Improved method of RNA production in fungi

Abstract

The invention relates to a method of recombinantly producing RNA, in particular messenger RNA (mRNA) of interest or long non-coding RNA (lncRNA) of interest, by recombinant fungi, for instance by yeasts with recombinant T-bodies. The invention also relates to means useful for implementing said method, or to products that may be obtained by said method, including kits, recombinant fungi cells, in particular recombinant yeast cells, as well as the complexes or T-bodies produced thereof. The invention further relates to a method for producing a pharmaceutical composition, and to the use of the products and compositions of the invention for medical applications, for instance for vaccines, antitumor, anti-infectious and pro-regenerative applications.

Description

IMPROVED METHOD OF RNA PRODUCTION IN FUNGIFIELD OF INVENTION

[0001] The present invention relates to the production of RNA of interest, more specifically of messenger RNA of interest or of long non-coding RNA of interest, by fungi. The recombinant fungi are genetically modified in order to produce the RNA of interest in the form of a recombinant complex containing the RNA of interest and proteins, particularly in the form of recombinant T-bodies.

[0002] Thus, the invention relates to a method of recombinantly producing RNA, in particular messenger RNA (mRNA) of interest or long non-coding RNA (IncRNA) of interest, by recombinant fungi, for instance by yeasts producing recombinant T-bodies. The invention also relates to means useful for implementing said method, and to products that may be obtained by said method, including kits, recombinant fungi cells, in particular recombinant yeast cells, as well as complexes or T-bodies produced thereof. The invention further relates to a method for producing a pharmaceutical composition, and to the use of the products and compositions of the invention for medical applications, for instance for vaccines, anti-tumor, anti-infectious and pro-regenerative applications.BACKGROUND OF INVENTION

[0003] Different methods of producing RNA, in particular RNA of more than 200 base pairs (bp), are known in the art. These methods include in vitro synthesis (IVT) methods that use RNA polymerases.

[0004] The production of RNA, in particular mRNA, which are competent for the translation into proteins in mammalian cells, is problematic for post-transcriptional modifications such as the presence of a polyA tail at the 3' end, a 7-m ethylguanosine (m7G) cap at the 5' end, or even certain methylations of adenines and cytosines.

[0005] Historical in vitro synthesis methods for example propose placing cap analogs in the transcriptional mixture, such as m7G5'ppp5'G. In order to stimulate the incorporation of the dinucleotide at the beginning of the chain, the in vitro synthesis methods of the prior art also propose to reduce the concentration of guanosine triphosphate (GTP) in the transcriptional mixture relative to the other nucleoside triphosphates (NTPs), which decreases transcription efficiency. In addition, the cap analog can then be incorporated in both orientations resulting in 50% of poorly capped RNA. These methods of in vitro synthesis of RNA are therefore not very effective.

[0006] Other in vitro synthesis methods use modified cap analogs which have been designed to ensure incorporation at the 5’ end only.

[0007] Other in vitro synthesis methods use a chimeric enzyme comprising the catalytic domains of an RNA triphosphatase, a guanylyltransferase, an N7-guanine methyltransferase, and a DNA-dependent RNA polymerase.

[0008] These in vitro RNA synthesis methods nevertheless remain expensive.

[0009] Moreover, in vitro methods are less efficient for production of large mRNA (of 5 kbp and above) of appropriate quality.

[0010] RNA recombinant production methods have been further developed. For example, one method uses the machinery of the mitochondria, to make it an RNA production and storage unit. The RNA thus produced do not, however, have a cap at the 5' end, which limits their medical applications.

[0011] Some authors proposed methods based on the yeast Tyl retrotransposon system allowing the combined production of the yeast Gag protein and mRNA. However, the proposed Gag sequences do not prevail the generation of Virus-Like Particles (VLP) entrapping mRNA, which reduces potential recovery of mRNA of interest.

[0012] The inventors provide methods for producing RNA that do not have all the drawbacks of the prior art.

[0013] Thus, the methods of the invention allow producing recombinant RNA-protein complexes, in particular in the form of T-bodies rather than in the form of VLP. The T- bodies containing RNAs of interest may be recovered by cell lysis. The methods of the invention thus allow the recombinant production of RNAs of interest, with high productivity and purity. mRNA thus produced may have a cap and poly- A tail.

[0014] The invention thus relates to the production of RNA, more particularly messenger RNA (mRNA) or long non-coding RNA (IncRNA), by recombinant fungi, more particularly by yeasts containing recombinant complexes containing the RNA of interest and proteins, or T-bodies.

[0015] Fungi are genetically engineered to produce recombinant complexes that contain the RNA to be produced. Recombinant complexes containing RNA and proteins are produced from certain fungus Ty retrotransposon elements, but do not retro-transcribe the RNA they contain.

[0016] The Ty retrotransposon elements used comprise in particular a Gag protein of a Ty retrotransposon, as well as a fragment of the RNA which corresponds to this protein. This RNA fragment is not intended to be translated into protein in fungi. However, it may serve as an addressing sequence, which guides an RNA (of interest) to the Gag protein, thereby forming a complex between this RNA and Gag protein, wherein the RNA is incorporated into a Gag protein-containing assembly. In particular, said complex may be a structured T-body.

[0017] The RNA thus produced can advantageously have a poly A tail at the 3 ' end and / or a 7-methylguanosine (m7G) cap at the 5' end, and / or methylation of adenine and / or cytosine and / or pseudo-uridine residue(s). They may therefore be competent for possible translation into mammalian cells. In addition, RNA can be produced in large amounts, and at a reduced cost.

[0018] The invention presents several advantages over the prior art including: improvement of the productivity of RNA of interest per cell, improvement of the purity of RNA of interest in the cell,facilitation of the process industrialization for production of large quantities of RNA of interest at lower cost.

[0019] The application further concerns the engineered fungi, in particular yeast, as well as the complexes, in particular T-bodies, that they produce. The application also relates to means for their production, including nucleic acids, vectors and kits, as well as medical applications, more particularly vaccine, anti-tumor, anti-infectious and pro-regenerative applications.SUMMARY

[0020] This invention thus relates to a method of recombinant production of an RNA of interest comprising: a) culturing fungus cells, which have been genetically modified to produce recombinant RNA-protein complexes comprising said RNA of interest, and b) recovering said RNA of interest comprised in the complexes thus produced, wherein genetically modifying the fungus cells to produce the complexes comprises transfecting said fungus cells with one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to the sequence SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof,- the second nucleotide sequence comprises a DNA sequence whose RNA transcript is:- the sequence of said RNA of interest, linked to- an addressing sequence comprising, or consisting of, the sequence SEQ ID NO: 3, wherein the first and second nucleotide sequences thereby allowing the formation, in said fungus cells, of recombinant complexes encapsulating said RNA of interest.

[0021] In some embodiments, step b) of recovering the RNA of interest comprised in the complexes produced comprises: lysing the fungus cells cultured at step a), preferably with glass beads, and centrifuging the lysed fungus cells and recovering the recombinant RNA- protein complexes from the pellet.

[0022] In some embodiments, the fungus is Thermothelomyces heterothallica (formerly Myceliophthora thermophilia), Saccharomyces paradoxus, Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Hansenula polymorpha (Pichia augusta), Yarrow ia lipolytica, Kluyveromyces marxianus, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.

[0023] In some embodiments, the fungus cell contains at least one mutated gene selected among the group consisting of the rpbl, spt21, srb2 and srb5 genes, wherein said at least one mutated gene comprises at least one mutation stimulating or increasing the production of the recombinant RNA-protein complexes.

[0024] In some embodiments, the RNA of interest comprises a sequence encoding:- a bacterial protein or polypeptide,- a genome editing enzyme,- a virus protein or polypeptide,- a transcription factor,- a growth factor,- a CFTR protein,- an antibody or antibody fragment,- a polypeptide comprising at least one antigen epitope, or- a combination thereof.

[0025] In some embodiments, the RNA of interest further comprises a sequence encoding a replicase allowing RNA self-amplification, and / or wherein the RNA of interest further comprises 3’ and 5’ sequences allowing the generation of circular RNA.

[0026] A further aspect of the invention is a kit suitable for carrying out the method according to the invention, wherein said kit comprises one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof, and- the second nucleotide sequence comprises a DNA sequence which comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3, wherein said kit optionally comprises cells of a fungus.

[0027] Another aspect of the invention is a recombinant fungus strain suitable for carrying out the method according to the invention, wherein the fungus strain has been genetically modified to comprise one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof, and- the second nucleotide sequence comprises a DNA sequence whose RNA transcript comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3.

[0028] Another aspect of the invention is a recombinant RNA-protein complex or T- body, which is obtained or obtainable by the method of the invention.

[0029] Another aspect of the invention is a method of producing a pharmaceutical composition which comprises at least one RNA of interest, said method comprising:- producing an RNA by the method of the invention, and- contacting said RNA with a pharmaceutically acceptable carrier.

[0030] In some embodiments, the pharmaceutical composition is for:- the prevention or treatment of microbiological infection,- the prevention or treatment of tumor proliferation,- the prevention or treatment of a chronic disease,- use as a vaccine,- tissue or cell regeneration therapy, or- gene therapy.

[0031] In some embodiments of the methods of the invention, or of the in vitro use of the kit of the invention, or of the recombinant fungus strain of the invention, or of the recombinant T-body of the invention, the RNA of interest is a messenger RNA (mRNA) or a non-coding long RNA (IncRN A).

[0032] In some embodiments of the methods of the invention, or of the in vitro use of the kit of the invention, or of the recombinant fungus strain of the invention, or of the recombinant T-body of the invention, the RNA of interest is an mRNA or IncRNA having a 3'- polyA tail and / or a 5'- methylguanosine cap.DEFINITIONS

[0033] In the present invention, the following terms have the following meanings:

[0034] The term prokaryotic is understood according to its usual meaning in the field. It relates more particularly to a bacterium, especially a bacterium with potential infectious and / or pathogenic power for the human species.

[0035] The term virus is understood according to its usual meaning in the field. It relates more particularly to a virus with potential infectious and / or pathogenic power for the human species, in particular Zika or Influenza.

[0036] The term eukaryotic is understood according to its usual meaning in the field. When it relates to the RNA, the mRNA or the mRNA of the application, it relates more particularly to a eukaryote other than fungus, more particularly a multicellular eukaryote such as a mammal (more particularly a human or a non-human mammal), more particularly a human.DETAILED DESCRIPTION

[0037] The application relates to a method of recombinant production of an RNA of interest comprising: a) culturing fungus cells, which have been genetically modified to produce recombinant RNA-protein complexes comprising said RNA of interest, and b) recovering said RNA of interest comprised in the complexes thus produced, wherein genetically modifying the fungus cells to produce the complexes comprises transfecting said fungus cells with one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising thesequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof,- the second nucleotide sequence comprises a DNA sequence whose RNA transcript is:- the sequence of said RNA of interest, linked to- an addressing sequence comprising, or consisting of, the sequence SEQ ID NO: 3, wherein the first and second nucleotide sequences thereby allowing the formation, in said fungus cells, of recombinant complexes encapsulating said RNA of interest.

[0038] The cells used in the method of the invention are fungus cells or cells derived from a fungus.

[0039] The fungus may for instance be Thermothelomyces heterothallica (formerly Myceliophthora the rm ophili a).

[0040] In particular, the fungus may be a yeast. More particularly, the fungus may be a yeast of the genus Saccharomyces or the genus Pichia.

[0041] In some embodiments, the fungus is Thermothelomyces heterothallica (formerly Myceliophthora thermophilia), Saccharomyces paradoxus, Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Hansenula polymorpha (Pichia augusta), Yarrow ia lipolytica, Kluyveromyces marxianus, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.

[0042] An example of S. paradoxus strain is the strain that is accessible from ATCC under the number 76528™. ATCC® is the American Type Culture Collection (10801 University Blvd.; Manassas, Va. 20110-2209; USA.).

[0043] An example of S. cerevisiae strain is strain YAM510, which does not contain functional Tyl retrotransposon, and which has been described in Rothstein et al. 1983 and Thomas et al. 1989.

[0044] In some embodiments, in addition to having been genetically modified to produce (recombinant) T-bodies, the fungus cell is a cell naturally devoid of Ty retrotransposon, or which comprises an inactive Ty retrotransposon, or which naturally comprises a Ty retrotransposon but whose Ty retrotransposon sequence has been modified so as not to produce or be deficient in the retrosome.

[0045] Alternatively, the fungus cell may be a cell devoid of functional Ty retrotransposons. In the context of the invention, a functional Ty retrotransposon is a Ty retrotransposon which may lead to the production of Virus-Like Particles (VLP) in the cell.

[0046] The term “Ty retrotransposon" relates to a Tyl, Ty2, Ty3, Ty4 or Ty5 retrotransposon. Preferably, the Ty retrotransposon is a Tyl, Ty2 or Ty3 retrotransposon, more preferably a Tyl retrotransposon.

[0047] The term “retrosome” herein refers to cytoplasmic foci in which Gag protein and retrotransposon RNA, e.g. Ty-retrotransposon RNA, colocalize.

[0048] Thus, in some embodiments, the fungus cell is naturally devoid of Tyl retrotransposon, or is a cell which comprises an inactive Tyl retrotransposon, or is a cell which naturally comprises a Tyl retrotransposon, but whose Tyl retrotransposon sequence has been modified so as not to produce or be deficient in the retrosome. Alternatively, the fungus cell may be a cell devoid of functional Tyl retrotransposon.

[0049] In some embodiments, the fungus cell is naturally devoid of Ty2, Ty3, Ty4 and / or Ty5 retrotransposon, or is a cell which comprises an inactive Ty2, Ty3, Ty4 and / or Ty5 retrotransposon, or is a cell which naturally comprises a Ty2, Ty3, Ty4 and / or Ty5 retrotransposon but which has been genetically modified so as not to produce a retrosome encoded by a Ty2, Ty3, Ty4 and / or Ty5 retrotransposon. Alternatively, the fungus cell may be a cell devoid of functional Ty2, Ty3, Ty4 and / or Ty5 retrotransposon.

[0050] Pichia pastoris (Komagataella phaffii) is an example of yeast which is naturally devoid of Ty retrotransposon.

[0051] Saccharomyces cerevisiae is an example of yeast which naturally comprises Ty retrotransposons, in particular Tyl retrotransposons, and which may be genetically modified so as not to produce a T-retrosome.

[0052] A genetic modification that allows not to produce T-retrosomes may, for example, includes the deletion in the chromosome of the fungus of the expression promoter of the Ty retrotransposons, and / or a mutation (deletion, replacement and / or insertion of one or more nucleotides) in the Ty retrotransposon sequence in the fungus chromosome.

[0053] The fungus cell may advantageously contain genetic mutations stimulating or increasing the production of the recombinant RNA-protein complexes or T-bodies.

[0054] The genetic mutations may be substitutions, deletions and / or insertions.

[0055] Preferably, the fungus cell contains at least one mutated gene selected among the group consisting of the rpbl, spt21, srb2 and srb5 genes. More preferably, said at least one mutated gene comprises at least one mutation stimulating or increasing the production of the recombinant RNA-protein complexes.

[0056] In some embodiments, the fungus cell contains at least one mutated gene selected among the group consisting of the rpbl, spt21, srb2 and srb5 genes, wherein said at least one mutated gene comprises at least one mutation stimulating or increasing the production of the recombinant RNA-protein complexes.

[0057] For example, the Rpbl gene may encode a polypeptide comprise at least one of the amino acid mutations C67Y, C70Y or H80Y, the amino acid positions being determined by reference to the exemplary Rpbl polypeptide sequence of SEQ ID NO: 4.

[0058] For example, one or more of the Spt21, Srb2 and Srb5 genes may be partially or totally deleted [ASpt21, ASrb2 and ASrb5],

[0059] In some embodiment, the fungus is a fungus of which: the Rpbl gene encodes a polypeptide comprising at least one of the amino acid mutations C67Y, C70Y and H80Y, and / or one or more of the Spt21, Srb2 and Srb5 genes are totally or partially deleted.

[0060] For example, the fungus may be a yeast strain of S. paradoxus (which is naturally free of Tyl in particular Tyl), which comprises at least one of the following genetic mutations: Rpbl C67Y, Rpbl C70Y, Rpbl H80Y, ASrb2, ASpt21, ASrb2 and ASpt21, C67Y ASrb2, C70Y ASrb2, Rpbl H80Y ASrb2, Rpbl C61Y ASpt21, Rpbl C70Y ASpt21, Rpbl H80Y ASpt21, Rpbl C67Y ASrb2 ASpt21, Rpbl C70Y ASrb2 ASpt21, or Rpbl H80Y ASrb2 ASpt21.

[0061] The fungus may for example be a yeast strain of S. cerevisiae which has been genetically modified not to produce T retrosomes (strain TyO), which comprises at least one of the following genetic mutations: Rpbl C67Y, Rpbl C70Y, Rpbl H80Y, ASrb2, ASpt21, ASrb2 ASpt21, C67Y ASrb2, C70Y ASrb2, Rpbl H80Y ASrb2, Rpbl C67Y ASpt21, Rpbl C70Y ASpt21, Rpbl H80Y ASpt21, Rpbl C67Y ASrb2 ASpt21, Rpbl C70Y ASrb2 ASpt21, or Rpbl H80Y ASrb2 ASpt21.

[0062] The fungus cell may advantageously be auxotroph for specific nutrients, complements, or antibiotics, such as for example L-histidine, L-tryptophan, L- methionine, uracil or L-leucine.

[0063] The fungus auxotrophy may for instance be used for selecting fungus cells or clones which have integrated specific recombinant genes or DNA sequences.

[0064] Preferably, the fungus cell is auxotroph for at least 2, 3, 4 or 5 nutrients, complements, or antibiotics.

[0065] The fungus cells cultured at step a) have been genetically modified to produce recombinant RNA-protein complexes.

[0066] The genetic modifications made to the fungus cells to produce the complexes comprises transfecting said fungus cells with one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising thesequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof,- the second nucleotide sequence comprises a DNA sequence whose RNA transcript is:- the sequence of said RNA of interest, linked to- an addressing sequence comprising, or consisting of, the sequence SEQ ID NO: 3.

[0067] In some embodiments, each sequence is associated with, or comprises, a DNA sequence coding for an auxotrophic gene. A non-limiting example of an auxotrophic gene is the URA3 gene, which encodes orotidine-5-phosphate decarboxylase, an essential enzyme in pyrimidine biosynthesis in Saccharomyces cerevisiae. Other examples of auxotrophic genes include the HIS3, LEU2, TRP1, and MET 15 marker genes, which encode essential enzymes for de novo synthesis of the amino acids L-histidine, L-leucine, L-tryptophan, and L-methionine, respectively.

[0068] The genetic modifications made to the fungus cells to produce recombinant RNA-protein complexes comprise the transfer into these cells (e.g. by transfection or transformation) of a first nucleotide sequence comprising a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon.

[0069] In the context of the invention, the Gag protein encoded by DNA sequence comprised in the first nucleotide sequence has the sequence SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to the sequence SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2. Alternatively, the Gag protein encoded by DNA sequence comprised in the first nucleotide sequence has the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof.

[0070] In some embodiments, the Gag protein has the amino acid sequence of SEQ ID NO: 1.

[0071] In some embodiments, the nucleic sequence coding for the Gag protein is codon- modified to eliminate binding of the Gag protein to its own mRNA.

[0072] In some embodiments, the Gag protein has the amino acid sequence of SEQ ID NO: 2.

[0073] Thus, in some embodiments, the invention relates to a method of recombinant production of an RNA of interest comprising: a) culturing fungus cells, which have been genetically modified to produce recombinant RNA-protein complexes comprising said RNA of interest, and b) recovering said RNA of interest comprised in the complexes thus produced, wherein genetically modifying the fungus cells to produce the complexes comprises transfecting said fungus cells with one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises, or consists of, a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having, or consisting of, the sequence SEQ ID NO: 2, or a sequence at least 90% identical to the sequence SEQ ID NO: 2,- the second nucleotide sequence comprises, or consists of, a DNA sequence whose RNA transcript is:- the sequence of said RNA of interest, linked to- an addressing sequence comprising, or consisting of, the sequence SEQ ID NO: 3, wherein the first and second nucleotide sequences thereby allowing the formation, in said fungus cells, of recombinant complexes encapsulating said RNA of interest.

[0074] In some embodiments, the Gag protein has an amino acid sequence consisting of a fragment of the sequence SEQ ID NO: 1, said fragment comprising the sequence SEQ ID NO: 2.

[0075] By "fragment" of a reference sequence is meant herein a sequence constituted by a chain of consecutive amino acids of a reference sequence and whose size is smaller than the size of the reference sequence.

[0076] In the context of the invention, the fragment of the sequence SEQ ID NO: 1 necessarily comprises the sequence SEQ ID NO: 2. Therefore, it may for example have a size of between 288 and 378 amino acids, between 288 and 370 amino acids, between 288 and 350 amino acids, between 288 and 330 amino acids, between 288 and 310 amino acids, between 288 and 300 amino acids, between 288 and 295 amino acids, between 288 and 290 amino acids, between 288 and 289 amino acids. Most preferably, the fragment of the sequence SEQ ID NO: 1 has a size of 288 amino acids.

[0077] The Gag protein of the invention may also be a "variant", "homologue" or "derivative" of the Gag protein of a fungus Ty retrotransposon which exhibits the same biological activity as said Gag protein.

[0078] Preferably, the polypeptides of the invention are naturally occurring variants of a Gag protein of a fungus Ty retrotransposon.

[0079] The Gag proteins of the invention thus include polypeptides having sequences derived from the amino acid sequence of SEQ ID NO: 1, or derived from a fragment of the amino acid sequence of SEQ ID NO: 1, defined by a percentage of sequence identity with the sequence of SEQ ID NO: 1 or of said fragment thereof.

[0080] "Variants", "homologues" or "derivatives" are defined as comprising a sequence at least 80%, preferably at least 85%, more preferably at least 90%, even at least 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% identical to the reference sequence.

[0081] In some embodiments, the Gag protein of the invention has an amino acid sequence at least 90% identical to SEQ ID NO: 1.

[0082] In some embodiments, the Gag protein has a sequence at least 90% identical to a fragment of the sequence of SEQ ID NO: 1, said fragment comprising the sequence SEQ ID NO: 2.

[0083] In some embodiments, the Gag protein has a sequence at least 90% identical to a fragment of the sequence of SEQ ID NO: 11. In some embodiments, the Gag protein has a sequence at least 90% identical to a fragment of the sequence of SEQ ID NO: 11.

[0084] These derived sequences may differ from the reference sequence (e.g. SEQ ID NO: 1 or a fragment thereof) by substitution, deletion and / or insertion of one or more amino acids, at positions such that these modifications do not have any significant negative impact on the biological activity of the polypeptides. The substitutions may in particular correspond to conservative substitutions or to substitutions of natural amino acids by non-natural amino acids or pseudo amino acids.

[0085] By "amino acid sequence having (for instance) at least 90% of identity with a reference sequence" is meant herein a sequence identical to the reference sequence but this sequence may comprise up to ten mutations (substitutions, deletions and / or insertions) per each part of one hundred amino acids of the reference sequence. Therefore, for a reference sequence of 100 amino acids, a fragment of 90 amino acids and a sequence of 100 amino acids comprising 10 substitutions compared with the reference sequence are two examples of sequences having 90% sequence identity with the reference sequence.

[0086] Percentage of identity is generally determined using sequence analysis software (for example the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). The amino acid sequences to be compared are aligned to obtain maximum percentage identity. For this purpose, it may be necessary to artificially add gaps in the sequence. The alignment can be performed manually or automatically. Automated alignment algorithms of nucleotide sequences are well known to persons skilled in the art and described for example in Altschul et al. (1997) Nucleic Acids Res. 25:3389 and implemented by a software, such as the BLAST software. One algorithm which can be isolated is the Needleman-Wunsch algorithm for example (Needleman and Wunsch (1970) J Mol Biol. 48:443-53). Once optimal alignment has been achieved, the percentage identity is established by recording all the positions at which the amino acids of the two compared sequences are identical, compared with the total number of positions.

[0087] Therefore, the Gag protein of the invention may consist of a sequence selected from:a) a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the amino acid sequence SEQ ID NO: 1, b) a fragment of a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the amino acid sequence SEQ ID NO: 1, c) a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the sequence of a fragment of the sequence of SEQ ID NO: 1 comprising the sequence SEQ ID NO: 2, or d) a fragment of a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the sequence of a fragment of the sequence of SEQ ID NO: 1 comprising the sequence SEQ ID NO: 2, e) a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the amino acid sequence SEQ ID NO: 11, f) a fragment of a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the amino acid sequence SEQ ID NO: 11, g) a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the sequence of a fragment of the sequence of SEQ ID NO: 11, or h) a fragment of a sequence having at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% or 100% of identity with the sequence of a fragment of the sequence of SEQ ID NO: 11.

[0088] In some embodiments, the sequence of the Gag protein differs from the reference sequence (e.g. SEQ ID NO: 1 or a fragment thereof) solely through the presence of conservative substitutions. Conservative substitutions are substitutions of amino acids of the same class, such as substitutions of amino acids with non-charged side chains (such as asparagine, glutamine, serine, cysteine, and tyrosine), of amino acids with basic side chains (such as lysine, arginine and histidine), of amino acids with acid side chains (such as aspartic acid and glutamic acid), of amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan).

[0089] The Gag protein encoded by the first nucleotide sequence may form protein polymers.

[0090] The Gag protein may be derived from a fungus Ty retrotransposon. For instance, the Gag protein may be derived from a Ty retrotransposon of S. cerevisiae. For example, the Gag protein may be derived from Tyl, Ty2 or Ty3 retrotransposon of S. cerevisiae.

[0091] In other embodiments, the Gag protein may be derived from a retrotransposon of another fungus, including e.g. Saccharomyces paradoxus, Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Hansenula polymorpha (Pichia augusta), Yarrow ia lipolytica, Kluyveromyces marxianus, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.

[0092] In addition to the DNA sequence encoding the Gag protein, the first nucleotide sequence may comprise a DNA sequence encoding a marker, e.g. allowing detection of the Gag protein, or a label, e.g. allowing purification of the Gag protein.

[0093] Non-limiting examples of markers or labels include fluorescent proteins, such as a green fluorescent protein (GFP), poly-histidine tags, bacteriophage MS2 sequences, etc.

[0094] The genetic modifications made to the fungus cells to produce recombinant RNA-protein complexes may also comprise the transfer into these cells (e.g. by transfection or transformation) of a nucleic acid construct which RNA transcript comprises:- the sequence of an RNA of interest, and- an addressing sequence.

[0095] For instance, the addressing sequence may address the RNA of interest to a Gag protein of a fungus Ty retrotransposon. In particular, the addressing sequence may address the RNA of interest to the Gag protein which is produced from the first nucleotide sequence.

[0096] Preferably, in the second nucleotide sequence, the sequence of the RNA of interest is linked to the addressing sequence. This linkage may be at the 5' or 3' end of the sequence of the RNA of interest. In some embodiments, the addressing sequence is linked to the 5' end of the sequence of the RNA of interest. In some embodiments, the addressing sequence is linked to the 3 ' end of the sequence of the RNA of interest.

[0097] The combination of the first and second nucleotide sequences thus produces recombinant RNA-protein complexes comprising said RNA of interest.

[0098] The second nucleotide sequence is intended to be transcribed into RNA in the transfected fungus cells.

[0099] In some embodiments, the second nucleotide sequence is devoid of an open reading frame comprising a start codon of translation. In such embodiments, the second nucleotide sequence may not be translated into a protein or polypeptide.

[0100] The second nucleotide sequence may further comprise a tag DNA sequence, allowing purification, such as e.g. one or more copies of the bacteriophage MS2 sequence.

[0101] The first and second nucleotide sequences may be contained within a single construct or within two or more distinct or separate constructs.

[0102] The first and second nucleotide sequences may integrate into the genome of the transfected fungus cell, or remain in the nucleus or cytoplasm, for example as an episome.

[0103] The first and second nucleotide sequences may for example be both in the form of episomes that replicate autonomously, or be both integrated within a chromosome of the fungus.

[0104] Alternatively, the first nucleotide sequence may be integrated into the fungus genome, while the second nucleotide sequence may be present in the nucleus and / or the cytoplasm of the fungus cell, in the form of episomes.

[0105] Still alternatively, the first nucleotide sequence may be present in the nucleus and / or the cytoplasm of the fungus cell in the form of episomes, while the second nucleotide sequence may be integrated into the fungus genome.

[0106] In some embodiments, the first and second nucleotide sequences are contained in a single construct. This construct may be integrated into the fungus genome, or may be present in the nucleus and / or the cytoplasm of the fungus cell in the form of episomes.

[0107] Advantageously, the first and second nucleotide sequences are each (independently of each other) present in multiple copies. Advantageously, the number of copies of the second nucleotide sequence is greater (for example 2, 3, 4, 5 or 6 times greater) than the number of copies of the first nucleotide sequence.

[0108] Advantageously, the first and second nucleotide sequences are comprised in one or more vectors, more particularly nucleic acid vectors suitable for the transfer of nucleic acids into fungus cells, for example suitable for transfection or transformation of these fungus cells.

[0109] The term “nucleic acid vector” for example relates to a plasmid, more particularly a replicative plasmid or an episomal plasmid. The first and second nucleotide sequences may (independently of each other) be comprised in one or more plasmids, especially episomal plasmids or replicative plasmids.

[0110] In some embodiments, the first and second nucleotide sequences are contained in two or more distinct or separate nucleic acid vectors, in particular two or more distinct or separate plasmids.

[0111] The first nucleotide sequence may for example be comprised in a nucleic acid vector, in particular a plasmid, more particularly an integrative episomal plasmid or non- integrative replicative plasmid.

[0112] The second nucleotide sequence may for example be comprised in a nucleic acid vector, in particular a plasmid, more particularly an integrative episomal plasmid or non- integrative replicative plasmid.

[0113] For example, the first nucleotide sequence may be comprised in an integrative plasmid, more particularly an episomal plasmid, while the second nucleotide sequence may be comprised in a non-integrative plasmid, more particularly a replicative plasmid.

[0114] Alternatively, the second nucleotide sequence may be comprised in an integrative plasmid, more particularly an episomal plasmid, while the first nucleotide sequence may be comprised in a non-integrative plasmid, more particularly a replicative plasmid.

[0115] In some embodiments, the first and second nucleotide sequences are contained in a single nucleic acid vector, in particular a single plasmid.

[0116] The nucleic acid comprising the first nucleotide sequence may advantageously comprise a promoter enabling the expression of the Gag protein of a fungus Ty retrotransposon. Advantageously, the promoter of the first nucleotide sequence is a promoter enabling the expression in a fungus cell.

[0117] The nucleic acid comprising the second nucleotide sequence may advantageously comprise a promoter enabling the transcription of the second nucleotide sequence. Advantageously, the promoter of the second nucleotide sequence is a promoter enabling the transcription in a fungus cell.

[0118] The promoter controlling the first nucleotide sequence may be the same as that controlling the second nucleotide sequence, or they may be different promoters.

[0119] The promoters may be (independently of each other) natural, synthetic or semisynthetic.

[0120] The promoters may be (independently of each other) constitutive promoters or inducible promoters (e.g., a galactose-inducible promoters).

[0121] Any inducible or constitutive promoter may be used. Examples of constitutive promoters comprise, in particular, the ADH, CYC, PGK1, GPD promoters. Examples of inducible promoters comprise, in particular, the Gall, GallO, Gal7, Tet07, Met promoters.

[0122] The promoters may be (independently of each other) weak or strong promoters.

[0123] Advantageously, the fungus cells do not comprise a nucleotide sequence allowing the retro-transcription of the RNA of interest. The aim here is to limit partial loss of the RNA of interest due to its retro-transcription into DNA.

[0124] Thus, advantageously, the one or more nucleic acid(s) comprising the first and second nucleotide sequences do not comprise a sequence encoding a reverse transcriptase, in particular a reverse transcriptase of a fungus Ty retrotransposon.

[0125] Alternatively or additionally, the one or more nucleic acid(s) comprising the first and second nucleotide sequences do not comprise a sequence encoding an integrase, in particular an integrase of a fungus Ty retrotransposon.

[0126] Alternatively or additionally, the one or more nucleic acid(s) comprising the first and second nucleotide sequences do not comprise a sequence encoding a protease, in particular a protease of a fungus Ty retrotransposon.

[0127] The RNA of interest may be any kind of RNA. For example, the RNA of interest may be a messenger RNA (mRNA) or a long non-coding RNA (IncRNA). In some embodiments, the RNA of interest is a messenger RNA (mRNA). In some embodiments, the RNA of interest is a long non-coding RNA (IncRNA).

[0128] Preferably, the RNA of interest is heterologous to fungus, in particular to yeast. In some embodiments, the RNA of interest is heterologous to the fungus that produces it.

[0129] One or more different RNA(s) of interest may be produced by the method of the invention in a same fungus cell. The RNA(s) of interest may be contained within a single nucleic acid molecule or within two or more distinct or separate nucleic acid molecules.

[0130] The RNA of interest may in particular consist of, or comprise:- a prokaryotic RNA, for example a bacterial RNA,- a virus RNA, for example a Zika or Influenza RNA,- a eukaryotic RNA, in particular of a eukaryote other than fungus, for example a human RNA,- an artificial or non-natural RNA, for example an RNA encoding one or more epitopes derived from one or more microorganisms (for example epitopes of different bacteria and / or viruses), in particular antigenic epitopes.

[0131] The RNA of interest is advantageously linked to an addressing sequence, e.g. a sequence which enables it to be addressed to the Gag protein as described hereinabove.

[0132] The RNA of interest may comprise at least 120 nucleotides, in particular at least 150 nucleotides, in particular at least 180 nucleotides, in particular at least 210nucleotides, in particular at least 240 nucleotides, in particular at least 270 nucleotides, in particular at least 300 nucleotides.

[0133] Alternatively or additionally, the RNA may comprise less than 20000 nucleotides, less than 10000 nucleotides, in particular less than 5000 nucleotides, in particular less than 4000 nucleotides, in particular less than 3000 nucleotides, in particular less than 2000 nucleotides, in particular less than 1800 nucleotides.

[0134] All combinations of minimum number and maximum number of nucleotides are here explicitly targeted. The RNA of interest may for example comprise at least 150 nucleotides and less than 10000 nucleotides.

[0135] In some embodiments, the RNA of interest comprises a sequence encoding: a bacterial protein or polypeptide, a genomic editing enzyme, such as for example a Cas9, or a transposase, a virus protein or polypeptide, a transcription factor, in particular a human transcription factor, a growth factor, in particular a human growth factor a CFTR (Cystic Fibrosis Transmembrane conductance Regulator) protein, in particular a human CFTR, an antibody, or an antibody fragment such as e.g. a Fv, Fab, or F(ab')2, a polypeptide comprising one or more epitopes, for example of bacterial or viral origin, or a combination thereof.

[0136] Advantageously, the RNA of interest may comprise a polyA tail at the 3' end.

[0137] Advantageously, the RNA of interest may comprise a cap in the 5' position, in particular a cap which comprises a 7-methylguanosine (m7G). Such cap may block the action of exonucleases and be necessary to stimulate translation of the RNA of interest into mammalian cells.

[0138] The cap present in the 5' position may be a cap-1 (having a methylated 2'-hydroxy group on the first ribose sugar), a cap-2 (having methylated 2'-hydroxy groups on the first two ribose sugars), or any other modified cap.

[0139] Advantageously, the RNA of interest may comprise a secondary structure in the 5' region, such as an Internal Ribosome Entry Site (IRES) or other similar configurations. This secondary structure may play a crucial role in the RNA functional capacities, for example by facilitating the initiation of translation in an alternative way, or by acting as an essential protective element, shielding RNA from degradation by exonucleases.

[0140] Advantageously, the RNA of interest may be methylated, in particular at the level of one or more of its adenine and / or cytosine and / or pseudo-uridine residue(s). This may increase its translational efficiency in mammalian cells.

[0141] In some embodiments, the RNA of interest further comprises a sequence encoding a replicase allowing RNA self-amplification, and / or wherein the RNA of interest further comprises 3’ and 5’ sequences allowing the generation of circular RNA.

[0142] The first and second nucleotide sequences allow the formation, in the fungus cells, of complexes which comprise: a protein polymer comprising the Gag protein produced from the first nucleotide sequence, and an incorporated RNA of interest transcribed from the second nucleotide sequence.

[0143] The recombinant complexes produced by the method of the invention may be structured. For instance, the recombinant RNA-protein complexes may be in the form of a structure, such as e.g. a complex, a granule or a T-body.

[0144] In some embodiments, the recombinant RNA-protein complexes may be in the form of granules.

[0145] In some embodiments, the recombinant RNA-protein complexes may be in the form of T-bodies. "T-bodies” herein designates microscopically distinct cytoplasmic foci in which Gag proteins and Ty transposon RNA colocalize.

[0146] Thus, another object of the invention is a recombinant RNA-protein complex or T-body, which is obtained or obtainable by the method of the invention.

[0147] The method of the invention comprises a step of culturing fungus cells, which have been genetically modified to produce recombinant RNA-protein complexes comprising said RNA of interest.

[0148] The culture medium and culture conditions, and / or the transformation medium and transformation conditions used within the context of the method of the invention may advantageously stimulate or increase the production of the recombinant RNA-protein complexes.

[0149] The culture medium used within the context of the method of the invention may also advantageously stimulate or increase the growth or multiplication of the fungus cells. It may in particular comprise one or more compound(s) chosen among fungus extract, glucose, peptone and NaCl.

[0150] The culture medium used within the context of the method of the invention may also advantageously comprise a compound suitable for the induction of the promoter(s) that may be present in the nucleic acids comprising the first and second nucleotide sequences.

[0151] The culture conditions used within the context of the method of the invention may also advantageously stimulate or increase the growth or multiplication of the fungus cells. It may in particular comprise the addition of feed nutrients at the beginning or during the culture, the application of specific physico-chemical conditions. In particular, these physico-chemical conditions may include pH, oxygen level and temperature.

[0152] In some embodiments, the complexes, granules or T-bodies produced by the recombinant fungus cells are recovered, for example by lysis of the cells. For example, the complexes, granules or T-bodies produced by the recombinant fungus cells may be recovered by chemical or mechanical lysis (such as e.g. with beads, in particular glass beads, or using high pressure).

[0153] In some embodiments, the step of recovering the RNA of interest comprised in the complexes produced comprises:- lysing the fungus cells cultured at step a) of the method of the invention, preferably with glass beads, and centrifuging the lysed fungus cells and recovering the recombinant RNA-protein complexes from the pellet.

[0154] In some embodiments, the RNA of interest contained in these complexes, granules or T-bodies are then purified.

[0155] Alternatively, the RNA of interest contained in the complexes, granules or T- bodies are directly extracted and / or purified from the fungus cells, without prior extraction or recovery of the complexes, granules or T-bodies that contain them.

[0156] Advantageously, large amounts of RNA may be produced, for example at least 0.9 gram of RNA per liter of fungus cell culture.

[0157] Another object of the invention is a kit suitable for carrying out the method of recombinant production of an RNA of interest of the invention, wherein said kit comprises one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein has the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof, and- the second nucleotide sequence comprises a DNA sequence which comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3, wherein said kit optionally comprises cells of a fungus.

[0158] The nucleic acids comprising the first nucleotide sequence and the second nucleotide sequence may be combined and used simultaneously, sequentially or separately.

[0159] The kit of the invention may further comprise one or more fungus cells as described hereinabove. The fungus cells contained in the kit may comprise one or more copies of the first and second nucleotide sequences as described hereinabove.

[0160] The kit may further comprise a leaflet containing instructions for its use in the transfection or transformation of fungus cells, and / or for its use in the recombinant production of RNA of interest.

[0161] Another object of the invention is a recombinant fungus strain suitable for carrying out the method of the invention, wherein:- the fungus strain has been genetically modified to comprise one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof,- the second nucleotide sequence comprises a DNA sequence whose RNA transcript comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3.

[0162] The disclosure also relates to a method for producing DNA which comprises producing an RNA according to the method of the invention, to retrotranscribe this RNA into DNA (in particular into cDNA), and optionally to recover said DNA.

[0163] The disclosure also relates to a method for producing a protein, which comprises producing an mRNA according to the method of the invention, and translating this mRNA into a protein, and optionally to recover said protein.

[0164] The invention also relates to a method for the production of a pharmaceutical composition comprising an RNA of interest and a pharmaceutically acceptable carrier, vehicle or excipient.

[0165] Thus, another object of the invention is a method of producing a pharmaceutical composition which comprises at least one RNA of interest, said method comprising:- producing an RNA by the method of recombinant production of an RNA of interest of the invention, and- contacting said RNA with a pharmaceutically acceptable carrier, vehicle or excipient.

[0166] The term «pharmaceutical composition» in particular includes vaccine compositions or immunogenic compositions.

[0167] The term “pharmaceutically acceptable excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Said excipient does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.

[0168] Pharmaceutically acceptable excipients that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0169] In some embodiments, the pharmaceutical compositions according to the present invention comprise vehicles which are pharmaceutically acceptable for a formulationcapable of being injected to a subject. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0170] Examples of pharmaceutically acceptable vehicles may for example comprise injectable fluids, such as water, physiological saline, buffers, emulsions.

[0171] A pharmaceutically acceptable carrier may have one or more of the following functions: diluent functions, excipient, additive, emulsifier, dispersing agent, pH adjusting agent, preservative, surfactant, gelling agent, buffering agent, stabilizing agent, an RNA stabilizing agent (especially an agent protecting the RNA from enzymatic degradation), a solubilizing agent.

[0172] In some embodiments, the pharmaceutical composition of the invention is for:- the prevention or treatment of microbiological infection, in particular bacterial and / or viral and / or parasitic infectious diseases,- the prevention or treatment of tumor proliferation,- the prevention or treatment of a chronic disease (e.g. cystic fibrosis)- use as a vaccine,- tissue or cell regeneration therapy (e.g. cardiovascular diseases or rare diseases), or- gene therapy.

[0173] For example, an RNA of interest produced according to the invention may be formulated as a vaccine or an immunogenic composition, an anti-tumor drug, a drug for the treatment or palliation of cystic fibrosis (for example by the introduction of RNA encoding CFTR), or a tissue or cell regenerative product.TABLE OF SEQUENCESBRIEF DESCRIPTION OF THE DRAWINGS

[0174] Figure 1 shows a Gag alpha-fold view.

[0175] Figure 2 shows the final structure of the Tyl-Gag full-length molecule of sequence SEQ ID NO: 5, upon MD simulation.

[0176] Figure 3 shows the final structure of the truncated Tyl-Gag molecule of sequence (Gag amino acids 101-440, SEQ ID NO: 10), upon MD simulation.

[0177] Figure 4 shows the microphotographs of transformed yeast cells expressing T- bodies visualized by eGFP fluorescence: overlay of bright field (exposure to white light) and fluorescence (exposure to 488 nm wavelength) images. Yeast clones transformed with Tyl Gag 101-440, Tyl Gag 63-440, Tyl GAG 114-401 or Ty3 Gag.

[0178] Figure 5 shows the graph of flow cytometry of transformed yeast cells expressing truncated Tyl Gag (Tyl Gag 101-440, Tyl Gag 63-440 or Tyl Gag 114-401). Acquisition of 500,000 events per sample, in ungated plot. Laser: 488 nm. Filter: 533 / 30. Figure 5A: clones expressing Gag 101-440; Figure 5B: clones expressing Gag 63-440; Figure 5C: clones expressing Gag 114-401. Black line represents non-transformed cells.

[0179] Figure 6 shows protein size by SDS-PAGE / Westem Blot of truncated Tyl Gag 101-440, Tyl Gag 63-440 and Tyl Gag 114-401. Gag were revealed with an anti-eGFP monoclonal antibody.

[0180] Figure 7 shows particle size analysis of T-bodies extracted from yeast cells expressing truncated Ty 1 Gag (Tyl 63-440, Tyl 101-440 or Tyl 114-401) or full-length Gag (Tyl). Figure 7A: histogram of particle size for each Gag sequence; in black is the calibration standard. Figure 7B: average size of T-bodies with truncated or full-length Gag sequences (T-body mean diameter as measured by TRPS).

[0181] Figure 8 shows bar chart of percent increase in luciferase purity in truncated Tyl Gag (Gag 114-401) vs full-length Gag (Tyl Gag). Percent of purity is the ratio of luciferase mRNA to sum of luciferase and Gag mRNA.

[0182] Figure 9 shows bar chart of luciferase mRNA specific activity in mRNA extracted from yeast clones transformed with full-length (Tyl Gag) or truncated Tyl Gag sequences (Gag 63-440, Gag 101-440 or Gag 114-401).EXAMPLES

[0183] The present invention is further illustrated by the following examples.Example 1: In silica determination of Gag structure by molecular dynamic (MD) simulationsMaterials and Methods

[0184] Molecular dynamics (MD) simulations are typically used in molecular biology and drug discovery to capture the behavior of proteins and other biomolecules in full atomic detail and at very fine temporal resolution. Simulations have proven valuable in deciphering functional mechanisms of proteins and other biomolecules, in uncovering the structural basis for disease, and in the design and optimization of peptides, and proteins. MD simulations predict how every atom in a protein or other molecular system will move over time based on a general model of the physics governing interatomic interactions. These simulations can capture a wide variety of important biomolecular processes, including conformational change, ligand binding, and protein folding, revealing the positions of all of the atoms at femtosecond temporal resolution.

[0185] The full-length Gag sequence (SEQ ID NO : 5) and truncated Gag sequences (Gag amino acids 101-440, SEQ ID NO: 10) were submitted to MD simulations in order to predict the 3D conformation of proteins and exposure of specific areas that are described to be of importance for Gag-mRNA binding (i.e. nucleic acid chaperone sequence) as well as impact of removal of the N-terminal amino acids 1-100.

[0186] As part of this analysis, the Gag structure was divided into several regions (Figure 1):- N-terminal region: Gag amino acids 1-100 (SEQ ID NO: 6),- unstructured region: Gag amino acids 101-170 (SEQ ID NO: 7),- region of major interest: Gag amino acids 171-375 (SEQ ID NO: 8), and C-terminal region: Gag amino acids 376-440 (SEQ ID NO: 9).Results

[0187] Alphafold prediction (www.alphafold.ebi.ac.uk) indicates that region of major interest (Gag amino acids 171-375) can be predicted to form beta-helix structures (Figure 1, central part) whereas the other 3 regions (N-terminal and C-terminal) remain unstructured (Figure 1, N-terminal region on bottom and left sides, C-terminal region on right side).

[0188] MD simulations of full-length Tyl-Gag (SEQ ID NO: 5) indicates that structure quickly converges in a conformational space where the movements are caused by the unstructured loops causing an important tension on the central helix (Figure 2). Simulation of truncated Tyl-Gag (Gag amino acids 101-440, SEQ ID NO: 10) led to a different structure (Figure 3) that seems more stable due to more favorable hydrogen bonds. Juxtaposition of both structures focusing on the region of major interest indicate that the C-terminal part of this region (involved in chaperone activity) remains similar between full-length (SEQ ID NO: 5) and truncated Gag (Gag amino acids 101-440, SEQ ID NO: 10).

[0189] In conclusion, the truncation of the N-terminal aa 1-100 Gag sequence leads to a new, stable protein whose structure is significantly different from the full-length Gag, but with the chaperone region being better exposed to solvents than full-length Gag.Example 2:Materials and MethodsPreparation of plasmid vectorsDigestion, purification and assembly of plasmid

[0190] A DNA fragment of interest (i.e., to be inserted in plasmid vector) was isolated either by PCR or by digestion with restriction enzymes. Plasmid was digested with appropriate restriction enzymes in digestion buffer. Digestion products were loaded on an agarose gel and were then purified using NucleoSpin™ Gel and PCR Clean-up Kit (Macherey -Nagel™) following manufacturer’s instructions. Purified DNA fragment ofinterest and purified plasmid were assembled using Gibson Assembly® Cloning Kit (New England Biolabs) or NEBridge® Golden Gate Assembly Kit (New England Biolabs) following manufacturer’s instructions.Transformation ofE. coli cells

[0191] Competent E. coli cells (NEB® 5-alpha Competent E. coli, New England Biolabs) were transformed with the plasmid containing the DNA of interest following manufacturer’s instructions, and cultured on petri dishes. Transformant colonies were selected on the petri dishes and cultured individually in bacterial tubes. Plasmid DNA was extracted using a NucleoSpin Plasmid, Mini kit for plasmid DNA (Macherey- Nagel™) following manufacturer’s instructions. Positive clones were analyzed: (1) by restriction analysis to confirm the presence and correct insertion of the DNA of interest into the plasmid, and / or (2) by colony PCR to confirm the presence of the DNA of interest into the plasmid, and / or (3) by Sanger sequencing to verify integrity of the sequence of interest.Production of plasmid

[0192] Positive clones selected were cultured in Erlenmeyer flasks. Plasmid DNA was extracted using NucleoBond Xtra Midi kit for transfection-grade plasmid DNA or NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA (Macherey-Nagel™) following manufacturer’s instructions.Yeast transformation

[0193] Yeast strain from working glycerol stock was thawed and streaked on YPD agar plates and grown at 24-30°C for 3 to 5 days until colonies form.Pre-culture inoculation

[0194] 10 mL of YPD media in a sterile 125 mL baffled flask were inoculated with a single colony picked up from the YPD agar plate, and incubated at 24-30°C with shaking at 300 rpm for 1 or 2 days.Culture inoculation

[0195] 100 mL of YPD media in a sterile 1 -liter flask was inoculated with the pre-culture to an OD600 of 0.2, and incubated at 24-30°C for 1 or 2 days.

[0196] OD600 of the culture was monitored until it reached between 0.7 and 1.0. Cells were transferred to a centrifuge bottle and centrifuged at 4,000 g at 4°C for 10 minutes.Preparation of competent fungus cells

[0197] Supernatant was removed and the fungus pellet was washed twice with 1 mL of distilled sterile water. Then, supernatant was removed and the fungus pellet was washed again twice with 0.1 M lithium acetate (LiAOc).Transformation with plasmid DNA

[0198] The following reagents were added to the pellet: 240 pL PEG 50%, 36 pL 1 M LiAOc, 4 pL plasmid helper (1 pg / pL), 4 pL plasmid RNA (1 pg / mL), 10 pL denaturated salmon sperm, 30 pL sterile DMSO, and sterile distilled water, before vortexing for 1 minute at full speed. This mix was then incubated for 30 minutes at 25°C, followed by 20 minutes at 37°C, before centrifugation for 30 seconds at 8,000 rpm in a microcentrifuge. Supernatant was removed, 200 pL of sterile water was added to gently resuspend the pellet. These steps of centrifugation, supernatant removal and pellet resuspension was repeated once more.Selection of transformants

[0199] Transformant fungi were selected by spreading the resuspended pellet on a selective plate and incubated for 4 days at 25°C. In order to control the quality of the transformation, 10 colonies were picked up with a sterile handle, each was spread on a new selective plate and incubated for 4 days at 25°C.ProductionPre-culture and culture pre-induction

[0200] One fungus colony was picked from the transformants selection plates, placed into 3 mL of selective liquid growth medium, and incubated for 16 hours at 30°C with stirring (200 rpm). The 3 mL of culture were then transferred into a sterile 250 mL Erlenmeyer with baffles containing 30 mL of selective liquid growth medium and incubated for 16 hours at 30°C with stirring (200 rpm). The 30 mL of culture were then transferred in a sterile 2-liters Erlenmeyer flask with baffles containing 500 mL of selective liquid growth medium, and incubated with stirring (200 rpm) for approximately 7 hours until optical density reached 0.6 to 0.7.Induction

[0201] The culture was centrifuged 20 min at 4,000g and the pellet was washed twice with 500 mL of sterile water. The pellet was then resuspended gently with 500 mL of selective liquid induction medium, transferred in a sterile 2-liters Erlenmeyer flask with baffles and incubated overnight at 30°C with stirring (200 rpm).T-bodies isolation

[0202] Yeast cells were harvested by centrifuging at 4,000 g for 5 minutes at 4°C. Supernatant was decanted and remaining media was carefully removed by aspiration.

[0203] Lysis buffer was added to the fungus pellet and vortexed in order to resuspend the pellet. The sample was added to the same volume of acid-washed glass beads, vortexed for 2 minutes and cooled on ice for 2 minutes until the cells were completely disrupted.

[0204] The previous step was repeated by transferring half of the sample volume in a 50 mL falcon centrifuge tube and adding new lysis buffer onto beads.

[0205] The fungus lysate was centrifuged at 4,000 g for 10 minutes. The supernatant was carefully transferred into 2 mL Eppendorf tubes and centrifuged at 14,000 rpm for 30minutes. Finally, T-bodies were recovered from the pellet, optionally after specific washes of the T-bodies (with PBS or Tris buffer + additives) using same centrifugation parameters.T-bodies analysis

[0206] T-bodies from the fungus were quantified and characterized using Tunable Resistive Pulse Sensing (TRPS) technology post-lysis and post-purification.

[0207] Samples were kept as raw material and measured through different pores (NP 150 to observe particles >55 nm and / or NP100 to observe particles above 35 nm).

[0208] Presence of T-bodies issued from different cell culture conditions and observation of T-body shape or more diffuse forms through fluorescence of the Gag-mRNA interaction was used as a qualitative output. Repartition of the T-body size of the particles can also be observed to ensure robustness of the process.

[0209] Concentration of particles that may be directly correlated to Gag protein and mRNA concentration was evaluated as a quantitative output.

[0210] Tunable Resistive Pulse Sensing (TRPS) technology was used to follow T-body size, distribution and stability allowing to better control the T-body during purification process.Modified Gag analysis

[0211] Quantity and characteristics of modified Gag protein against other proteins was analyzed by absorbance at 280 nm throughout the purification process SDS-PAGE and western-blot. Typically, Gag particle size was analyzed. Gag protein concentration directly correlated to mRNA concentration provided an evaluation of the purity.SDS-PAGE

[0212] Stain free acrylamide gels were 4 to 20% acrylamide for high molecular weight proteins (>50 kDa), 12% acrylamide for mid-range molecular weight proteins (15-50 kDa), and 15% acrylamide for low molecular weight proteins (<15 kDa). 5 pL of pre-stained protein marker or 50 pL protein sample was loaded in each lane of the acrylamide gel.

[0213] Samples were run in gel running buffer (19.3 mM Glycine, 2.5 mM Tris base, 0.1% SDS) at 200V. Electrophoresis was complete when the dye front migrated about 2 mm from the bottom of the gel.

[0214] Gels are stain-free gels allowing direct imaging of the gels using Imager.

[0215] A piece of nitrocellulose membrane and 6 absorber papers were cut and wet in 15 mL of transfer buffer (25 mM Tris-HCL, 192 mM Glycine, 0.1% SDS, 20% ethanol) for 15 min at room temperature.

[0216] Transfer was done in semi-dry mode using a Transblot Transfer System at 25V for 40 min.

[0217] Then, the membrane was saturated for 1 hour at RT or at 2-8°C overnight with 15 mL IX TBS-T (150 mM NaCl, 20 mM Tris-HCl pH7.5, 1% Tween) + 3% milk (saturation buffer). The membrane was then incubated with primary anti-EGFP antibody at 2 mg / L in saturation buffer for 1.5 hour at RT or overnight at 2-8°C. The membrane is then washed 5 times with 20 mL for 12.5 min per wash in saturation buffer. The secondary antibody is then applied (0.2 mg / L in saturation buffer) for 45 to 1 hour. The membrane is finally washed 4 times with 20 mL for 10 min per wash in saturation buffer.

[0218] The membrane was placed in developing solution, prepared beforehand by adding 1 mL of luminol and 1 mL H2O2 for 5 min at RT. Visualization is performed through chemidoc station with exposition from 1.0 to 20.0 seconds with 1 image / s. mRNA analysis

[0219] mRNA of interest was quantified and characterized using absorbance at 260 nm and RT-qPCR methods.

[0220] mRNA was reverse-transcribed into cDNA using RevertAid RT reverse transcription kit (ThermoFischer). cDNA concentration was estimated through optical density analysis. qPCR was performed using QuantiFast SYBR® Green RT-PCR Kit (QIAGEN) following manufacturer’s protocol. Primers used were specific for the RNA of interest and for the polyA tail included. A standard plasmid was used as a control to produce a linear curve.

[0221] This RT-qPCR method allows to quantify specific mRNA and polyA mRNA. In addition, thanks to the primers, purity of the mRNA of interest can be calculated taking into consideration prior oligo DT purification during the process.Process-related impuritiesQuantification of host-derived proteins using ELISA HCP

[0222] Quantification of host-derived proteins was performed by ELISA HCP (Host Cell Proteins), which is an ELISA directed against proteins for host cell of choice.

[0223] Sample was loaded onto a multi -well plate coated with polyclonal IgG antibodies directed against host cell proteins, and washed. Then an anti-IgG antibody conjugated with alkaline phosphatase was added to the plate. Plate reading was performed on a spectrophotometer, after incubation of the plate with PNPP (p-nitrophenyl phosphate in a Diethanolamine).Quantification of host-cell derived DNA by qPCR assay

[0224] Quantification of host-cell derived DNA was performed by qPCR assay.

[0225] qPCR was performed using QuantiFast SYBR® Green RT-PCR Kit (QIAGEN) following manufacturer’s protocol, using primers specific to the host DNA and standard DNA used as a control to produce a linear curve. The level of DNA was quantified as compared to the standard curve.Example 3: Generation of yeast clones co-expressing either one of the truncated Gag proteins or Ty3 Gag and exogenous luciferase mRNAMaterials and MethodsPreparation of plasmid vectors List of plasmids and yeast strains

[0226] Tables 1 and 2 below show the lists of plasmids and yeast strains generated.Table 1: List of plasmids generatedTable 2: List of yeast strains generatedDigestion, purification and assembly of plasmid

[0227] Plasmid pOlO (Ty3 Gag-eGFP) was constructed by isolating DNA fragments of Ty3 Gag and eGFP by PCR. PCR-amplified fragments of interest and PCR-amplified plasmid were assembled using Gibson Assembly® Cloning Kit (New England Biolabs), following manufacturer’s instructions.

[0228] Truncated Gag plasmids (p014, p015 and p016) were constructed from pOOl plasmid using Gibson Assembly® Cloning Kit (New England Biolabs), following manufacturer’s instructions.Transformation ofE. coli cells

[0229] Competent E. coli cells (NEB® 5-alpha Competent E. coli, New England Biolabs) were transformed with the plasmid containing the DNA of interest following manufacturer’s instructions. Transformant colonies were selected on Petri dishes and cultured individually in bacterial tubes. Plasmid DNA was extracted using Monarch®Plasmid Miniprep kit (New England Biolabs, T1010), following manufacturer’s instructions. Positive clones were analyzed: (1) by colony PCR to confirm the presence of the DNA of interest into the plasmid, and / or (2) by Sanger sequencing or whole plasmid sequencing to verify integrity of the sequence of interest.Production of plasmid

[0230] Positive clones selected were cultured in Erlenmeyer flasks. Plasmid DNA was extracted using NucleoBond Xtra Midi kit (Macherey-Nagel, 740420), following manufacturer’s instructions.Yeast transformationPre-culture inoculation

[0231] One vial (1 mL) of SP-001 yeast strain working glycerol stock was thawed and inoculated in 25 mL of YPD medium in 125 mL baffled flask for about 16 hours.Culture inoculation

[0232] 50 mL of YPD media in a sterile 250 mL baffled flask was inoculated with the pre-culture to an OD600 of 0.2, and incubated at 30°C.

[0233] OD600 of the culture was monitored until it reached 0.7 or above. 3 mL of cell suspension were transferred to a centrifuge sterile tube and centrifuged at 4,000g at RT for 10 minutes.Preparation of competent cells

[0234] Supernatant was removed and the cell pellet was washed twice with 1 mL of distilled sterile water. Then, the supernatant was removed, and the cell pellet was washed again twice with 0.1 M lithium acetate (LiAOc).Transformation with plasmid DNA

[0235] The following reagents were added to the pellet: 240 pL PEG 50%, 36 pL 1 M LiAOc, 4 pL plasmid helper (1 pg / pL), 4 pL plasmid DNA (1 pg / mL), 10 pL denaturated salmon sperm (10 mg / mL), 30 pL sterile DMSO, and sterile distilled water, before vortexing for 1 minute at full speed. This mix was then incubated for 30 minutes at 30°C, followed by 20 minutes at 42°C, before centrifugation for 30 seconds at 10,000 g in a microcentrifuge. Supernatant was removed, 200 pL of sterile water was added togently resuspend the pellet. These steps of centrifugation, supernatant removal and pellet resuspension was repeated once more.Selection of transformants

[0236] Transformant cells were selected by spreading the resuspended pellet on a selective plate and incubated for 3-4 days at 30°C. Positive clones were analyzed by colony PCR to confirm the presence of the DNA of interest.ProductionPre-culture and culture pre-induction

[0237] Individual clones were picked from single colonies and inoculated in 5 mL of selection growth medium, in bacterial tubes. Tubes were incubated at 30°C, 200 rpm (25 mm / 1 inch orbital diameter), for 16-20 hours.

[0238] 25 mL of selection growth medium were inoculated at 0.1 unit of OD600 nm with pre-culture in 125 mL Erlenmeyer flasks. Erlenmeyer flasks were incubated at 30°C, 150 rpm (25 mm / 1 inch orbital diameter) or 120 rpm (50 mm / 2 inches orbital diameter), until OD600 nm reaches 0.6-0.7.Induction

[0239] Cell suspension was centrifuged 20 minutes at 4,000 g. Pellet was washed twice with 50 mL of sterile water. The pellet was then resuspended gently with 25 mL of prewarmed selection induction medium, transferred in a sterile 125 mL baffled Erlenmeyer flask, and incubated overnight at 30°C, 150 rpm (25-mm / l-inch orbital diameter). mRNA analysis

[0240] After total RNA extraction using Rneasy® mini prep Kit. Post extraction material was then quantified and characterized through absorbance at 260 nm and RT-qPCR methods.mRNA extraction

[0241] 200 pL of yeast cells were harvested by centrifuging at 4,000 g for 5 minutes at 4°C. Supernatant was decanted and remaining media was carefully removed by aspiration.

[0242] Total RNA was extracted from yeast cells by using RNeasy® mini prep Kit, according to the following protocol:

[0243] Lysis buffer was added to the fungus pellet and vortexed in order to resuspend the pellet. The sample was added to the same volume of acid-washed glass beads, vortexed for 2 times 5 minutes.

[0244] The previous step was repeated by transferring half of the sample volume in a 50 mL falcon centrifuge tube and adding new lysis buffer onto beads.

[0245] The fungus lysate was centrifuged at 4,000 g for 10 minutes to remove membrane debris. The supernatant was carefully transferred into 2 mL Eppendorf tubes. At this stage, T-bodies were disrupted through the action of lysis buffer (detergent + guanidine isothiocyanate).

[0246] Total RNA was then purified from host cell proteins, DNA and buffer components by following manufacturer instructions that consist in purification through silica column using Ethanol precipitation, specific buffers for washing and 10 mM Tris pH 7.5 to elute total RNA. mRNA analysis

[0247] mRNA was reverse-transcribed into cDNA using RevertAid RT reverse transcription kit (ThermoFischer). cDNA concentration was estimated through optical density analysis. qPCR was performed using QuantiFast SYBR® Green RT-PCR Kit (QIAGEN) following manufacturer’s protocol. Primers used were specific for the RNA of interest and for the included poly A tail. A standard plasmid was used as a control to produce a linear curve.

[0248] This RT-qPCR method allows to quantify specific mRNA and polyA mRNA. In addition, thanks to the primers, purity of the mRNA of interest can be calculated taking into consideration prior analysis of total RNA and protein observed by spectrophotometric method (absorbance at 260 and 280 nm).Results

[0249] The following plasmids were generated: plasmids based on pYES2 vector coding for truncated Tyl Gag protein (SEQ ID NO: 1, SEQ ID NO: 10 or SEQ ID NO: 2) (under control of an inducible promoter), fused with fluorescent tag (eGFP, SEQ ID NO: 12), plasmids coding for Ty3 Gag protein (SEQ ID NO: 11) (under control of an inducible promoter), fused with fluorescent tag (eGFP, SEQ ID NO: 12), plasmid coding for exogenous mRNA linked to Gag addressing sequence (under control of an inducible promoter), for example a mRNA coding for luciferase (SEQ ID NO: 13) linked (in 3’) to Gag DNA partial sequence SEQ ID NO: 3 and a poly-A tail of 120 nucleotides.

[0250] Yeast clones were generated and evaluated starting from a yeast strain void of Tyl retrotransposon (e.g. Saccharomyces paradoxus YAM13) co-transformed with 1) plasmids coding for truncated forms and 2) plasmids coding for exogenous mRNA. Clones were selected by growing cells under selection pressure on Petri dishes and then in shake flasks.

[0251] Cells transformed with clones expressing Tyl Gag 101-440-eGFP, Tyl Gag 63- 440-eGFP or Ty3 Gag-eGFP produced T-bodies. As illustrated on the microphotographs of Figure 4, the T-bodies are revealed by fluorescence of GFP protein co-expressed with the truncated Gag. Produced T-bodies, visible as bright round structures, about 50 to 100 nM large and spread within the cytoplasm of the cells, are observed in all pictures.

[0252] As shown in the flow cytometry graphs of Figures 5A-5B, after transformation with clones expressing Tyl Gag 101-440 (Figure 5A), Tyl Gag 63-440 (Figure 5B) andTyl Gag 114-401 (Figure 5C), the transformed cells display a majority of Gag-eGFP expressing cells.

[0253] Confirmation of protein expression of the three truncated Tyl Gag by transformed yeast strains SP-006 (Tyl Gag 63-440), SP-007 (Tyl Gag 101-440) and SP- 052 (Tyl Gag 114-401) was performed by running SDS-PAGE / Western Blot analysis of lysed yeast strains expressing truncated Gag sequences (SEQ ID NO: 10, SEQ ID NO: 1 or SEQ ID NO: 2). Tyl Gag proteins were revealed using an anti-eGFP antibody. As shown in Figure 6, truncated bands were visible at the appropriate molecular weight (70.1 kDa for SEQ ID: 1, 66.1 kDa for SEQ ID: 10 and 60.1 kDa for SEQ ID:2) demonstrating that the right proteins were expressed in yeast strains.Example 4: Purification and characterization of T-bodies isolated from transformed yeast cells producing truncated GagMaterials and MethodsYeast lysis and particle recovery

[0254] Yeast cells were washed two times with water by centrifugating cell suspension at 4,000 g for 5 minutes at 4°C. Supernatant was discarded and remaining media was carefully removed by aspiration. Cell pellet was resuspended in equal volume of 50 mM Tris pH 7.5, lO mM EDTA.

[0255] Yeast cells are disrupted by using a High Pressure Homogenization system (Panda plus from GEA Westfalia) at pressures ranging from 1500 to 2000 bars.

[0256] Membrane debris were removed by centrifugation at 4,000 g for 10 min, supernatant was then collected in a sterile bottle.

[0257] T-bodies were further purified from other particles using a combination of ion exchange chromatography and tangential filtration using hollow fiber.Ion exchange chromatography

[0258] Supernatant was loaded in an ion exchange membrane equilibrated with 50 mM Tris, 100 mM NaCl pH 7.5, 10 mM EDTA. Retained material was then washed with 4 volumes of equilibration buffer.

[0259] Elution was performed in buffer containing high NaCl content. Elution peak was collected following UV-280 / 260 nm for further analysis. Enrichment of T-bodies was confirmed by measuring mRNA content of the elution peak using RTqPCR technique described below.T-body concentration by Tangential filtration

[0260] Post-ion exchange eluate was concentrated through tangential filter (hollow fiber) with pore size adapted to the size of T-bodies. The material was concentrated by recirculation. Material was then dialyzed against 50 mM Tris, 100 mM NaCl, 10 mM EDTA.T-body characterization

[0261] T-bodies were quantified and characterized in size using Tunable Resistive Pulse Sensing (TRPS) technology. TRPS is a single-particle technique used to measure the size, concentration and zeta potential of particles as they pass through a size-tunable nanopore. The technique adapts the principle of resistive pulse sensing, which monitors current flow through an aperture, combined with the use of tunable nanopore technology, allowing the passage of ionic current and particles to be regulated by adjusting the pore size. The addition of the tunable nanopore allows for the measurement of a wider range of particle sizes and improves accuracy.Results

[0262] T-bodies extracted from yeast strains SP-006 (Tyl Gag 63-440), SP-007 (Tyl Gag 101-440) and SP-052 (Tyl Gagl 14-401) were analyzed by TRPS for particle size and content. The concentration of particles obtained ranged from 2.2 to 3.4xlO10particles per mL of concentrated post-ion exchange eluates. T-bodies were further characterizedfor particle size. The size of T-bodies measured in all truncated forms ranges from 50 to 250 nm with an average diameter of 100 to 130 nm (Figure 7A). Similar size range was observed among the three Tyl Gag truncated forms as well as for full-length Tyl Gag. In conclusion, the studied truncated Tyl Gag were able to generate T-bodies in spite of the truncation of their N-terminal sequence for Tyl Gag 63-440 and Tyl Gag 114-440 and both N- and C-terminal sequences for Tyl Gag 101-401. The measured average diameter of T-bodies was found to be on the same range among all Gag truncated forms (value between 100 and 130 nm) with a slightly higher size compared to full-length Gag (Figure 7B). These results indicated that the characteristics of T-bodies remained similar in truncated Gag forms despites removal of a significant part of their sequence.Example 5: Improved mRNA purity obtained from yeast strains expressing truncated Tyl GagMaterials and MethodsProduction of mRNA by transformed yeast strainsPre-culture and culture pre-induction

[0263] Clones from SP-006 (Tyl Gag 63-440), SP-007 (Tyl Gag 101-440) and SP-052 (Tyl Gag 114-401) strains were picked from single colonies and inoculated in 5 mL of selection growth medium, in bacterial tubes. Tubes were incubated at 30°C, 200 rpm (25 mm / 1 inch orbital diameter), for 16-20 hours.

[0264] 25 mL of selection growth medium were inoculated at 0.1 unit of OD600 nm with pre-culture in 125 mL Erlenmeyer flasks. Erlenmeyer flasks were incubated at 30°C, 150 rpm (25 mm / 1 inch orbital diameter) or 120 rpm (50 mm / 2 inches orbital diameter), until OD600 nm reaches 0.6-0.7.Induction

[0265] Cell suspension was centrifuged 20 minutes at 4,000 g. Pellet was washed twice with 50 mL of sterile water. The pellet was then resuspended gently with 25 mL of pre-warmed selection induction medium, transferred in a sterile 125 mL baffled Erlenmeyer flask, and incubated overnight at 30°C, 150 rpm (25-mm / l-inch orbital diameter). mRNA analysis mRNA extraction

[0266] 200 pL of yeast cells were harvested by centrifuging at 4,000 g for 5 minutes at 4°C. Supernatant was decanted and remaining media was carefully removed by aspiration.

[0267] Total RNA was extracted from yeast cells by using RNeasy® mini prep Kit, according to the following protocol:

[0268] Lysis buffer was added to the fungus pellet and vortexed in order to resuspend the pellet. The sample was added to the same volume of acid-washed glass beads, vortexed for 2 times 5 minutes.

[0269] The previous step was repeated by transferring half of the sample volume in a 50 mL falcon centrifuge tube and adding new lysis buffer onto beads.

[0270] The fungus lysate was centrifuged at 4,000 g for 10 minutes to remove membrane debris. The supernatant was carefully transferred into 2 mL Eppendorf tubes. At this stage, T-bodies were disrupted through the action of lysis buffer (detergent + guanidine isothiocyanate).

[0271] Total RNA was then purified from host cell proteins, DNA and buffer components by following manufacturer instructions that consist in purification through silica column using ethanol precipitation, specific buffers for washing and 10 mM Tris pH 7.5 to elute total RNA. mRNA characterization and purity assessment

[0272] Samples were pre-treated with proteinase K used to digest Gag protein and consequently release mRNA from T-bodies.mRNA analysis

[0273] mRNA of interest was characterized using RT-qPCR methods.

[0274] mRNA was reverse-transcribed into cDNA using RevertAid RT reverse transcription kit (ThermoFischer). qPCR was performed using QuantiFast SYBR® Green RT-PCR kit (QIAGEN) following manufacturer’s protocol. Primers used were specific for the RNA of interest, Gag mRNA and for the included poly A tail.

[0275] By using primers specific to their sequences, purity of the mRNA of interest can be calculated by ratioing the quantity of mRNA of interest with the total quantity of mRNA (sum of mRNA of interest and Gag mRNA).Results

[0276] The purity of luciferase mRNA was expressed as the ratio of luciferase mRNA to the sum of luciferase mRNA and Tyl Gag mRNA.Tyl Gag 114-401 induced higher purity than full-length Tyl Gag. This result indicates that purity of mRNA of interest (in this case luciferase) can be increased in T-bodies generated with truncated Tyl Gag compared to T-bodies generated with full-length Tyl Gag. This result is coherent with the fact that Tyl Gag mRNA 5’ upstream sequence (corresponding to N-term sequence of the Gag protein) is responsible for binding of the Tyl Gag mRNA to the Gag protein. Higher mRNA purity obtained with truncated Tyl Gag forms provides a tremendous advantage for the industrial production of mRNA from a quality and cost point of view.Example 6: Luciferase activity from mRNAMaterials and MethodsProduction of mRNA by transformed yeast strainsPre-culture and culture pre-induction

[0277] Clones from SP-006 (Tyl Gag 63-440), SP-007 (Tyl Gag 101-440) and SP-052 (Tyl Gag 114-401) strains were picked from single colonies and inoculated in 5 mL ofselection growth medium, in bacterial tubes. Tubes were incubated at 30°C, 200 rpm (25 mm / 1 inch orbital diameter), for 16-20 hours.

[0278] 25 mL of selection growth medium were inoculated at 0.1 unit of OD600 nm with pre-culture in 125 mL Erlenmeyer flasks. Erlenmeyer flasks were incubated at 30°C, 150 rpm (25 mm / 1 inch orbital diameter) or 120 rpm (50 mm / 2 inches orbital diameter), until OD600 nm reaches 0.6-0.7.Induction

[0279] Cell suspension was centrifuged 20 minutes at 4,000 g. Pellet was washed twice with 50 mL of sterile water. The pellet was then resuspended gently with 25 mL of prewarmed selection induction medium, transferred in a sterile 125 mL baffled Erlenmeyer flask, and incubated overnight at 30°C, 150 rpm (25-mm / l-inch orbital diameter). mRNA analysis

[0280] After total RNA extraction using Rneasy® mini prep Kit. Post extraction material was then quantified and characterized through absorbance at 260 nm and RT-qPCR methods. mRNA extraction

[0281] 200 pL of yeast cells were harvested by centrifuging at 4,000 g for 5 minutes at 4°C. Supernatant was decanted and remaining media was carefully removed by aspiration.

[0282] Total RNA was extracted from yeast cells by using RNeasy® mini prep Kit, according to the following protocol:

[0283] Lysis buffer was added to the fungus pellet and vortexed in order to resuspend the pellet. The sample was added to the same volume of acid-washed glass beads, vortexed twice for 5 minutes.

[0284] The previous step was repeated by transferring half of the sample volume in a 50 mL falcon centrifuge tube and adding new lysis buffer onto beads.

[0285] The fungus lysate was centrifuged at 4,000 g for 10 minutes to remove membrane debris. The supernatant was carefully transferred into 2 mL Eppendorf tubes. At this stage, T-bodies were disrupted through the action of lysis buffer (detergent + guanidine isothiocyanate).

[0286] Total RNA was then purified from host cell proteins, DNA and buffer components by following manufacturer instructions that consist in purification through silica column using ethanol precipitation, specific buffers for washing and 10 mM Tris pH 7.5 to elute total RNA.Luciferase mRNA potency assessment

[0287] The potency of luciferase mRNA was assessed by 1) generating luciferase protein from sample mRNA using Rabbit reticulocyte lysate system kit and 2) measuring luminescence induced by luciferase protein using a Firefly luciferase glow assay kit (Pierce).

[0288] 1 pL of purified mRNA from yeast strains transformed with Tyl truncated Gag and 0.5 pL of control mRNA (supplied in Rabbit reticulocyte lysate system kit) were first translated onto proteins by adding them to a preaction pool. For one reaction, 35 pl of rabbit reticulocyte lysate were mixed with 0.5 pL of 1 mM amino acid mixture minus leucine, 0.5 pL of 1 mM amino acid mixture minus methionine, 1 pL of Ribolock, and 9 pL of rnase free water. Material was then incubated for 90 to 180 min at 30°C.

[0289] In each sample, 50 pL of D-Luciferin were added and luminescence was measured after 10 min using a Fluostar Omega system (BMG Labtech) using specific 96 black-well plate.Results

[0290] Results are expressed in RFU / pg of mRNA of interest (luciferase) of the two independent experiments. Similar translation / activity potency of the mRNA produced in truncated Tyl Gag sequences could be observed whatever the Gag truncation (Figure 9). Furthermore, truncated Gag sequences yielded a mRNA that were more potent thanmRNA from full-length Tyl Gag. These results demonstrate the ability of truncated Gag to yield mRNA of higher potency than full-length Gag.

Claims

CLAIMS1. A method of recombinant production of an RNA of interest comprising: a) culturing fungus cells, which have been genetically modified to produce recombinant RNA-protein complexes comprising said RNA of interest, and b) recovering said RNA of interest comprised in the complexes thus produced, wherein genetically modifying the fungus cells to produce the complexes comprises transfecting said fungus cells with one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to the sequence SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof,- the second nucleotide sequence comprises a DNA sequence whose RNA transcript is:- the sequence of said RNA of interest, linked to- an addressing sequence comprising, or consisting of, the sequence SEQ ID NO: 3, wherein the first and second nucleotide sequences thereby allowing the formation, in said fungus cells, of recombinant complexes encapsulating said RNA of interest.

2. The method of claim 1 , wherein step b) of recovering the RNA of interest comprised in the complexes produced comprises: lysing the fungus cells cultured at step a), preferably with glass beads, and centrifuging the lysed fungus cells and recovering the recombinant RNA- protein complexes from the pellet.

3. The method of any one of claims 1 to 2, wherein the fungus is Thermothelomyces heterothallica (formerly Myceliophthora thermophilia), Saccharomyces paradoxus, Saccharomyces cerevisiae, Pichia pastoris (Komagataella phaffii), Hansenula polymorpha (Pichia augusta), Yarrowia lipolytica, Kluyveromyces marxianus, Arxula adeninivorans, Kluyveromyces lactis, or Schizosaccharomyces pombe.

4. The method of any one of claims 1 to 3, wherein the fungus cell contains at least one mutated gene selected among the group consisting of the rpbl, spt21, srb2 and srb5 genes, wherein said at least one mutated gene comprises at least one mutation stimulating or increasing the production of the recombinant RNA-protein complexes.

5. The method of any one of claims 1 to 4, wherein the RNA of interest comprises a sequence encoding:- a bacterial protein or polypeptide,- a genome editing enzyme,- a virus protein or polypeptide,- a transcription factor,- a growth factor,- a CFTR protein,- an antibody or antibody fragment,- a polypeptide comprising at least one antigen epitope, or- a combination thereof.

6. The method of claim 5, wherein the RNA of interest further comprises a sequence encoding a replicase allowing RNA self-amplification, and / or wherein the RNA of interest further comprises 3’ and 5’ sequences allowing the generation of circular RNA.

7. A kit suitable for carrying out the method according to any one of claims 1 to 6, wherein said kit comprises one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof, and- the second nucleotide sequence comprises a DNA sequence which comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3, wherein said kit optionally comprises cells of a fungus.

8. A recombinant fungus strain suitable for carrying out the method according to any one of claims 1 to 6, wherein the fungus strain has been genetically modified to comprise one or more nucleic acid(s) comprising a first nucleotide sequence and a second nucleotide sequence, wherein:- the first nucleotide sequence comprises a DNA sequence encoding the Gag protein of a fungus Ty retrotransposon, said Gag protein having the sequence of SEQ ID NO: 1, or a fragment thereof comprising the sequence SEQ ID NO: 2, or a sequence at least 90% identical to SEQ ID NO: 1 or to said fragment thereof comprising the sequence SEQ ID NO: 2, or said Gag protein having the sequence SEQ ID NO: 11 or a fragment thereof, or a sequence at least 90% identical to the sequence SEQ ID NO: 11 or to said fragment thereof, and- the second nucleotide sequence comprises a DNA sequence whose RNA transcript comprises, or consists of, the sequence SEQ ID NO: 3 or a sequence at least 90% identical to SEQ ID NO: 3.

9. A recombinant RNA-protein complex or T-body, which is obtained or obtainable by the method of any one of claims 1 to 6.

10. A method of producing a pharmaceutical composition which comprises at least one RNA of interest, said method comprising:- producing an RNA by the method of any one of claims 1 to 4, and- contacting said RNA with a pharmaceutically acceptable carrier.

11. The method of claim 8, wherein the pharmaceutical composition is for:- the prevention or treatment of microbiological infection,- the prevention or treatment of tumor proliferation,- the prevention or treatment of a chronic disease,- use as a vaccine,- tissue or cell regeneration therapy, or- gene therapy.

12. The method of any one of claims 1 to 4, or the in vitro use of the kit of claim 5, or the recombinant fungus strain of claim 6, or the recombinant T-body of claim 7, or the method of claim 8 or 9, wherein the RNA of interest is a messenger RNA (mRNA) or a non-coding long RNA (IncRNA).

13. The method of any one of claims 1 to 4, or the in vitro use of the kit of claim 5, or the recombinant fungus strain of claim 6, or the recombinant T-body of claim 7, or the method of claim 8 or 9, wherein the RNA of interest is an mRNA or IncRNA having a 3'- polyA tail and / or a 5'- methylguanosine cap.