably linked" to an ADN sequence encoding an ARN or protein if the two sequences are positioned in such a way that the regulatory ADN sequence affects expression of the ADN coding sequence (in other words, the ADN coding sequence is under the transcriptional control of a promoter). The coding sequence may be operably linked to regulatory sequences in sense and antisense orientations. Preferably, the coding sequence of the invention is operably linked to the regulatory sequence in sense orientation.

By "regulatory sequence" or "regulatory element" is meant herein a polynucleotide sequence necessary to affect the expression and maturation of the coding sequences to which they are ligated. Such regulatory sequences include, inter alia, 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 increase translation efficiency (e.g., Kozak sequences); sequences that increase the stability of the protein; and, if necessary, a sequence that increases protein secretion.

Preferably, the regulatory sequences of the present invention comprise a promoter sequence, i.e. the gene encoding the ARNm of the present invention is preferably operably linked to a promoter allowing expression of said corresponding ARNm. Preferably, when the gene encoding the ARNm of the invention is located downstream of a promoter (i.e. promoter 3'), it is operably linked to the promoter, thereby forming an expression cassette.

As used herein, the term "promoter" refers to a nucleotide sequence, most often located upstream (5') of a coding sequence, which is recognized by ARN polymerase and other factors necessary for transcription, and thereby controls the expression of the coding sequence. As used herein, "promoter" specifically includes the minimal promoter, i.e., a short ADN sequence consisting of a TATA box and other sequences, which allows for transcription initiation site specificity. "promoter" within the meaning of the present invention also includes nucleotide sequences which include a minimal promoter and regulatory elements capable of controlling the expression of the coding sequence. For example, the promoter sequences of the present invention may comprise regulatory sequences, such as enhancer sequences which may influence the level of gene expression.

Advantageously, the promoters according to the invention are those which function together with the ARN polymerase used in cell-free transcription systems. For example, promoters recognized by the ARN polymerase of SP6 and T7 phages are well known to those skilled in the art. Thus, in the experimental part below, the pMBx-luc2 vector carrying the promoter recognized by the T7 ARN polymerase was used (Rog e and Betton, 2005). Furthermore, vectors containing such promoters are commercially available.

In a specific embodiment, the invention comprises ADN molecules encoding ARNm of the invention, operably linked to at least one regulatory element. Preferably, the ADN molecule encoding the ARNm of the invention is operably linked to an upstream located promoter sequence, thereby forming an expression cassette. In another embodiment, the invention consists of an ADN molecule encoding an ARNm of the invention operably linked to an upstream located promoter sequence, thereby forming an expression cassette.

Even more preferably, the ADN molecule of the invention comprises:

a promoter recognized by the T7 ARN polymerase, said promoter comprising the sequence of SEQ ID NO: 46;

a 5' -UTR region comprising the sequence defined by SEQ ID NO: 50. 71, 72, 73, 85, or 86;

an open reading frame; and

a 3' -UTR region comprising a sequence selected from SEQ ID NO: 53. 54, 55 and 56, respectively.

Advantageously, the regulatory sequences of the invention comprise a transcription terminator sequence, that is to say the gene encoding the ARNm of the invention is preferably operably 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 mechanism of transcription termination differs between prokaryotes and eukaryotes. Those skilled in the art know the signals used according to different cell types. For example, if they wish to express an ARNm of the invention in bacteria, they will use a Rho independent terminator (inverted repeat sequence followed by a series of T (uracil in transcribed ARN) or Rho dependent terminator (consisting of a consensus sequence recognized by Rho proteins). when the gene encoding an ARNm of the invention is located upstream of the terminator (i.e. 5' to the terminator), it is preferably operably linked to the terminator, thereby forming an expression cassette.

Advantageously, the terminators according to the present invention are those which function together with the ARN polymerase used in cell-free transcription systems. For example, terminators recognized by ARN polymerase of SP6 and T7 phage are well known to those skilled in the art. Vectors containing such terminators are commercially available.

In a specific embodiment, the invention includes ADN molecules encoding ARNm of the invention operably linked to at least one regulatory element and at least one transcription terminator.

In a third aspect, the invention also relates to a vector comprising at least an ADN or ARNm molecule according to the invention.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double loop of single-chain ADN into which additional ADN fragments can be ligated. Another type of vector is a viral vector, wherein additional ADN fragments can be ligated into the viral genome. Alternatively, the viral vector may comprise an ARNm (e.g., a retrovirus or ARN virus) in the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., integrating mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby replicated together with the host genome.

It is known to those skilled in the art that nucleic acid molecules of interest can be inserted into a number of vectors for the purpose of introducing and maintaining the nucleic acid molecule of interest in a eukaryotic or prokaryotic host cell. The choice of an appropriate vector will depend on the intended use of the vector (e.g., replication of the sequence of interest, expression of the sequence, maintenance of the sequence extrachromosomally, even integration into the chromosomal material of the host), and the nature of the host cell (e.g., the plasmid is preferably introduced into a bacterial cell, while the YAC is preferably used in yeast). These expression vectors may be plasmids, YACs, cosmids, retroviruses, episomes derived from EBV, and any vector deemed suitable by one skilled in the art for expression of the sequences. 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 comprises a promoter recognized by an ARN polymerase used in cell-free transcription systems, such as the promoters of SP6 and T7 phage. Even more preferably, the promoter carried by the plasmid is capable of directing expression of an ARNm of the invention in the presence of at least the ARN polymerase.

Preferably, the vectors of the present invention comprise an origin of replication to allow propagation of the vector in a host cell. The term "origin of replication" (also known as ori) is a unique ADN sequence that allows for the initiation of replication. Unidirectional or bidirectional replication begins with the sequence. Those skilled in the art know that the origin of replication varies in structure from one species to another; thus, although they all have certain characteristics, they are specific. Protein complexes form on this sequence and allow initiation of ADN opening and replication.

Advantageously, the vector comprising the expression cassette of the invention also comprises a selection marker to facilitate the identification of the cells comprising said vector, in particular after transformation. A "selection marker" according to the invention is a polynucleotide sequence carried by a vector, which allows the identification and selection of cells bearing said vector. Selection markers are well known to those skilled in the art. Preferably, it is a gene encoding a protein conferring antibiotic resistance.

The vector of the present invention comprising the nucleic acid of interest of the present invention is prepared by methods generally used by those skilled in the art. To introduce a polynucleotide into a host cell, the resulting clone can be introduced into a suitable host by standard methods known to those skilled in the art. Such methods may be transformation using dextran, precipitation using calcium phosphate, transfection using polypropylene, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, biolistic injection, and direct microinjection of ADN into the 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, such as a transporter (e.g., a nanotransporter) or a liposomal formulation, or a cationic polymer. In addition, these methods may be advantageously combined, for example, by using electroporation in connection with liposomes.

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

According to another preferred embodiment of the invention, the vector comprises an ARNm molecule of the invention. Preferably, the vector of the invention is a viral or synthetic ARN vector.

According to another aspect, the invention relates to a host cell comprising said vector. The term "host cell" as used herein refers to a cell into which a recombinant expression vector for expressing an ARNm of the invention has been introduced. The term should be understood to include not only the particular host cell described, but also its progeny. It is understood that certain modifications may exist over multiple generations due to mutation or environmental influences. As a result, progeny may not be identical to the parent cell, but are still included in the term "host cell" as used herein.

The ADNs and/or vectors described above are particularly useful for generating large quantities of ARNm of the invention.

According to another aspect, the present invention relates to a method for producing ARNm of the invention using the ADN or the vector described above. Therefore, ARNm can be produced by any method known to those skilled in the art. This can be done, for example, by chemical synthesis, by expression in vivo or by expression in vitro.

Preferably, the ARNm is expressed in vitro using a cell-free ARNm expression system. By "cell-free ARNm expression system" is meant in the context of the present invention a biochemical system that allows the synthesis of an ARNm of the invention in the absence of cells. Cell-free systems are based on the use of the organism's transcription machinery to produce specific ARNm from exogenous genetic information. Thus, in the context of the present invention, a cell-free ARNm expression system comprises all the elements required for producing ARNm in the absence of cells. The organisms from which this mechanism is extracted are many and varied, and are derived from both prokaryotic and eukaryotic organisms.

In particular, the system comprises in particular a transcription mechanism of cellular origin. More specifically, the system comprises an ARN polymerase capable of recognizing the promoter of the expression cassette described above. Thus, such an ARN polymerase is capable of directing transcription of an ARNm gene encoding the invention in the presence of suitable nucleotides and under suitable ionic conditions. Such systems have been well known to those skilled in the art for decades (see, for review, e.g., Beckert and Masquida, 2011). In cell-free systems, there are many ways to transcribe ADN into ARN. Kits available from a number of companies may also be used: new England Biolabs, Sigma Aldrich, Thermo Scientific, Promega, Roche Diagnostics, Ambion, Invitrogen, and the like.

In addition to the ARN polymerase, the cell-free in vitro transcription system also contains a reaction buffer. Advantageously, the system comprises each of the 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. Thus, the in vitro transcription yields of the ARNm of the invention are much higher than those of capped ARNm produced by in vitro reactions, which greatly reduces the costs of ARNm synthesis.

Preferably, the production process comprises a step of purifying the ARNm.

In a preferred embodiment, plasmid ADN (comprising a promoter recognized by a bacteriophage ARN polymerase, followed by an ADN sequence encoding an ARNm of interest) is contacted with a bacteriophage ARN polymerase in a cell-free in vitro transcription system. The ARNm synthesized by the method can then be purified. Preferably, the plasmid ADN is linearized prior to contact with an ARN polymerase in a cell-free in vitro transcription system. More preferably, the ADN is linearized by enzymatic digestion downstream of the 3' -UTR region. Even more preferably, the ADN is linearized by enzymatic digestion with Ssp1 or Eco53 kI.

Preferably, the ARN polymerase is bacteriophage T7 ARN polymerase.

ARNm molecules are capable of directing the production of a polypeptide of interest in a eukaryotic organism into which it is introduced. It is therefore particularly suitable for gene therapy or genetic vaccination. Thus, ARNm molecules of the invention may be used as a medicament or vaccine.

According to another aspect, the invention also relates to a pharmaceutical or vaccine composition.

Thus, more specifically, the present invention relates to pharmaceutical or vaccine compositions comprising the ARNm of the present invention. The ARNm contained in the composition is used to transfect cells, which can then translate the ARNm into protein. Preferably, these proteins have prophylactic or therapeutic activity.

Indeed, the inventors have surprisingly demonstrated that ARNm according to the present invention is more effective than conventional capped ARNm during tissue transfection. Indeed, the inventors have surprisingly demonstrated that the expression levels and durations obtained after in vivo transfection are at least as high as the capped ARNm. Specifically, the ARNm of the invention is expressed in vivo in the dermis or muscle of the mouse more than the capped ARNm of the control (figure 5, figure 15). In addition, the inventors have surprisingly shown that ARNm according to the invention persists in the cell over a long period (i.e. over 7 weeks). Finally, the inventors surprisingly show that co-transfecting a cell with a first ARNm according to the invention encoding a 2Apro protein and a second ARNm according to the invention encoding a second protein makes it possible to increase the translation of this second protein.

Thus, according to another aspect of the invention, a pharmaceutical composition comprises a 2Apro protein or an ARNm encoding said protein. Preferably, the 2Apro protein has the amino acid sequence of SEQ ID NO: 81, in sequence. Preferably, the ARNm encoding said protein has the amino acid sequence of SEQ ID NO: 80 in a sequence of seq id no. According to a particular embodiment of the invention, the pharmaceutical composition comprises at least two different cap-free messenger ribonucleic acid (ARNm) molecules comprising, from 5 'to 3':

5' -UTR region comprising at least one copy of GUCAGRYC (N)_7-19)GCCA(N_12-19) UGCNRYCUG (xrRNA) consensus sequence,

one copy of an Internal Ribosome Entry Site (IRES) ARN sequence; and

an Open Reading Frame (ORF),

wherein at least one ARNm molecule comprises an open reading frame encoding a 2Apro protein.

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

preferably, the composition will be supplemented with excipients and/or pharmaceutically acceptable carriers. In the present specification, the term pharmaceutically acceptable carrier refers to a compound or a combination of compounds included in a pharmaceutical composition which does not cause side effects and allows, for example, to improve the ease of administration of the active compound, which increases the lifetime and/or effectiveness in vivo, which increases the solubility in solution, or even improves storage. Such pharmaceutically acceptable carriers are well known and will be adjusted by the person skilled in the art according to the nature and mode of administration of the active compound selected. In the present specification, the term "pharmaceutically acceptable excipient" refers to a compound or a combination of compounds included in a pharmaceutical composition which does not cause side effects and allows, for example, to improve the ease of administration of the active compound, which increases its lifetime and/or effectiveness in an organism, which increases its solubility in solution or even improves storage. The excipient may in particular be added to the composition immediately prior to administration, for example if the ARN is stored in lyophilized form. Pharmaceutically acceptable excipients/carriers are well known and will be adjusted by the person skilled in the art according to the nature and mode of administration of the active compound selected. The Science and Practice of Pharmacy, Remington, 22 nd edition, Pharmaceutical Press, London, UK (2013), describes various excipients specifically. For example, pharmaceutically acceptable compositions comprise sterile water and/or chloroquine as excipients. "chloroquine" excipients also include any variant or analog thereof, such as primaquine.

Indeed, the inventors have surprisingly shown that under certain conditions, co-injection of chloroquine with ARNm makes it possible to increase the transfection efficiency. Specifically, the transfection efficiency of ARNm complexed with a "CPP" -type peptide was improved (see figure 10).

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 of 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 μ g ARN: 2.5-20 mug chloroquine.

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

Preferably, these compositions will be administered by intramuscular, intradermal, intraperitoneal or subcutaneous routes, by respiratory routes, or by topical routes. These compositions are preferably for injection into mammalian tissue, even more preferably, humans. These compositions are preferably intended for injection by intramuscular, intravenous, intradermal, intraperitoneal or subcutaneous routes, according to methods known to those skilled in the art. The pharmaceutical composition of the invention may be administered several times staggered over time. The mode of administration, dosage and optimal galenical form can be determined according to the criteria usually considered when establishing a treatment suitable for a patient, for example, the age or weight of the patient, the severity of the patient's general condition, tolerance to the treatment, and the side effects observed.

Parenteral administration forms include aqueous suspensions, isotonic saline solutions, or sterile and injectable solutions, which may contain pharmacologically compatible dispersing and/or wetting agents. Forms that can be administered by the respiratory route include aerosols. Topical administration forms include patches, gels, creams, ointments, lotions, sprays, eye drops.

Methods of making compounds for parenteral administration will be known or apparent to those skilled in the art and are described in more detail, for example, in Pharmaceutical Sciences of Remington, 17 th edition, Mack Publishing Company, Easton, Pa, (1985), and 18 th and 19 th editions thereof. The use of media and agents for pharmaceutically active substances 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.

The effective dosage of the compounds of the invention will vary with a number of parameters, such as the chosen route of administration, the body weight, the age, the sex, the state of progression of the pathology to be treated, and the sensitivity of the individual to be treated.

According to a particular aspect, the invention relates to the use of said composition in gene therapy. The compositions of the present invention are useful for treating or modulating a variety of diseases, such as: cancer, hereditary 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.

According to another aspect, the invention relates to said composition and its use in genetic vaccines. For example, the compositions of the present invention may 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.

The inventors specifically show that the ARNm of the invention is stable in vivo. Indeed, the ARNm of the invention induced an amount of protein synthesis in vivo that was at least as high as that of capped ARNm, indicating that its resistance to Xrn1 is at least as effective as that of capped ARNm. In addition, the inventors specifically showed that the ARNm of the present invention is present in cells for a long period of time. Such ARNm molecules are also highly advantageous because their production costs are much lower than those of capped ARNm molecules. Finally, the inventors have shown that ARNm comprising an ARN aptamer that penetrates cells directly (e.g., ARN aptamer C), or that penetrates cells via a CPP peptide (e.g., ARN aptamer a), transfects cells more efficiently, which advantageously improves production of one or more proteins of interest.

The present invention will be more specifically described by way of the following examples.

Drawings

FIG. 1: structure of messenger ARN.

(A) The capped MB5-luc 2ARNm corresponds to the traditional messenger ARN encoding luciferase, having a cap analogue at its 5 'end and a 3' -UTR region comprising a poly (A) tail. (B) MB5-luc 2ARNm is identical to (A) except for the absence of the cap analogue. (C) MB7-luc 2ARNm lacks a cap and has a stem-loop at its 5' end and an Internal Ribosome Entry Site (IRES) for EMCV virus. (D) MB8-luc 2ARNm lacks a cap and has a stem-loop with two Xrn1 resistant sequences (xrna 1 and xrna 2) from West Nile Virus (WNV) and an IRES from EMCV virus in the 5' UTR region. (E) MB9-luc 2ARNm is similar to (D) except that the 3'-UTR and poly (A) of (D) have been replaced by the 3' -UTR from Kunjin virus (KUN). (F) The uncapped MB11-luc 2ARNm was similar to (D) except that aptamer a was added in the 5' region upstream of the two Xrn1 resistant sequences (xrna 1 and xrna 2) of West Nile Virus (WNV) and the IRES of EMCV virus, and it did not have a stem-loop. MB13-luc2(G) and MB14-luc2(H) ARNm have this same configuration, but contain 5' stem-loops followed by ARN aptamers B and C, respectively. (I) The capped MB15-luc 2ARNm was similar to (A) except aptamer A was added in the 5' region behind the cap. (J) MB17-luc 2ARNm is similar to (D) except aptamer A is added 5' between the stem-loop and xrRNA sequences. (K) MB18-luc 2ARNm 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.

FIG. 2: luciferase expression kinetics in Caco-2 cells over four days.

Fused (confluents) Caco-2 cells were transfected with MB5-luc2 capped ARNm (diamonds) and uncapped ARNm (circles) and MB7-luc 2ARNm (triangles) and MB8-Iuc 2ARNm (squares). Expression kinetics were followed for four days.

FIG. 3: kinetics of luciferase expression in Caco-2 cells within ten days.

Fused Caco-2 cells were transfected with a capped MB5-luc 2ARNm (diamonds), MB8-luc 2ARNm (squares), and MB9-luc 2ARNm (triangles). Expression kinetics were followed for ten days.

FIG. 4: long-term luciferase expression kinetics in human mesenchymal stem cells.

MB8-Iuc 2ARNm was complexed with pepMB1 peptide (positive charge: ARNm negative charge ratio of pepMB1 peptide about 2.2: 1) and mesenchymal stem cells were transfected in 48-well plates at 2. mu.g of the above complex per well for 1 hour. Luciferase activity was then monitored for approximately 48 days.

FIG. 5: mouse muscle and dermis were transfected.

The muscle and dermis of male BALB/cByJ mice were transfected with 10. mu.g and 5. mu.g of uncapped MB8-luc 2ARNm or capped MB5-luc 2ARNm, respectively. Luciferase activity was measured in muscle and skin 16 hours and 18 hours after injection, respectively.

FIG. 6: toxicology studies of MB8-2Apro ARNm.

To evaluate the cytotoxic effect of 2Apro protein expression, C2C12 cells were transfected with MB8-luc 2ARNm alone, or MB8-Iuc 2ARNm and MB8-2Apro ARNm at 465: 1 or 9: 1 to transfect C2C12 cells. 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: non-lysed untransfected cells or MB8-luc 2ARNm transfected cells alone. Positive control: untransfected cells lysed with Triton X-100.

FIG. 7: effect of 2Apro protein on luciferase expression as function of MB8-luc2 ARNm: the molar ratio of MB8-2Apro ARNm varied.

In the presence of increased amounts of MB8-2Apro ARNm, C2C12 cells were transfected with MB8-luc2 ARNm. MB8-luc2 ARNm: the molar ratio of MB8-2Apro ARNm is 540: 1 to 240: 1. the total amount of ARNm transfected per well was 750 ng. Luciferase activity was measured 18 hours after transfection.

FIG. 8: luciferase expression kinetics in seven days with or without MB8-2Apro ARNm.

Fused C2C12 cells were transfected with MB8-luc 2ARNm (black line) alone or fused C2C12 cells were transfected with MB8-luc 2ARNm in combination with MB8-2Apro ARNm (grey dashed line). MB8-luc2 ARNm: the molar ratio of MB8-2Apro ARNm is 465: 1. expression kinetics were measured by luciferase activity levels for 7 days.

FIG. 9: effect of 2Apro protein on expression of the different messenger ARN luciferases.

The effect of the 2Apro protein on the expression of luciferase from the different messenger ARN was evaluated. In each case, ARNm encoding luciferase was transfected (-) alone or in co-transfection (+) with a second ARNm encoding 2Apro protein at a molar ratio of 465: 1 have the same characteristics. (1) MB8-luc 2ARNm alone; (2) MB8-luc 2ARNm co-transfected with MB8-2Apro ARNm; (3) separately capped MB5-luc2 ARNm; (4) co-transfection of the capped MB5-luc 2ARNm with the uncapped MB8-2Apro ARNm; (5) MB5-luc 2ARNm alone lacking the cap analog; (6) co-transfecting MB5-luc 2ARNm lacking the cap analogue with MB8-2Apro ARNm lacking the cap analogue; (7) MB7-luc 2ARNm alone; (8) MB7-luc 2ARNm was co-transfected with MB8-2Apro ARNm. Luciferase activity was measured 18 hours after transfection.

FIG. 10: effect of different aptamers on transfection efficiency of capped ARNm molecules in muscle.

Muscles from male BALB/cByJ mice were transfected with: 5 μ g of MB8-luc 2ARNm, MB13-luc 2ARNm, MB14-luc 2ARNm, MB11-luc 2ARNm complexed with the M12-H6 peptide, or MB11-luc 2ARNm complexed with the M12-H6 peptide in the presence of 5 μ g of chloroquine. Luciferase activity was measured 16 hours after injection.

FIG. 11: according to CPP3-H6 peptide in dermis: transfection efficiency of ARNm at molar ratio of MB11-luc2 ARNm.

The dermis OF male OF1 mice was transfected with 5.6. mu.g OF MB11-luc 2ARNm complexed with CPP3-H6 peptide, at an increased molar ratio (CPP 3-H6: MB11-luc 2ARNm between 1: 8 and 1125: 1). Luciferase activity was measured 18 hours after injection. The molar ratio allowing optimal transfection was 1CPP 3-H6: 4MB11-luc2 ARNm.

FIG. 12: according to CPP1-H6 peptide in dermis: transfection efficiency of ARNm at molar ratio of MB11-luc2 ARNm.

The dermis OF male OF1 mice was transfected with 5.6. mu.g OF MB11-luc 2ARNm complexed with CPP1-H6 peptide, at an increased molar ratio (CPP 1-H6: MB11-luc 2ARNm between 1: 8 and 3: 1). Luciferase activity was measured 18 hours after injection. The molar ratio allowing optimal transfection was 1CPP 1-H6: 4MB11-luc2 ARNm.

FIG. 13: according to CPP2-H6 peptide in dermis: transfection efficiency of ARNm at molar ratio of MB11-luc2 ARNm.

The dermis OF male OF1 mice was transfected with 5.6 μ g OF MB11-luc 2ARNm complexed with CPP2-H6 peptide at an increased molar ratio (CPP 2-H6: MB11-luc 2ARNm molar ratio between 1: 4 and 2.75: 1). Luciferase activity was measured 18 hours after injection. The molar ratio allowing optimal transfection was 2 CPP 2-H6: 1MB11-luc2 ARNm.

FIG. 14: effect of aptamer a on transfection efficiency of capped ARNm molecules in dermis.

ARN aptamer a was inserted into the 5' -UTR of a conventional capped MB5-luc 2ARNm to produce a capped MB15-luc2 ARNm. In view of the results previously obtained (see fig. 12), 5.6 μ g was mixed at a molar ratio of 2 CPP 2-H6: 1MB15-luc 2ARNm or alternatively this molar ratio OF capped MB15-luc 2ARNm transfected male OF1 mice complexed with CPP2-H6 peptide. Transfection was not improved with this construct in the presence or absence of aptamer A or CPP2-H6 peptide.

FIG. 15: effect of 5' -SL on transfection efficiency of ARNm molecules in dermis.

Influence of stem-loops (here "5' -SL" with the sequence of SEQ ID NO: 87) when the stem-loop is followed 5 nucleotides downstream by the xrRNA1 sequence (MB8-luc2 and MB18-luc2 ARNm) on transfection efficiency compared to ARNms lacking stem-loops (MB11-luc2 ARNm) and those with stem-loops located more than 70 nucleotides upstream of xrRNA1 (MB17-luc2 ARNm). Conventional MB5-luc2 capped ARNm was used as a control for efficacy of the 5' -SL followed by the two xrRNA sequences. The dermis OF male OF1 mice was transfected with 5.6 μ g OF each different ARNm and luciferase activity was measured 18 hours after injection. Aptamer a of MB11-luc2 was not linked to the peptide and therefore had no effect on the transfection efficiency of this ARNm.

Detailed Description

The invention is illustrated by the following non-limiting examples. As will be appreciated by those skilled in the art, the teachings include alternatives, modifications, and equivalents.

Example 1: plasmid construction

By chemical synthesis, a peptide corresponding to SEQ ID NO: 49. SEQ ID NO: 50. or SEQ ID NO: 51, integrated into the ADN vector and sequenced by ProteoGenix. The ADN fragment was excised with restriction enzymes. Plasmids of the pMBx-luc2 series were digested with the same restriction enzymes and the ADN fragment was integrated into these plasmids by the action of T4 ADN ligase. These plasmids contain a gene encoding luciferase (SEQ ID NO: 62) inserted downstream of the bacteriophage T7 promoter. A short non-coding 3' -UTR sequence followed by a sequence according to SEQ ID NO: 53, downstream of the luciferase gene. Different non-coding 5' -UTR sequences separate the promoter from the luciferase gene. The plasmids thus constructed were amplified, verified and linearized downstream of the transcription polyadenylation sequence by restriction enzymes.

Example 2: plasmid linearization and in vitro transcription of ARNm

The Ssp1 restriction site is located immediately downstream of poly (A) of each plasmid (see SEQ ID NO: 54). 10. mu.g of plasmid were digested with 20 units of Ssp1-HF restriction enzyme (New England Biolabs) in 1 XCutSmart buffer and digested at 37 ℃ for 4 hours.

Then, 8. mu.l of the plasmid linearized with Ssp1-HF was mixed with 2. mu.l of 10 XT 7 ARN polymerase reaction buffer, 2. mu.l each of the four nucleoside triphosphates (ATP, GTP, CTP and UTP) and 2. mu.l of T7 ARN polymerase solution (New England Biolab). The cap analog is included in the reaction mixture for the synthesis of capped ARNm. Synthesis of the cap-less ARNm was omitted.

The transcription was carried out for three to ten hours and in a block heater at 37 ℃. Then, 1. mu.L of TURBO DNase (Thermo Fisher) was added to degrade the plasmid, and the mixture was incubated at 37 ℃ for 15 minutes.

MB5-luc 2ARNm is a conventional ARNm encoding luciferase. It has an amino acid sequence according to SEQ ID NO: 57, selected for optimal initiation of translation and a 3' -UTR region comprising a poly (a) tail according to SEQ ID NO: 60. The ARNm was synthesized with or without capping (see fig. 1(a) and 1 (B)).

The uncapped MB7-luc 2ARNm had a stem-loop at its 5 'end to replace the 5' -UTR of the MB5-luc 2ARNm, followed by the IRES sequence of the EMCV virus. Thus, although there is no cap, the IRES sequence will allow it to efficiently recruit ribosomes. However, the ARNm is sensitive to Xrn1 enzyme (see fig. 1 (C)). The 5' -UTR region of MB7-luc 2ARNm corresponds to the sequence of SEQ ID NO: 58 and the 3' -UTR region correspond to SEQ ID NO: 60.

the uncapped MB8-luc 2ARNm had a stem-loop at its 5' end, followed by two contiguous sequences of anti-Xrn 1 from the flavivirus WNV 3' -UTR in the 5' -UTR region, called xrna 1 and xrna 2(Kieft et al, 2015). These sequences are followed by the IRES sequence of EMCV virus, and thus are protected by two anti-Xrn 1 sequences (see fig. 1 (D)). The 5' -UTR region of MB8-luc 2ARNm corresponds to the sequence of SEQ ID NO: 59 and the 3' -UTR region correspond to SEQ ID NO: 60. the cost of producing uncapped MB8-luc 2ARNm was about 30 times lower than the cost of producing capped ARNm.

The capped MB9-luc 2ARNm differed from the capped MB8-luc 2ARNm 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 the kungunya virus. This region lacks the poly (A) sequence (see FIG. 1 (E)). The 3' -UTR region of MB9-luc 2ARNm corresponds to the sequence of SEQ ID NO: 61.

the capped MB11-luc 2ARNm differed from the capped MB8-luc 2ARNm only in the absence of a stem-loop at the 5 'end and the presence of aptamer in the upstream 5' region of two consecutive sequences resistant to Xrn1 (see fig. 1 (F)). MB11-luc 2ARNm comprises SEQ ID NO:64 aptamer A; the 5' -UTR region of MB11-luc 2ARNm thus corresponds to SEQ ID NO: 67.

the cap-free MB13-luc2 and MB14-luc 2ARNm differ from the cap-free MB8-luc 2ARNm only in the presence of aptamers in the upstream 5' region of two consecutive sequences resistant to Xrn1 (see FIG. 1(G, H)). MB13-luc 2ARNm comprises SEQ ID NO: 65 aptamer B; the 5' -UTR region of MB13-luc 2ARNm thus corresponds to the nucleotide sequence of SEQ ID NO: 68. MB14-luc 2ARNm comprises SEQ ID NO: 66 aptamer C; thus, the 5' -UTR region of MB14-luc 2ARNm corresponds to the nucleotide sequence of SEQ ID NO: 69.

MB15-luc 2ARNm corresponds to a capped MB5-luc 2ARNm in which aptamer A (SEQ ID NO: 64) has been inserted into the 5' -UTR region (see FIG. 1 (I)); thus, the 5' -UTR region of MB15-luc 2ARNm corresponds to the nucleotide sequence of SEQ ID NO: 70.

MB17-luc 2ARNm differs from uncapped MB11-luc 2ARNm only in the presence of a stem-loop at the upstream 5' end of aptamer A (see FIG. 1 (J)). MB17-luc 2ARNm comprises SEQ ID NO:64 aptamer A; thus, the 5' -UTR region of MB17-luc 2ARNm corresponds to the nucleotide sequence of SEQ ID NO: 83.

the difference between the capped MB18-luc 2ARNm and the capped MB8-luc 2ARNm was only the presence of aptamers in the 5' region between two consecutive sequences resistant to Xrn1 and the IRES sequence (see FIG. 1 (K)). MB18-luc 2ARNm comprises SEQ ID NO:64 aptamer A; thus, the 5' -UTR region of MB18-luc 2ARNm corresponds to the nucleotide sequence of SEQ ID NO: 84.

example 3: purification of messenger ARN

The different luciferases ARNm were purified using the MegaClear kit (Ambion). Mu.l of the elution solution, 350. mu.l of the binding solution concentrate and 250. mu.l of 100% ethanol were added to 21. mu.l of the previous mixture. This 700. mu.l was placed on a Filter Cartridge (Filter Cartridge) and centrifuged at 10,000 Xg for 1 minute. The filter retains messenger ARN. Two washes with 500. mu.l of wash solution were performed and centrifuged at 10,000 Xg for 1 minute. The ARN was then eluted from the filter by adding 50 μ Ι of elution solution twice and heated to 70 ℃ for ten minutes in a block heater. The eluate was obtained by centrifugation at 10,000 Xg for 1 minute.

A second purification step was performed by precipitation with lithium chloride. Mu.l LiCl precipitation solution was added to 100. mu.l of the eluate. The mixture was cooled at-20 ℃ for 1 hour and then centrifuged at 4 ℃ for 15 minutes at maximum speed. The pellet was washed with 500 μ l of 70% ethanol and finally centrifuged at 4 degrees for 5 minutes at maximum speed. Messenger ARN precipitates were air dried for a few minutes and then resuspended in sterile deionized water. The concentration of ARNm solution was determined by measuring the absorbance at 260nm using a spectrophotometer.

Example 4: assembly of messenger ARN/pepMB1 Complex

The cationic peptide pepMB1 was synthesized by ProteGenix, purified and lyophilized. The amino acid sequence is as follows: CRRRRRRRRC are provided. The lyophilizate was resuspended in sterile deionized water.

Mu.g luciferase ARNm was mixed with 5. mu.g pepMB1 to a final ARN concentration of 20. mu.g/ml. The mixture was incubated at room temperature (20-25 ℃) for 15 minutes and then frozen at-80 ℃. The ARNm/pepMB1 complex was then lyophilized for about 20 hours.

Example 5: transfection of Caco-2 or C2C12 cells

Materials and methods:

a) culture and inoculation of Caco-2 or C2C12 line cells

All cells were operated in laminar flowThe process is carried out under a purifying hood. The Caco-2 cell line (ECACC) was cultured in dmem (gibco) supplemented with a mixture of non-essential amino acids, antibiotics and antifungals, and fetal bovine serum (final 15%). At 75cm²The flask (Corning) was cultured at 37 ℃.

When the number of cells required for seeding 48-well plates (Corning) was reached, the cells were detached from the bottom of the flask by treatment with 3ml of TryPLE Select 1X (Gibco) for 5 minutes at 37 ℃. 7ml DMEM was added to neutralize TryPLE Select 1X. Cells were centrifuged at 100Xg for 10 min at room temperature. The cell pellet was then resuspended in 10ml of culture medium. 250. mu.l of this cell suspension were introduced into each well of a 48-well plate and the latter was placed at 37 ℃ in a medium containing 5% CO₂In an incubator.

After seeding, C2C12 cells can remain confluent (confluence) in the wells of a 48-well plate for about 12 days. After this period, these cells differentiate into the intestinal epithelium, thereby affecting ARNm translation. In contrast, human mesenchymal stem cells can be preserved in confluent cultures for more than 7 weeks. Thus, mesenchymal stem cells (Millipore, human mesenchymal stem cells (bone marrow)) were cultured in 48-well plates (Corning) in ready-to-use media (Millipore, mesenchymal stem cell expansion media) for up to 48 days. For cells that will be lysed more than 5 days after transfection, the culture medium was changed 3 times per week.

b) Transfection of Caco-2 or C2C12 cells

For Caco-2 cells, transfection was performed in five different wells for each ARNm. A lyophilisate of the ARNm/pepMB1 complex (Proteogenix) was resuspended in 750. mu.l transfection buffer (20mM Hepes, 40mM KCl and 100mM trifluoroacetic acid).

For C2C12 cells, transfection of MB8 ARNm was performed as indicated below. By contacting a peptide with a positively charged peptide of about 2.2: ratio of negatively charged ARNms, ARNms were incubated for 30 minutes at room temperature to assemble MB8-luc2 ARNm/pepMB1 complex (Proteogenix). The solution was then diluted with 3 × DMEM to obtain 1 × final DMEM.

In both cases, the wells were emptied of culture medium to introduce 150. mu.l of ARN/pepMB1 complex solution (1. mu.g of ARNm per well for Caco-2 cells; and C2C12 cell pair)Should be 2. mu.g per well). Exposing the cells to CO₂Incubate at 37 ℃ for 30 minutes (Caco-2 cells) or 1 hour (C2C12 cells). The ARNm/pepMB1 complex solution was then aspirated and replaced with 250. mu.l of culture medium. Then, the cells were CO at 37 ℃ depending on the cell type₂Incubate in incubator for 6 hours to 48 days.

c) Caco-2 or C2C12 cells were lysed, luciferase activity was measured six hours to 48 days after transfection, and cells were lysed to perform expression kinetics of luciferase protein. The culture medium was aspirated and replaced with 250. mu.l of lysis buffer (Luciferase Assay System, Promega). Mu.l of each cell lysate were placed in a tube suitable for luminometers (Berthold Technologies). 100 μ l of luciferase substrate (Promega) was added to the cell lysate by luminometer. Then, the photometer measures the amount of light emitted by the enzymatic reaction catalyzed by luciferase. The results are expressed in Relative Light Units (RLU). The amount of luciferase protein produced by Caco-2 cells or C2C12 cells was normalized by means of luciferase ARNm by analyzing total cellular protein using a 660nm protein assay kit (Pierce). For this, 100. mu.l of cell lysate were mixed with 1.5ml of reagent and the absorbance was measured at 660 nm. The calibration range was performed using bovine serum albumin solution. Thus, luciferase activity is expressed as RLU per mg of protein.

As a result:

the results are shown in fig. 2, 3 and 4. The cap-free MB5-luc 2ARNm induced low and short expression of luciferase protein in Caco-2 cells. Without the cap, ARNm is rarely translated into protein and is rapidly degraded by Xrnl (see figure 2).

The capped MB7-luc 2ARNm provided stronger and more durable luciferase protein expression in Caco-2 cells than the capped MB5-luc2 ARNm. The IRES region recruits ribosomes, but no apparent resistance to Xrn1 (see fig. 2).

The kinetics of luciferase expression induced by the uncapped MB8-luc 2ARNm were similar to that obtained by the capped MB5-luc 2ARNm (see FIGS. 2 and 3). This means that, similar to the cap of MB5-luc 2ARNm, the addition of two sequences of WNV virus with Xrn1 resistance confers resistance of ARNm to Xrn 1. The expression of luciferase induced by MB7-luc 2ARNm, lacking the cap and sequences resistant to Xrn1, was between that of the cap-free MB8-luc 2ARNm and that of the cap-free MB5-luc 2ARNm (see FIG. 2).

MB9-luc 2ARNm differs from MB8-luc 2ARNm in that the 3' -UTR end lacks poly (A). It induced significantly lower and consistently shorter expression of luciferase in Caco-2 cells than that induced by MB8-luc 2ARNm (see figure 3).

In human mesenchymal stem cells, MB8-luc 2ARNm unexpectedly and advantageously induced the expression of luciferase, which lasted for a long time. Indeed, even though expression decreased over time, it was detectable 48 days after transfection (see fig. 4).

Example 6: transfection of mouse muscle and dermis

Materials and methods:

a) animal feeding:

for muscle, 8-week old male BALB/cByJ mice were housed in open cages, with 5 animals per cage. The day/night cycle is managed by an automatic device (12h day/12 h night). They were kept and they were able to drink filtered water ad libitum. For skin, 6-week-old male OF1 mice were housed in open cages OF four animals per cage.

b) Extemporaneous preparation of ARNm samples:

for each intramuscular injection, 100. mu.l of naked ARNm solution containing 230mM NaCl was prepared.

For each intradermal injection, 17. mu.l of naked ARNm solution containing 160mM NaCl was prepared.

When CPP-H6 peptide was used, it was mixed with ARNm and MgCl at 5mM Hepes pH 7.5 and 0.7mM at room temperature₂Incubate in the presence for 30 minutes.

c) Intradermal and intramuscular injection of ARNm:

anesthetizing mice was achieved by using isoflurane. For intramuscular injection, analgesia is given by buprenorphine injection. For intradermal injections, the skin of the back was shaved three to four days ago (for details see example 10).

Mu.l and 17. mu.l of ARNm solutions were injected into the biceps femoris and skin, respectively. The animals were returned to their cages until the next morning.

d) Skin and muscle samples:

for muscle, mice were CO administered 16 hours after injection₂No pain and death. For skin, at 18 hours post injection, mice were anesthetized with isoflurane and killed painlessly by cervical dislocation. Skin and muscle injection sites were obtained. These biopsies were washed with physiological saline, cut into fine pieces, and then placed in tubes containing lysis buffer (Promega). The tubes were immediately frozen in liquid nitrogen.

e) Lysis of skin and muscle biopsied cells:

three freeze/thaw cycles were performed for each skin and muscle biopsy. In practice, tubes containing biopsy and lysis buffer were frozen at-80 ℃ for 10 min. They were then thawed in a water bath at room temperature for two minutes and mixed briefly using vortexing. The tubes were then centrifuged at 5000x g for 5 minutes at 20 ℃ to pellet the tissue debris and obtain a clear cell lysate supernatant.

f) Measurement of luciferase Activity:

20 μ l of each cell lysate was used to measure luciferase expression in each biopsy. The tube luminometer added 100. mu.l of luciferase substrate (Promega) to each sample, and measured the luminescence amount for 10 seconds. Results are expressed in relative light units or RLUs.

The cell lysates were then diluted 8 to 20 fold using 660nm protein assay kit (Pierce) for protein analysis. 100 μ l of the diluted cell lysate was mixed with 1.5ml of the reagent for 6 minutes and the absorbance was measured at 660 nm. Bovine serum albumin was calibrated in the range of 0 to 750. mu.g/ml.

As a result:

the results are shown in FIG. 5. At 16 hours (muscle) or 18 hours (dermis) post-injection, uncapped MB8-luc 2ARNm induced higher luciferase expression in skeletal muscle (a) and skin (B) than capped MB5-luc2 ARNm. These results indicate that the presence of two xrna sequences (here from WNV virus) and IRES (here from EMCV) renders ARNm resistant to Xrn1 in vivo and that the translation efficiency is superior to that of the MB5-luc 2ARNm cap. Indeed, it was very surprising that transfection of 10 μ g of MB8-luc 2ARNm into mouse muscle tissue resulted in 2.6-fold higher luciferase expression than that obtained with the same dose of capped MB5-luc 2ARNm (see FIG. 5A). Similarly, transfection of 5. mu.g of MB8-luc 2ARNm in skin produced 9.3-fold higher luciferase expression than that obtained at the same dose of capped MB5-luc 2ARNm (see FIG. 5B).

Thus, MB8-luc 2ARNm may completely replace the capped MB5-luc 2ARNm, and may even be more advantageous.

Example 7: cytotoxicity of MB8-2Apro ARNm

Materials and methods:

expression of the 2Apro protein in mammalian cells can induce toxicity by apoptosis or necrosis until cell death results (Goldstaub et al, 2000). During these processes, the cell releases Lactate Dehydrogenase (LDH) into the extracellular environment. LDH activity can be measured using a commercial kit Promega CytoTox96 Non-Radioactive Cytoxicity Assay.

MB8-2Apro ARNm (SEQ ID NO: 80) is an ARNm encoding the 2A non-structural protein (2Apro, sequence with SEQ ID NO: 81) from the genome of human rhinovirus 2(HRV 2). It has an amino acid sequence according to SEQ ID NO: 57, which is identical to MB8-luc 2ARNm and comprises a 5' non-coding sequence (UTR) according to SEQ ID NO: the 3' -UTR region of the poly (A) tail of 60.

750ng of MB8-luc 2ARNm alone or a mixture of MB8-luc 2ARNm and MB8-2Apro ARNm were complexed with pepMB1 peptide as described in example 4.

MB8-luc 2ARNm alone or MB8-luc 2ARNm mixed with MB8-2Apro ARNm at two different molar ratios were transfected into C2C12 cells. Specifically, C2C12 cells in wells from 48-well plates were incubated with ARNm/pepMB1 complex for one hour. After 18 hours, luciferase activity was measured (fig. 6). Untransfected C2C12 cells that were not lysed 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.

As a result:

the ARNm blend did not induce cytotoxicity even at the highest molar ratio (MB8-luc2 ARNm: MB8-2Apro ARNm ratio of 9: 1). This indicates that expression of viral protease does not induce cytotoxicity (fig. 9).

Example 8: optimization of luciferase expression kinetics by Co-transfection of MB8-luc2 and MB8-2Apro ARNm

Materials and methods:

MB8-luc2 and MB8-2Apro ARNm were subsequently co-transfected into C2C12 cells at varying ratios, according to the method described above, without toxicity. After 18 hours, luciferase activity was measured (fig. 7). Kinetics were then measured from 6 hours to 7 days post-transfection according to the method described above to determine whether improved luciferase expression was observed only at 18 hours post-transfection.

As a result:

unexpectedly, co-transfection of MB8-luc2 and MB8-2Apro ARNm increased luciferase expression of MB8-luc 2ARNm in C2C12 cells by at least 2.5-fold in all ratios tested. The best MB8-luc2 ARNm: MB8-2Apro ARNm molar ratio 465: 1 and increased luciferase expression by 3.4-fold.

Kinetics performed 6 hours to 7 days after transfection unexpectedly demonstrated that improved luciferase expression was observed for at least one week. In fact, luciferase expression increased by an average of 2.4-fold (FIG. 7).

Example 9: effect of ARN aptamers on ARNm transfection efficiency in muscle

To improve the internalization of ARNm molecules according to the invention, different aptamers are incorporated into ARN molecules, as detailed below. The effect of these aptamers was then evaluated in vivo to determine whether an improvement in luciferase expression could be observed.

Materials and methods:

aptamer selection

Aptamers were selected that penetrated C2C12 cells. First, a 5' primer is hybridized, followed by extension of single-stranded ADN from the single-stranded ADN library, to generate double-stranded ADN. The double-stranded ADN thus obtained is then precipitated and purified according to methods well known to the person skilled in the art.

Then, an ARN aptamer library was obtained by transcribing the purified fragment using the T7 DuraScribe transcription kit (20 μ l/round), followed by ARN purification using ssDNA and ARN purification kits. Finally, the solution was treated with DNAseI to remove contaminating ADN. Aptamers were dissolved in 1 × DMEM + ITS at 8 μ M ARN (1288 μ g/5 ml). For selection of aptamers, 5ml of DMEM/ITS/ARN solution was added to cells previously washed twice with DMEM without antibiotics or serum. Cells were incubated at 37 ℃ for 1 hour, and flasks containing the mixture were briefly shaken every 15 minutes. Then, the flasks containing the cells were placed on ice and the cells were washed 5 times with 15 mL of cold 1 × PBS to remove ARN aptamers that did not penetrate the cells. Then, the cells were lysed with trizol (invitrogen), and total ARN was extracted by a phenol-chloroform method. Endogenous ARNs were digested with RNase A, the remaining ARNs hybridized to the 3' primer, and reverse transcribed by Superscript III enzyme (ThermoFisher) before PCR amplification in the presence of 5' primer (100. mu.M), 3' primer (100. mu.M), Q5 High Fidelity ADN polymerase (NEB) and 1 Xconcentration of Q5 High-Fidelity Master Mix buffer. All these steps allowing to obtain a library of aptamer ARNs were repeated. Thus, two rounds of selection of ARN aptamers that penetrate C2C12 cells were performed.

Two ARN aptamers that penetrated C2C12 cells (B and C) were selected and sequenced (SEQ ID NOS: 65 and 66, respectively). ARN aptamers B and C were then inserted into the 5' -UTR of MB8-luc 2ARNm upstream of xrRNA1, yielding MB13-luc2 and MB14-luc 2ARNm, respectively.

Aptamers that tightly bind polyhistidine peptide motifs

A second strategy aimed at improving ARNm internalization consists of: in the 5' -UTR upstream of xrRNA1 of MB8-luc 2ARNm, another ARN aptamer (aptamer A) capable of binding strongly to the poly-histidine peptide motif in the presence of magnesium was inserted. The ARNm incorporated with ARN aptamer A was designated MB11-luc 2. The mouse muscle fiber penetrating peptide M12 (see Gao et al, 2014) was linked into the hexahistidine motif by a spacer (here containing glycine and serine amino acids). The different spacers are illustrated by way of the following sequence example: SEQ ID NO: 75 (PRQPPRSISSHP)GGGGSGGGGSGGGGSGGGGSGGHHHHHH)、SEQ ID NO： 76(PQRDTVGGRTT PPSWGPAKAGGGGSGGGGSGGGGHHHHHH)、SEQ ID NO：77(GPFHFYQFLFPPVGGGGSGGGGSGGG GSGGGGSGHhhhhhhh) or SEQ ID NO: 78 (GSPWGLQHHPPRT)GGGGSGGGGSGGGGSGGGGSGHhhhhhhh) (spacer sequences are underlined). The peptide thus formed was named M12-H6(SEQ ID NO: 75). MB11-luc 2ARNm was incubated with M12-H6 peptide for 30 minutes at room temperature before re-injection into the biceps femoris of mice.

In vivo transfection

Biceps femoris of male BALB/cByJ mice were transfected with 5. mu.g of MB8-luc 2ARNm, MB13-luc 2ARNm, MB14-luc 2ARNm, or MB11-luc 2ARNm complexed with M12-H6 peptide, in the presence or absence of 5. mu.g of chloroquine. Luciferase activity was measured 16 hours after injection.

As a result:

MB13-luc 2ARNm transfected muscle as MB8-luc 2ARNm (FIG. 10). Very advantageously, MB14-luc 2ARNm comprising the C aptamer was 2.1 times more efficient than MB8-luc 2ARNm in transfecting biceps femoris. Therefore, ARN aptamer C improves internalization of ARNm molecules into the muscle fiber into which it is inserted.

The transfection efficiency of MB11-luc 2ARNm was moderate (FIG. 10). However, it was unexpected that co-injection of 5. mu.g of MB11-luc 2ARNm, M12-H6 peptide and 5. mu.g of chloroquine could increase transfection efficiency by 31-fold compared to MB11-luc 2ARNm in the presence of peptide without chloroquine. In addition, the expression of luciferase obtained from MB11-luc 2ARNm in the presence of M12-H6 and chloroquine was very advantageously 2.7-fold higher than that obtained from MB8-Iuc2 ARNm.

Without being bound by theory, it can be hypothesized that the high transfection efficiency of M12-H6 peptide complexed MB11-luc 2ARN in the presence of chloroquine benefits from slowing endosomal acidification by chloroquine after ARNm penetration into cells. Chloroquine may slow the protonation of hexahistidine at acidic pH, thus preventing the instability of the complex between MB11-luc 2ARNm and M12-H6 peptides. As a result, ARNm can enhance escape of ARNm from endosomes, into the cytoplasm and subsequent translation therein, by facilitating passage through the endosomal membrane by virtue of M12-H6 still complexed therewith.

Example 10: mouse intradermal injection protocol

a-solution preparation

A60. mu.l solution containing 20. mu.g of ARNm was prepared for each mouse. For this purpose, deionized water, 50mM Hepes (1/8 Hepes, 7/8 Hepes sodium), NaCl (final 160mM), MgCl were mixed₂ARNm and optionally a peptide. The peptide was allowed to bind to the ARN by incubation at room temperature for 30 minutes. No incubation is required when ARNm is not complexed with a peptide. The ARNm solution was cryopreserved at-80 ℃ until injection.

b-intradermal injection and biopsy samples

Male OF1 mice (Charles River) 6 weeks old were used. They were shaved three to four days prior to intradermal injection. Anesthesia was performed using a mask. Induction of anesthesia was achieved with 4% isoflurane (Piramal healthcare). Anesthesia was maintained at 2% isoflurane percentage. Before injection, the shaved back skin before cleaning was wiped with alcohol. For filtration, 0.3mm x 8mm U-100(30G) insulin syringe (Becton-Dickinson), the ARNm solution was slowly thawed at room temperature. Approximately 17 μ l of ARN solution was injected three times into the shaved back skin of each mouse. Before the (resorber) is resolved, a papule forms. These are demarcated using permanent markers so that the skin area to be biopsied is identified the next day. The tail was also labeled with a marker to distinguish animals in the same cage.

At 18 hours post-injection, skin biopsies were taken from the injection area. For this purpose, mice were anesthetized and then euthanized by cervical dislocation. The biopsy was cut into small pieces with a pair of scissors to facilitate cell lysis and placed into tubes containing 500. mu.l of1 × lysis buffer (5 × luciferase cell culture lysate (Promega), diluted with water). Each tube was frozen at-20 ℃ until the next step.

c-cell lysis, luciferase and protein assays

The lysed collected tissue was obtained by performing three freeze/thaw cycles: leave at-80 ℃ for 10 minutes, leave in a room temperature water bath for 3 minutes, and then mix for a few seconds using a vortex mixer. The tubes were then centrifuged at 5000Xg for 5 minutes at 20 ℃ to pellet the tissue debris. The supernatant was transferred to another tube.

Luciferase assays were performed using the Luciferase Assay System kit (Promega). 20 μ l of each sample was placed in a photometer-specific tube (Berthold AutoLumat Plus LB 953). 100 μ l of substrate was injected into the apparatus and the amount of emitted light (RLU) was measured.

Protein analysis was performed using Pierce 660nm Protein Assay kit (Thermo Scientific). The range was calibrated using bovine serum albumin (Thermo science) and 1 × lysis buffer as diluents. The coverage was 100 to 500. mu.g protein per ml. 100. mu.l of1 Xlysis buffer was used as a blank. In some cases, the lysate is diluted with 1 × lysis buffer. 100 μ l of each sample was used for protein analysis. 1.5ml of reagent was added to the blank and sample. After incubation at room temperature for exactly 5 minutes in the dark, the absorbance of each sample was measured using a spectrophotometer at 660 nm.

Example 11: effect of CPP on the transfection efficiency of ARNm in skin

As described in example 9 above, the strategy to improve naked ARNm internalization by binding Cell Penetrating Peptides (CPPs) to ARN aptamer a is not limited to muscle. This strategy of using different CPPs has been applied here in mouse skin.

Materials and methods:

three CPPs are used here: CPP1, CPP2, and CPP3 (see Kamada et al, 2007 and Lee et al, 2012). They are linked to hexahistidine through a spacer consisting of glycine and serine to form a peptide having the sequence SEQ ID NO: 76. 77 and 78, CPP1-H6, CPP2-H6 and CPP 3-H6.

In a pre-optimized container containing 160mM NaCl, 0.7mM MgCl₂And 5mM Hepes, MB11-luc 2ARNm was incubated with increasing amounts of CPP3-H6, CPP1-H6, or CPP2-H6 peptide for 30 minutes. MB11-luc 2ARNm alone, as well as MB8-luc 2ARNm diluted in sterile ultrapure deionized water, supplemented with 160mM NaCl were also injected, and used here as controls.

An intradermal injection OF 5.6 μ g OF MB8-luc 2ARNm or 5.6 μ g OF MB11-luc2 was made in OF1 mice according to the protocol detailed in example 10. Mice were also injected with 1: a mixture of MB11-luc 2ARNm and MB8-luc 2ARNm in a ratio of1, and CPP3-H6 in an amount equal to 0.5 molar ratio, to determine whether the CPP3-H6 peptide can be isolated after intradermal injection from ARN aptamer a present in MB11-luc2 ARNm. Luciferase activity was measured 18 hours after injection.

As a result:

as expected, injection of MB8-luc 2ARNm resulted in efficient skin transfection. CPP3-H6, 0.7mM MgCl₂And injection of 5mM Hepes with MB8-luc 2ARNm did not significantly affect transfection efficiency (FIG. 11).

The optimal amount of CPP3-H6 peptide, which advantageously increases the transcription efficiency by 9.2-fold compared to MB11-luc 2ARNm alone, corresponds to 1CPP3-H6 peptide: 2ARNm molar ratio (fig. 11). In addition, it is very advantageous to have 2.1 times higher luciferase activity than that obtained with MB8-luc 2ARNm and 19.5 times higher luciferase activity than that obtained with conventional MB5-luc2 capped ARNm.

Transfection decreased 12.3-fold when one of every two ARNms did not bind CPP3-H6 (FIG. 11, MB8-luc2 and MB11-luc2 ARNms, 2.8. mu.g each). This means that after intradermal injection, the CPP3-H6 peptide can be isolated from the ARN aptamer a present in MB11-Iuc2 ARNm. CPP3-H6 did not effectively improve transfection if half of the ARNm lacked ARN aptamer A (MB8-luc2 ARNm).

MB11-luc 2ARNm was also incubated with CPP1-H6 peptide at different molar ratios. The optimal molar ratio is 1CPP1-H6 peptide: 4MB11-luc 2ARNm (FIG. 12). Transfection efficiency was increased 5.4 fold compared to MB11-luc 2ARNm alone.

Therefore, CPP1-H6 gave poorer results than CPP 3-H6. This can be illustrated 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.

Finally, MB11-luc 2ARNm was also incubated with CPP2-H6 at various molar ratios. The optimal molar ratio is 2 CPP2-H6 peptide: 1MB11-luc 2ARNm (FIG. 13). The transfection efficiency was increased by 10.6 fold compared to MB11-Iuc 2ARNm alone, and also higher than that obtained with CPP3-H6 peptide. Therefore, luciferase expression was 22.8 times higher than that of conventional MB5-luc2 ARNm.

Example 12: effect of CPP-H6 on transfection efficiency of capped ARNm in skin

Materials and methods:

to assess the effect of CPP (here CPP2-H6) on the transfection efficiency of capped ARNm, aptamer a was inserted into the 5' -UTR of a conventional capped MB5-luc 2ARNm to generate a vector with SEQ ID NO: 70 sequences of cap MB15-luc2 ARNm. As described in example 11 above, in the presence of 0.7mM MgCl₂And 5mM Hepes for 30 min MB15-luc 2ARNm was incubated with CPP 2-H6.

An intradermal injection OF 5.6 μ g OF MB15-luc 2ARNm was performed in OF1 mice according to the protocol detailed in example 10. Luciferase activity was measured 18 hours after injection.

As a result:

in the absence of CPP2-H6 peptide, the expression of capped MB15-Iuc 2ARNm was 1.68-fold lower than that obtained with capped MB5-luc2 ARNm.

Co-injection of the capped MB15-luc 2ARNm and CPP2-H6 peptides at a molar ratio of 2 peptides per ARNm resulted in only a 1.48-fold increase in transfection efficiency as compared to MB15-luc 2ARNm alone. This indicates that insertion of the ARN aptamer a into the 5' -UTR of a conventional capped ARNm and attachment of the CPP-H6 peptide to the ARN aptamer did not improve transfection efficiency compared to that observed with the capped ARNm MB11-luc2 (10.6 fold). Regardless of the capped ARNm molecule (with or without aptamer, whether linked to CPP2-H6 CPP), the transfection efficiency is still much lower than that observed for MB8-luc2 and MB11-luc2 ARNm. Thus, it is highly advantageous to use the ARNm molecules of the invention rather than capped ARNm molecules.

Example 13: effect of 5' -SL on in vivo luciferase expression in skin

Materials and methods:

the following ARNm: MB8-luc2, MB11-luc2, MB17-luc2, MB18-luc2 and capped MB5-luc2 were injected into the skin according to the protocol detailed in example 10 above, and luciferase activity was measured 18 hours after injection.

As a result:

as shown in fig. 15, ARNm according to the present invention comprising at least one xrna sequence and one IRES sequence (MB11-luc2) increased luciferase expression in skin by about 2-fold compared to capped ARNm (MB5-luc 2). Surprisingly, when the stem-loop was located five nucleotide positions upstream of the xrna 1 sequence (MB8-luc2 and MB18-luc2), the addition of the stem-loop (here 5' -SL has the sequence of SEQ ID NO: 87) resulted in an approximately 4.3-fold increase in luciferase expression in skin over the xrna and IRES sequences alone (MB11-luc 2).

However, when the stem-loop is located about 70 nucleotides upstream of the xrna sequence, the stem-loop does not improve efficiency except as observed in the absence of the stem-loop. Indeed, the translation efficiencies of MB11-luc2 and MB17-luc 2ARNm were similar. Surprisingly, placing aptamer a between the xrna sequence and the IRES sequence (MB18-luc2) resulted in even more improved translation efficiency compared to MB11-luc2 and MB17-luc2 ARNm. Thus, MB8-luc2 and MB18-luc 2ARNm have similar translation efficiencies.

And (4) conclusion:

the synthesis cost of the ARNm molecules of the invention is reduced by about 30-fold compared to capped ARNm molecules, while the yield of protein expression is increased by at least 2-fold, preferably by about 10-fold. Furthermore, the ARNm of the invention is at least as stable as a capped ARNm and its production by in vitro transcription is simplified due to the absence of a cap molecule or analogue thereof. When the ARNm of the invention encoding the HRV2 2A protease is co-transfected with an ARNm encoding a protein of interest, the ARNm of the invention encoding the HRV2 2A protease may increase translation of the ARNm encoding the protein of interest. In addition, for example, the transfection efficiency of a tissue (e.g., muscle or skin) can be improved by inserting an ARN aptamer that directly penetrates cells (CPP (linked to the ARN aptamer described above)) in the 5' -UTR region of an ARNm molecule according to the present invention, and/or by adding a stem-loop at the 5' end of the 5' -UTR region upstream of the xrna sequence.

Reference to the literature

Ausubel et al.，2001，Current Protocols in Molecular Biology，Wiley&Sons，Hoboken N.J.，USA.

Beckert et Masquida，2011，Methods Mol Biol.703：29-41.

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Dunn et al.，2017，Nat Rev Chem.1.s41570-017.10.1038/s41570-017-0076.

Gao et al.，2014，Molecular Therapy 22(7)：1333-41.

Goldstaub et al.，2000，Mol CellBiol，20：1271-1277

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Sequence listing

<110> Mexingjie biopharmaceuticals

<120> replacement of messenger RNA Cap by two RNA sequences introduced at the 5' end of messenger RNA

<130> 375816D38002

<150> FR 1854052

<151> 2018-05-15

<160> 89

<170> PatentIn version 3.5

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<400> 38

gucaggccaa gauugagaaa aucuugccac agcuuggcag acugugcagc cug 53

<210> 39

<211> 51

<212> RNA

<213> Dongkong 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 ugugaggcag ugcaggcug 59

<210> 41

<211> 48

<212> RNA

<213> Roxio virus

<400> 41

gucaggccgu ccuuggacgc cacccaaagc augggagggu gcugccug 48

<210> 42

<211> 49

<212> RNA

<213> Alfre Virus

<400> 42

gucaggccag ugaaaacugc caccggaugu ugguagacgg ugcugccug 49

<210> 43

<211> 43

<212> RNA

<213> Statratefavirus

<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 in RNAs from pMB8-luc2 and pMB9-luc2

<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 sequence 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

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 tgtatgggat 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> 3' -UTR of pMB5-luc2, pMB7-luc2 and pMB8-luc2 do not have 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> 3' -UTR of pMB5-luc2, pMB7-luc2 and pMB8-luc2 do not have a restriction site

<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 (with SspI restriction sites) of pMB5-luc2, pMB7-luc2 and pMB8-luc2

<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 300

agtctgcgac agtgccccag gaggactggg tgaacaaagg cgaatcaacg tcccacgcgg 360

ccctagctct ggcaatggtg ttaaccagag tgaaaggact agaggttaga ggagaccccg 420

cgttctgaag tgcacggccc agcctggctg aagctgtagg tcaggggaag gactagaggt 480

tagtggagac cccgtgccgc aaaacaccac aacaacacag catattgaca cctgggatag 540

actaggagat cttctgctct gcacaaccag ccacacggca cagtgcgccg acaatggtgg 600

ctggtggtgc gagaacacag gatct 625

<210> 56

<211> 718

<212> DNA

<213> Artificial sequence

<220>

<223> 3' UTR (Kunjin Virus) -Eco53kI (pMB9-luc2)

<400> 56

ttctagaata ctttgttaat tgtaaataaa tattgttatt atgtgtagaa gtttagcttt 60

ataatagtgt ttagtgtgtt tagagttaga aaaattttag tgaggaagtc aggccggaaa 120

attcccgcca ccggaagttg agtagacggt 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 RNA from 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 RNA from 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 ccaaaaacat 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

catgcccgcg 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

aaggaggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020

ggcctgacag aaacaaccag cgccattctg atcacccccg 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

aagaagcuac cgaucauaca aaagaucauc aucauggaua gcaagaccga cuaccagggc 480

uuccaaagca uguacaccuu cgugacuucc cauuugccac ccggcuucaa cgaguacgac 540

uucgugcccg agagcuucga ccgggacaaa accaucgccc ugaucaugaa caguaguggc 600

aguaccggau ugcccaaggg cguagcccua ccgcaccgca ccgcuugugu ccgauucagu 660

caugcccgcg accccaucuu cggcaaccag aucauccccg acaccgcuau ccucagcgug 720

gugccauuuc accacggcuu cggcauguuc accacgcugg gcuacuugau cugcggcuuu 780

cgggucgugc ucauguaccg cuucgaggag gagcuauucu ugcgcagcuu gcaagacuau 840

aagauucaau cugcccugcu ggugcccaca cuauuuagcu ucuucgcuaa gagcacucuc 900

aucgacaagu acgaccuaag caacuugcac gagaucgcca gcggcggggc gccgcucagc 960

aaggagguag gugaggccgu ggccaaacgc uuccaccuac caggcauccg ccagggcuac 1020

ggccugacag aaacaaccag cgccauucug aucacccccg 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

cccgccgcag ucgucgugcu ggaacacggu aaaaccauga ccgagaagga gaucguggac 1500

uauguggcca gccagguuac aaccgccaag aagcugcgcg gugguguugu guucguggac 1560

gaggugccua aaggacugac cggcaaguug gacgcccgca agauccgcga gauucucauu 1620

aaggccaaga agggcggcaa gaucgccgug uaauaa 1656

<210> 64

<211> 33

<212> RNA

<213> Artificial sequence

<220>

<223> aptamer A

<400> 64

gguauauugg cgccuucgug gaaugucagu gcc 33

<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 cggaaggagg accccacgug cuuuagccuc aaagcccagu 240

gucagaccac acuuuaaugu gccacucugc ggagagugca gucugcgaua gugccccagg 300

uggacugggu uaacaaaggc aaaacauuac uggccgaagc cgcuuggaau aaggccggug 360

ugcguuuguc uauauguuau uuuccaccau auugccgucu uuuggcaaug ugagggcccg 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> capping of the 5' -UTR of MB15-luc2 RNA

<400> 70

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 actcgcccga 60

aaaaaatcta gagcatgcaa aaaagtcagg ccagattaat gctgccaccg gaagttgagt 120

agacggtgct gcctgcggct caaccccagg aggactgggt gaccaaagct gcgaggtgat 180

ccacgtaagc cctcagaacc gtctcggaag gaggacccca 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

aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa 540

gacaaacaac gtctgtagcg accctttgca ggcagcggaa 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> capping of the 5' -UTR of 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 cuuuacaugu guuuagucga gguuaaaaaa cgucuaggcc ccccgaacca 780

cggggacgug guuuuccuuu gaaaaacacg augauaauau ggugacaagg cccaucauca 840

caacagccgg cccuuccgau auguacgugc acgugggcaa ccugaucuac aggaaccugc 900

accuguucaa cagcgagaug cacgagagca uccuggugag cuacagcagc gaucugauca 960

ucuacagaac caacacagug ggcgacgauu acauccccag cugugacugc acacaggcca 1020

ccuacuacug caagcacaag aacagauacu ucccuaucac agugacaagc cacgauuggu 1080

acgagaucca ggagagcgag uacuacccua agcacaucca guacaaccug cugaucggcg 1140

agggcccuug cgagcccgga gacuguggcg gaaagcugcu guguaagcac ggcgugaucg 1200

gcaucgugac cgccggcgga gauaaccacg uggccuucau cgaccugagg cacuuccacu 1260

gcgccgagga gcaguaauaa uucuagaaug uccgaauggu ugacacuuga ucucggcaac 1320

gcauaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 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

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> consensus sequences, wherein the "N" at position 9 corresponds to 7 to 19 nucleotides of any sequence, and

the "N" at position 14 corresponds to 12 to 19 nucleotides of 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