TRANSIENT EXPRESSION SYSTEM FOR RNA

FIELD OF INVENTION

[0001] The present invention relates to means and methods for robust transient RNA expression.

BACKGROUND OF INVENTION

[0002] Transferring genetic information to a cell typically requires the design of vectors capable of delivering this genetic information to the cell. There exist two main types of vectors: non-viral vectors and viral vectors, each of which is capable of transferring information, either in the form of DNA or RNA.

[0003] When transient - rather than sustained - effects are desired, non-integrating DNA vectors or RNA vectors are excellent candidates. However, non-integrating DNA vectors can induce adverse genotoxic effects due to the non-zero probability of any DNA molecule to recombine with another DNA molecule, for instance, with the DNA genome of the host cell. RNA vectors do not exhibit this risk of genotoxicity, since RNA cannot recombine with DNA. Nonetheless, current RNA vectors are not devoid of drawbacks, in particular in terms of efficacy. For instance, non-viral RNA vectors are underperforming, due to a low degree of RNA protection against degradation, and often, a low transfection rate. Hence, the few RNA molecules which ultimately reach the cytoplasm of a cell induce only poor transgene expression.

[0004] Retroviridae, or retroviruses, is a family of RNA viruses which are capable of inserting a copy of their genome - after reverse transcription using their own reverse transcriptase enzyme (RT) - into the chromosomal DNA of the host cells that they invade. The host cells then treat the viral DNA as part of their own genome, transcribing and translating the viral genes along with their own genes. This ability of Retroviridae has made them (and more particularly viruses from the Gammaretrovirus and Lentivirus genera) a benchmark tool for therapeutic gene delivery and transfer into cells since the early 1980’ s. More recently, these RNA vectors have been engineered to allow transient expression of therapeutic proteins. Such engineered vectors were described in WO 2005/116225 Al, WO 2013/060819 A2 or Mock etal., 2014 (Sci Rep. 4:6409) relating to retroviral vectors with detective RT activity.

[0005] Yet, these RT-defective retroviral vectors, although offering a transient expression of therapeutic proteins from their mRNA without the need of a DNA intermediate, are not devoid of drawbacks. Indeed, they typically package only up to two copies of their RNA genome, into which the transgene of interest (e.g., a therapeutic mRNA) is inserted. In other words, one retroviral vector transducing a host cell can only deliver two copies of a transgene of interest. Given that RNAs are very labile molecules - and thus rapidly degraded in the host cell, this solution was considered somehow ineffective, unless high vector doses are repeatedly administered in order to outweigh these issues. For in vivo applications, this is not desirable. For instance, Mock et al. (2014. Sci Rep. 4:6409) came to this conclusion, stating that they “observed very weak cap-dependent translation initiation from standard lentiviral vector genomes”, and called for further improvements.

[0006] There remains thus a need for RNA vectors, capable of inducing high transient expression levels in host cells, but without requiring the administration of high loads of vector.

[0007] Here, the Inventors have identified an artificial 9-nucleotide sequence, that they have named “RNA Booster”. The Inventors have surprisingly observed that the presence of this RNA Booster upstream or downstream of a transgene highly improved transgene expression in various host cells.

SUMMARY

[0008] The present invention relates to a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule comprising: an RNA Booster sequence comprising or consisting of the ribonucleic acid or deoxyribonucleic acid sequence mmsknkkkm or its reverse sequence mkkknksmm, wherein:

“m” indicates an adenine (a) or cytosine (c); “s” indicates a guanine (g) or a cytosine (c);

“k” indicates a guanine (g) or a uracyl/thymine (u/t);

“n” indicates any nucleotide; and a sequence of interest.

[0009] In some embodiments, the RNA Booster sequence comprises or consists of a ribonucleic acid or a deoxyribonucleic acid sequence mmskngkkm or its reverse sequence mkkgnksmm, preferably mmskngkgm or its reverse sequence mgkgnksmm, more preferably cmskhgkgm or its reverse mgkghksmc, even more preferably cmskwgkgm or its reverse sequence mgkgwksmc, yet even more preferably ccsuwgggm or its reverse sequence mgggwuscc, wherein:

■ “m” indicates an adenine (a) or cytosine (c);

■ “s” indicates a guanine (g) or a cytosine (c);

■ “k” indicates a guanine (g) or a uracyl/thymine (u/t);

■ “h” indicates an adenine (a) or a cytosine (c) or a uracyl/thymine (u/t);

■ “w” indicates an adenine (a) or a uracyl/thymine (u/t);

■ “n” indicates any nucleotide;

[0010] In some embodiments, the RNA Booster sequence is selected from the group consisting of:

RNA Booster 9 comprising or consisting of the sequence ccguaggga or its reverse sequence agggaugcc;

RNA Booster 8 comprising or consisting of the sequence cccuugggc or its reverse sequence cggguuccc;

RNA Booster 7 comprising or consisting of the sequence cacgugugc or its reverse sequence cgugugcac;

RNA Booster 6 comprising or consisting of the sequence cccucgggc or its reverse sequence cgggcuccc;

RNA Booster 5 comprising or consisting of the sequence aacuggggc or its reverse sequence cggggucaa;

RNA Booster 4 comprising or consisting of the sequence ccguggugc or its reverse sequence cguggugcc; RNA Booster 3 comprising or consisting of the sequence cccuaggua or its reverse sequence auggauccc;

RNA Booster 2 comprising or consisting of the sequence aaguuuggc or its reverse sequence cgguuugaa; and

RNA Booster 1 comprising or consisting of the sequence cccgugugc or its reverse sequence cgugugccc.

[0011] In some embodiments, the RNA molecule is comprised within a non-viral vector.

[0012] In some embodiments, the RNA molecule is packaged into an RNA virus vector derived from a Group III, Group IV, Group V or Group VI RNA virus. Preferably, the RNA molecule is packaged into RNA virus vector derived from a Group VI RNA virus. More preferably, the RNA molecule is packaged into a Retroviridae vector.

[0013] In some embodiments, the Retroviridae vector is an Orthoretrovirinae or a Spumaretrovirinae . Preferably, the Retroviridae vector is an Orthoretrovirinae. More preferably, the Retroviridae vector is selected from the group consisting of human immunodeficiency viruses (HIV), simian immunodeficiency viruses (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), puma lentivirus (PLV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus, Jembrana disease virus, avian sarcoma leukosis virus (ASLV), Rous sarcoma virus (RSV), avian myeloblastosis virus (AMV), mouse mammary tumor virus (MMTV), Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor viruses (ENTV), simian retroviruses (SRV), Mason-Pfizer monkey virus (M-PMV), human T-lymphotropic viruses (HTLV), simian T-lympho tropic viruses (STLV), bovine leukemia virus (BLV), Walleye dermal sarcoma virus (WDSV), Walleye epidermal hyperplasia viruses (WEHV), murine leukemia viruses (MLV), Abelson murine leukemia virus (AMLV), Friend virus (FV), feline leukemia virus (FeLV), koala retrovirus (KoRV), xenotropic murine leukemia virus-related virus (XMRV), chick syncytial virus (CSV), murine sarcoma viruses (MSV), feline sarcoma viruses (FSV), Gibbon ape leukemia virus (GaLV), guinea pig type-C oncovirus, porcine type-C oncovirus, reticuloendotheliosis virus, Trager duck spleen necrosis virus, viper retrovirus, and Woolly monkey sarcoma virus. [0014] In some embodiments, the Retroviridae vector is a lentiviral vector. Preferably, the Retroviridae vector is a lentiviral vector selected from the group consisting of human immunodeficiency virus-1 (HIV-1) and HIV-2. More preferably, the Retroviridae vector is HIV- 1.

[0015] In some embodiments, the Group VI RNA virus vector (preferably the Retroviridae vector) is reverse transcriptase (RT)-defective. Preferably, the Group VI RNA virus vector (preferably the Retroviridae vector) does not comprise a gene encoding a reverse transcriptase or wherein the retroviral vector comprises a gene encoding a mutated reverse transcriptase with abolished reverse transcription activity.

[0016] In some embodiments, when the RNA molecule is packaged into an RNA virus vector, the RNA molecule further comprises one or several of: a 5’ long terminal repeat (LTR), a packaging sequence, a Rev-response element sequence, a post-transcriptional regulation element sequence, and a 3’ LTR.

[0017] The present invention also relates to a pharmaceutical composition comprising the RNA or DNA molecule of the invention, optionally comprised within a non-viral vector or packaged into an RNA virus vector, and at least one pharmaceutically acceptable excipient or carrier.

[0018] The present invention also relates to the RNA or DNA molecule of the invention, optionally comprised within a non-viral vector or packaged into an RNA virus vector, or to the pharmaceutical composition comprising the same, for use in therapy.

[0019] The present invention also relates to an in vitro method of transiently expressing a sequence of interest in a cell, comprising transfecting or transducing the cell with the RNA molecule of the invention, optionally comprised within a non-viral vector or packaged into an RNA virus vector.

[0020] The present invention also relates to a nucleic acid system comprising: (i) a t least one first nucleic acid sequence encoding a Retroviridae genome comprising at least a retroviral gag and pol sequence; optionally wherein the pol sequence encodes a defective reverse-transcriptase (RT) or wherein the pol sequence lacks a RT gene;

(ii) at least one second nucleic acid sequence encoding a viral envelope glycoprotein; and

(iii) at least one third nucleic acid sequence encoding an expression cassette comprising: a packaging sequence,

- an RNA Booster sequence comprising or consisting of the following ribonucleic acid or deoxyribonucleic acid sequence mmsknkkkm or its reverse sequence mkkknksmm, wherein:

■ “m” indicates an adenine (a) or cytosine (c);

■ “s” indicates a guanine (g) or a cytosine (c);

■ “k” indicates a guanine (g) or a uracyl/thymine (u/t);

■ “n” indicates any nucleotide, optionally, a multiple cloning site, optionally, a sequence of interest; wherein the first and second nucleic acid sequences are trans-complementation sequences lacking a functional packaging sequence; preferably wherein the RNA Booster sequence comprises or consists of a nucleic acid sequence mmskngkkm or its reverse sequence mkkgnksmm, preferably of mmskngkgm or its reverse sequence mgkgnksmm, more preferably cmskhgkgm or its reverse sequence mgkghksmc, even more preferably cmskwgkgm or its reverse sequence mgkgwksmc, yet even more preferably ccsuwgggm or its reverse sequence mgggwuscc, wherein:

■ “m” indicates an adenine (a) or cytosine (c);

■ “s” indicates a guanine (g) or a cytosine (c);

■ “k” indicates a guanine (g) or a uracyl/thymine (u/t);

■ “h” indicates an adenine (a) or a cytosine (c) or a uracyl/thymine (u/t);

■ “w” indicates an adenine (a) or a uracyl/thymine (u/t);

■ “n” indicates any nucleotide; more preferably the RNA Booster sequence is selected from the group consisting of:

RNA Booster 9 comprising or consisting of the sequence ccguaggga or its reverse sequence agggaugcc;

RNA Booster 8 comprising or consisting of the sequence cccuugggc or its reverse sequence cggguuccc;

RNA Booster 7 comprising or consisting of the sequence cacgugugc or its reverse sequence cgugugcac;

RNA Booster 6 comprising or consisting of the sequence cccucgggc or its reverse sequence cgggcuccc;

RNA Booster 5 comprising or consisting of the sequence aacuggggc or its reverse sequence cggggucaa;

RNA Booster 4 comprising or consisting of the sequence ccguggugc or its reverse sequence cguggugcc;

RNA Booster 3 comprising or consisting of the sequence cccuaggua or its reverse sequence auggauccc;

RNA Booster 2 comprising or consisting of the sequence aaguuuggc or its reverse sequence cgguuugaa; and

RNA Booster 1 comprising or consisting of the sequence cccgugugc or its reverse sequence cgugugccc.

[0021] In some embodiment, each at least one nucleic acid sequence (i), (ii) and (iii) is independently from each other a linear nucleic acid or a plasmid.

[0022] The present invention also relates to a cell or cell population comprising the RNA or DNA molecule of the invention or the nucleic acid system of the invention.

[0023] The present invention also relates to a method of producing a Retroviridae vector comprising the RNA molecule of the invention, comprising: transfecting a cell or cell population with at least one RNA or DNA molecule of the invention, with the nucleic acid system of the invention, or providing a cell or cell population comprising the nucleic acid system of the invention; culturing the cell or cell population of a period of time sufficient for the production of the Retroviridae vector; recovering, and optionally purifying, the Retroviridae vector.

[0024] The present invention also relates to another nucleic acid system comprising at least one nucleic acid sequence encoding a recombinant expression cassette comprising:

- optionally, a promoter, and

- an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, and

- a multiple cloning site (MCS) or a sequence of interest, and

- optionally, an origin of replication (ORI), and

- optionally, a selectable marker.

DEFINITIONS

[0025] In the present invention, the following terms have the following meanings:

[0026] The terms “a” and “an” refer to one or to more than one (z.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0027] “About” preceding a number encompasses plus or minus 10%, or less, of the value of said number. It is to be understood that the value to which the term “about” refers is itself also specifically, and preferably, disclosed.

[0028] “Antigen”, also termed “immunogen”, refers to any substance that induces a state of sensitivity and/or immune responsiveness after any latent period (normally, days to weeks in humans) and that reacts in a demonstrable way with antibodies and/or immune cells of the sensitized subject in vivo or in vitro.

[0029] “Collagen induction therapy”, also known as “microneedling”, “dermarolling”, or “skin needling”, refers to a cosmetic procedure which involves repeatedly puncturing the skin of a subject with microneedles (z.e., needles having a length typically ranging from about 100 pm to 1 000 pm). [0030] “Consist essentially of’ and any declension thereof, with reference to a composition, means that the RNA or DNA molecule (or the vector comprising said RNA or DNA molecule), is the only one therapeutic agent or agent with a biological activity within said composition or pharmaceutical composition.

[0031] “Encoding”, as in encoding sequence, refers to the inherent property of a specific sequence of nucleotides in a nucleic acid, such as a gene, a complementary DNA (cDNA), or a messenger RNA (mRNA), to serve as template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., ribosomal RNA (rRNA), transfer RNA (tRNA) and mRNA) or a defined sequence of amino acids (e.g., polypeptide or protein) and the biological properties resulting therefrom.

[0032] “Exogenous” refers to a molecule that is not naturally present in a cell, but can be introduced into the cell by one or more genetic, biochemical or other methods. Natural presence in the cell may be determined with respect to the particular developmental stage and environmental conditions of the cell. For instance, a molecule that is present only in a cell during embryonic development is exogenous with respect to a cell in an adult subject. Similarly, a molecule induced by heat shock of a cell is exogenous with respect to a non-heat- shocked cell.

[0033] “Expression” refers to the transcription and/or translation of a particular nucleotide sequence, such as a gene.

[0034] As used herein, when referring to a given sequence, the expression “has/having a sequence as set forth in SEQ ID NO: X” means that the given sequence comprises or consists of the sequence as set forth in SEQ ID NO: X. The given sequence implicitly refers to a nucleic acid sequence (DNA or RNA sequence) or an amino acid sequence.

[0035] "Isolated'' with reference to a nucleic acid refers to a nucleic acid altered or removed from the natural state. For example, a nucleic acid naturally present in a living organism is not "isolated" but the nucleic acid partially or completely separated from the coexisting materials of its natural state is “isolated”. An “isolated nucleic acid” is thus a nucleic acid that is substantially separated from other nucleic acid sequences, such as genomic DNA or RNA, as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An isolated nucleic acid can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. Typically, a preparation of an isolated nucleic acid may comprise the nucleic acid at least about 80% pure, at least about 85% pure, at least about 90% pure, at least about 95% pure, greater than about 95% pure, greater than about 96% pure, greater than about 97% pure, greater than about 98% pure, or greater than about 99% pure. Isolated nucleic acids thus include nucleic acids purified by standard purification methods, and encompass nucleic acid sequences that have been removed from their naturally occurring environment. Isolated nucleic acids also include chemically synthesized nucleic acids and nucleic acids biologically synthesized by heterologous systems.

[0036] “Long-terminal repeats” refer to sequences of several hundred base pairs long. In RNA viruses, their genome is flanked by LTRs (a 5’ LTR and a 3’ LTR), typically having identical sequences.

[0037] “Rev-response element” refers to a highly structured RNA segment interacting with the Rev protein, allowing the viral genome to be exported to the cytoplasm for downstream processing, including virion packaging. Rev-response elements are typically characteristic of lentiviruses, but other RNA viruses of Group VI comprise similar systems, such as the Rem-response element in Betaretroviruses, the Rex-response element in Deltaretroviruses, or the constitutive transport element (CTE). These are also encompassed when mentioning Rev-response element herein, even if not explicitly cited.

[0038] “Nucleic acid” refers to a polymer of nucleotides (z.e., polynucleotides) covalently linked by phosphodiester bonds, such as deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), in either single- or double- stranded form. A used herein, a nucleic acid may thus be single-stranded, partially double-stranded, or fully doublestranded. The nucleotides making up nucleic acids of the present disclosure may be unmodified (natural) nucleotides or non-natural or modified nucleotides. Unmodified (or natural or naturally occurring) nucleotides include adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), cytidine monophosphate (CMP), deoxycytidine monophosphate (dCMP), guanosine monophosphate (GMP), deoxyguanosine monophosphate (dGMP), thymidine monophosphate (TMP), deoxythymidine monophosphate (dTMP), and uridine monophosphate (UMP). The term “nucleic acid” also encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.

[0039] “Nucleic acid sequence” or “nucleotide sequence” refers to a contiguous sequence of nucleotides in a single nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs (single-nucleotide polymorphisms), and complementary sequences as well as the sequence explicitly indicated. Notably, a particular nucleic acid sequence described herein implicitly comprises its corresponding complementary sequence, named reverse sequence. It should be noted that a particular nucleic acid sequence described herein implicitly comprises the DNA sequence and the corresponding RNA sequence.

[0040] “Operatively linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, e.g., a gene, resulting in a regulation of the expression of the latter by the former. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some embodiments, a promoter is operably linked to a gene if the promoter affects the transcription or expression of the gene. In some embodiments, an RNA Booster is operably linked to a gene if the RNA Booster affects the expression of the gene. Similarly, a regulatory sequence is operably linked to a gene if the regulatory sequence affects (z.e., either induces or inhibits (or represses)) the expression of the gene. Operably linked sequences can be contiguous with each other.

[0041] “Packaging sequence” refers to a stem-loop structured cz'.s- acting nucleic acid sequence, which regulates the process of packaging inside a viral capsid. This packaging sequence may be referred in the art to as “packaging signal” denoted “y”, or “encapsidation signal” denoted “E”. Packaging sequences may be of viral origin (z.e., wild-type viral packaging sequences) or may be synthetic. [0042] “Pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. The excipient or carrier does not produce any adverse, allergic or other untoward reaction when administered to a subject, preferably to a human. A pharmaceutically acceptable excipient or carrier is typically a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, the FDA (US Food and Drug Administration) or EMA (European Medicines Agency).

[0043] “Post-transcriptional regulation elements” refer to czs-acting RNA sequences that can increase the accumulation of cytoplasmic mRNA by promoting mRNA exportation from the nucleus to the cytoplasm, enhancing 3’-end processing and stability.

[0044] “Gene” broadly refers to an encoding nucleic acid sequence that can be transcribed into an RNA molecule, either a coding RNA molecule such as a mRNA which can be subsequently translated into a polypeptide or protein, or a non-coding RNA molecule such as a rRNA or a tRNA. Thus, as used herein, the term “gene” may refer to an encoding nucleic acid sequence comprising a coding sequence (or CDS) and at least one regulatory element that is transcribed but not translated, such as a 3’-UTR, a 5’-UTR and/or an intron.

[0045] “Sequence of interest” refers to as “transgene (of interest)”, refers to any nucleic acid sequence encoding a product of interest. The product of interest may be a protein or a fragment thereof; in this case, the sequence of interest is said to be a coding nucleic acid sequence. In this embodiment, the transgenes refers in particular to a gene originating from one species which is to be introduced into an organism belonging to a different species. It should thus be noted that a gene may or may not encompass a coding sequence (or CDS), that is to say a nucleic acid sequence that actually codes for a protein. A gene, in particular a gene encompassing a CDS, may also preferably encompass untranslated transcribed regions (UTRs), such as a 3’-UTR and/or a 5’-UTR and other sequences, such as regulatory elements and/or introns, which are transcribed but not translated. However, the term also encompasses non-coding nucleic acid sequences, i.e., nucleic acid sequences that do not encode a protein or a fragment thereof, but rather express an “RNA gene” (or “non-coding RNA”), such as, e.g., a transfer RNA, a ribosomal RNA, a small RNA, a long non-coding RNA, etc. The context will indicate whether the term “sequence of interest” refers to a DNA or an RNA sequence.

[0046] “Transgenesis” refers to the process of introducing one or several transgenes from one organism into another, with the intent of enabling the latter to transmit this transgene to its offspring.

[0047] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic measures; wherein the object is to slow down (lessen) the targeted pathologic condition or disorder. A subject or mammal is successfully "treated" for an infection if, after receiving a therapeutic amount of an RNA or DNA molecule of the present invention, the patient shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of pathogenic cells; reduction in the percent of total cells that are pathogenic; and/or relief to some extent, one or more of the symptoms associated with the specific disease or condition; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0048] “Vector” refers to a vehicle by which a nucleic acid sequence e.g., a DNA or RNA molecule), for example a nucleic acid encoding an RNA or a polypeptide or protein of interest, can be introduced into a host cell, so as to transform, transfect or transduce the host cell and promote expression (e.g., transcription and/or translation) of the introduced nucleic acid sequence.

[0049] “Expression vector” refers to a vector comprising regulatory elements (or regulatory sequences) operatively linked or to be operatively linked to a nucleic acid sequence of interest to be expressed, such as a gene of interest. An expression vector thus comprises sufficient cis-acting regulatory elements for controlling the expression of a nucleic acid sequence of interest (present or to be inserted in the expression vector); other elements that may be required for controlling the expression of the nucleic acid sequence of interest may be supplied by a host cell or an in vitro expression system.

DETAILED DESCRIPTION

[0050] A first object of the invention is a ribonucleic acid (RNA) molecule or deoxyribonucleic acid (DNA) molecule, corresponding to nucleic acid molecule.

[0051] According to the invention, the RNA or DNA molecule comprises, from 5’ to 3’: an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, and a sequence of interest.

[0052] According to the invention, the RNA or DNA molecule comprises, from 3’ to 5’:

- an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, and

- a sequence of interest.

[0053] According to the invention, the RNA or DNA molecule comprises an RNA Booster sequence. The term “RAA Booster” as used herein refers to an artificial 9-nucleotide sequence, which comprises or consists of a sequence mmsknkkkm or its reverse sequence mkkknksmm, wherein:

“m” indicates an adenine (a) or a cytosine (c);

“s” indicates a guanine (g) or a cytosine (c);

“k” indicates a guanine (g) or a thymine/uracyl (t/u);

“n” indicates any nucleotide.

[0054] In some embodiments, the RNA Booster sequence comprises or consists of a sequence mmskngkkm or its reverse sequence mkkgnksmm, wherein:

“m” indicates an adenine (a) or a cytosine (c);

“s” indicates a guanine (g) or a cytosine (c); “k” indicates a guanine (g) or a thymine/uracyl (t/u);

“n” indicates any nucleotide.

[0055] In some embodiments, the RNA Booster sequence comprises or consists of a sequence of mmskngkgm or its reverse sequence mgkgnksmm, wherein:

“m” indicates an adenine (a) or a cytosine (c);

“s” indicates a guanine (g) or a cytosine (c);

“k” indicates a guanine (g) or a thymine/uracyl (t/u);

“n” indicates any nucleotide.

[0056] In some embodiments, the RNA Booster sequence comprises or consists of a sequence cmskhgkgm or its reverse sequence mgkghksmc, wherein:

“m” indicates an adenine (a) or a cytosine (c);

“s” indicates a guanine (g) or a cytosine (c);

“k” indicates a guanine (g) or a thymine/uracyl (t/u);

“h” indicates an adenine (a) or a cytosine (c) or a thymine/uracyl (t/u).

[0057] In some embodiments, the RNA Booster sequence comprises or consists of a sequence cmskwgkgm or its reverse sequence mgkgwksmc, wherein:

“m” indicates an adenine (a) or a cytosine (c);

“s” indicates a guanine (g) or a cytosine (c);

“w” indicates an adenine (a) or a thymine/uracyl (t/u);

“h” indicates an adenine (a) or a cytosine (c) or a thymine/uracyl (t/u).

[0058] In some embodiments, the RNA Booster sequence comprises or consists of a sequence ccsuwgggm or its reverse sequence mgggwuscc, wherein:

“s” indicates a guanine (g) or a cytosine (c);

“w” indicates an adenine (a) or a thymine/uracyl (t/u);

“m” indicates an adenine (a) or a cytosine (c).

[0059] Exemplary RNA Booster sequences in RNA molecule include, but are not limited to:

RNA Booster 1: cccgugugc, or its reverse sequence RNA Booster 10: cgugugccc

RNA Booster 2: aaguuuggc, or its reverse sequence RNA Booster 11: cgguuugaa RNA Booster 3: cccuaggua, or its reverse sequence RNA Booster 12'. auggauccc

RNA Booster 4: ccguggugc, or its reverse sequence RNA Booster 13: cguggugcc

RNA Booster 5: aacuggggc, or its reverse sequence RNA Booster 14: cggggucaa

RNA Booster 6: cccucgggc, or its reverse sequence RNA Booster 15: cgggcuccc

RNA Booster 7: cacgugugc, or its reverse sequence RNA Booster 16: cgugugcac

RNA Booster 8: cccuugggc, or its reverse sequence RNA Booster 17: cggguuccc

RNA Booster 9: ccguaggga, or its reverse sequence RNA Booster 18: agggaugcc

[0060] Exemplary RNA Booster sequences in DNA molecule include, but are not limited to:

RNA Booster 1: cccgtgtgc, or its reverse sequence RNA Booster 10: cgtgtgccc

RNA Booster 2: aagtttggc, or its reverse sequence RNA Booster 11: cggtttgaa

RNA Booster 3: ccctaggta, or its reverse sequence RNA Booster 12: atggatccc

RNA Booster 4: ccgtggtgc, or its reverse sequence RNA Booster 13: cgtggtgcc

RNA Booster 5: aactggggc, or its reverse sequence RNA Booster 14: cggggtcaa

RNA Booster 6: ccctcgggc, or its reverse sequence RNA Booster 15: cgggctccc

RNA Booster 7: cacgtgtgc, or its reverse sequence RNA Booster 16: cgtgtgcac

RNA Booster 8: cccttgggc, or its reverse sequence RNA Booster 17: cgggttccc

RNA Booster 9: ccgtaggga, or its reverse sequence RNA Booster 18: agggatgcc

[0061] In some embodiments, the RNA Booster is described as forward sequence, meaning the RNA Booster sequence is oriented from 5’ to 3’.

[0062] The sequence of RNA Booster forward are selected in the group of RNA Booster 1 to 9.

[0063] In some embodiments, the RNA Booster is described as reverse sequence, meaning the RNA Booster sequence is oriented from 3’ to 5’.

[0064] The sequence of RNA Booster reverse are selected in the group of RNA Booster 10 to 18.

[0065] In order of preference, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting RNA Booster 9, RNA Booster 8, RNA Booster 7, RNA Booster 6, RNA Booster 5, RNA Booster 4, RNA Booster 3, RNA Booster 2, and RNA Booster 1.

[0066] In order of preference, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10,

[0067] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, RNA Booster 8, RNA Booster 7 , RNA Booster 6, RNA Booster 5, RNA Booster 4, RNA Booster 3, and RNA Booster 2.

[0068] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, RNA Booster 8, RNA Booster 7 , RNA Booster 6, RNA Booster 5, RNA Booster 4, and RNA Booster 3.

[0069] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, RNA Booster 8, RNA Booster 7 , RNA Booster 6, RNA Booster 5, and RNA Booster 4.

[0070] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, RNA Booster 8, RNA Booster 7 , RNA Booster 6, and RNA Booster 5. [0071] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11 , RNA Booster 10, RNA Booster 9, RNA Booster 8, RNA Booster 7 , and RNA Booster 6.

[0072] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, RNA Booster 8, and RNA Booster 7.

[0073] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10, RNA Booster 9, and RNA Booster 8.

[0074] In some embodiments, the RNA or DNA molecule comprises RNA Booster 9. In some embodiments, the RNA or DNA molecule comprises RNA Booster 8.

[0075] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11, RNA Booster 10 and RNA Booster 9.

[0076] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12, RNA Booster 11 and RNA Booster 10.

[0077] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13, RNA Booster 12 and RNA Booster 11. [0078] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14, RNA Booster 13 and RNA Booster 12.

[0079] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15, RNA Booster 14 and RNA Booster 13.

[0080] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16, RNA Booster 15 and RNA Booster 14.

[0081] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17, RNA Booster 16 and RNA Booster 15.

[0082] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18, RNA Booster 17 and RNA Booster 16.

[0083] In some embodiments, the RNA or DNA molecule comprises an RNA Booster sequence selected from the group comprising or consisting of RNA Booster 18 and RNA Booster 17.

[0084] In some embodiments, the RNA or DNA molecule comprises RNA Booster 18. In some embodiments, the RNA or DNA molecule comprises RNA Booster 17.

[0085] In some embodiments, the RNA Booster is located in 5’ of a sequence of interest, meaning that the RNA Booster is located upstream of a sequence of interest.

[0086] According to the invention, the RNA Booster is located in 5’ of a sequence of interest. [0087] In some embodiments, the RNA Booster is located in 3’ of a sequence of interest, meaning that the RNA Booster is located downstream of a sequence of interest.

[0088] According to the invention, the RNA Booster is capable to increase the expression of a gene of interest, as the transgene. The RNA Booster is operably linked to the gene of interest.

[0089] In some embodiments, the RNA Booster is located from 50 ribonucleotides or deoxyribonucleotide (50 nt) to 1000 ribonucleotides or deoxyribonucleotide (1 knt) upstream or downstream of a sequence of interest; such as, e.g., about 50+25 nt, 100+25 nt, 150+25 nt, 200+25 nt, 250+25 nt, 300+25 nt, 350+25 nt, 400+25 nt,

450+25 nt, 500+25 nt, 550+25 nt, 600+25 nt, 650+25 nt, 700+25 nt, 750+25 nt,

800+25 nt, 850+25 nt, 900+25 nt, 950+25 nt or 1+0.025 knt upstream of a sequence of interest. In some preferred embodiments, the RNA Booster is located about 500+25 nt upstream or downstream of a sequence of interest.

[0090] In some embodiments, the RNA Booster is located from 5 ribonucleotides or deoxyribonucleotide (5 nt) to 2000 ribonucleotides or deoxyribonucleotide (2 knt) upstream or downstream of a sequence of interest; such as, e.g., about 50+25 nt, 100+25 nt, 150+25 nt, 200+25 nt, 250+25 nt, 300+25 nt, 350+25 nt, 400+25 nt,

450+25 nt, 500+25 nt, 550+25 nt, 600+25 nt, 650+25 nt, 700+25 nt, 750+25 nt,

800+25 nt, 850+25 nt, 900+25 nt, 950+25 nt or 1+0.025 knt or 1.1+0.025 knt or 1.2+0.025 knt or 1.3+0.025 knt or 1.4+0.025 knt or 1.5+0.025 knt or 1.6+0.025 knt or 1.7+0.025 knt or 1.8+0.025 knt or 1.9+0.025 knt or 2+0.025 knt upstream of a sequence of interest, preferably from about 10 to about 2000, more preferably from about 10 to about 1000; even more preferably from about 50 to about 1000. In some preferred embodiments, the RNA Booster is located about 2+0.025 knt upstream or downstream of a sequence of interest.

[0091] According to the invention, the RNA or DNA molecule comprises a sequence of interest.

[0092] The sequence of interest can be a sequence encoding a peptide or protein (e.g., without limitation, an enzyme, a transcription factor, a growth factor, a trophic factor, a hormone, a cytokine, an antibody, an antigen, a receptor, an immune regulator, a differentiation factor, a suicide protein, a cell-cycle modifying protein, an anti-proliferative protein, an angiogenic factor, an anti-angiogenic factor, a genome editor, a nuclease, a recombinase, a transposase, a neurotransmitter, and a reporter, including any precursor thereof, as well as fusion proteins). In this case, the sequence of interest may typically be (or be derived from) an mRNA, a cDNA, a synthetic nucleic acid, or any combinations thereof.

[0093] The sequence of interest can alternatively be a sequence of a non-coding RNA.

[0094] Examples of non-coding RNAs include, but are not limited to, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), SmY RNAs, small Cajal body-specific RNAs (scaRNAs), guide RNAs (gRNAs), Y RNAs, telomerase RNA component (TERC), spliced leader RNAs (SL RNAs), catalytic RNAs (z.e., ribozymes; such as, e.g., ribonuclease P, ribonuclease MRP, and the like), antisense RNAs (aRNAs), c/.y-natural antisense transcript (cis-NAT), CRISPR RNAs (crRNAs), long non-coding RNAs (IncRNAs), microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), Zrans-acting siRNAs (tasiRNAs), repeat- associated siRNAs (rasiRNAs), 7SK RNAs (7SK), enhancer RNAs (eRNAs), pegRNA, base editing RNA and RNA aptamers.

[0095] Examples of sequences of interest include any nucleic acid sequence encoding a molecule of therapeutic interest, such as any nucleic acid sequence encoding a peptide or protein, or a non-coding RNA, that is lacking, deficient and/or non-functional in a subject affected with a disease or condition.

[0096] Some specific, non-limiting, examples of sequences of interest include nucleic acid sequence encoding a CRISPR element (such as, e.g., Cas9, Casl2 or a gRNA), a zinc finger protein, a transcription activator-like effector nuclease (TALEN), a meganuclease, a spike protein of an enveloped virus (such as the spike protein of a Coronaviridae, e.g., of SARS-CoV-2; of an Orthomyxoviridae, e.g., of an Influenza virus; or of a Retroviridae), a fibroblast growth factor (such as, e.g., the glia-activating factor FGF9), a vascular endothelial growth factor (VEGF, including its isoforms, e.g., VEGF121, VEGFmb, VEGF145, VEGF165, VEGFiesb, VEGF189, VEGF206), a neutrophic factor (such as, e.g., a brain-derived neurotrophic factor BDNF, a ciliary neurotrophic factor CNTF, or a glial cell line-derived neurotrophic factor GDNF), an antibody or an antigen-binding fragment thereof, a chimeric antigen receptor, a fluorescent reporter (such as, e.g., GFP, RFP or YFP), luciferase, an alternative oxidase (AOX), a recombinase (such as, e.g., Cre or Flp), a glutathione peroxidase, a myoblast determination protein 1 (MyoD), a superoxide dismutase (SOD), a transcription factor (such as, e.g., Oct3/4, c-Myc, Kruppel-like factor 4 KLF4, or Sox2), a tumor antigen (such as, e.g., p53), an immune checkpoint (such as, e.g., PD-1 or PD-L1), a microbial rhodopsin (such as, e.g., a halorhodopsin, in particular the halorhodopsin from Natronomonas [NpHR] and more particularly the engineered halorhodopsin from Natronomonas [eNpHR3.0], or a channelrhodopsin, in particular ChR2), a MAP kinase (such as, e.g., MAPK10, in particular the MAPK10 from Leishmania donovani [LmjMAPKIO]), and an interleukin (such as, e.g., IL-7 or IL-2).

[0097] In some preferred embodiments, the sequence of interest is an antigen, and the RNA or DNA molecule of the invention is particularly suitable for vaccination purposes.

[0098] In some preferred embodiments, the sequence of interest is a genome editor, and the RNA or DNA molecule of the invention is particularly suitable for genome editing purposes.

[0099] In some preferred embodiments, the sequence of interest is FGF9 or FGF7, preferably FGF7, and the RNA or DNA molecule of the invention is particularly suitable for cosmetic purposes.

[0100] In some embodiments, the expression of the sequence of interest is transient (i.e., not permanent or sustained). The duration and amount of expression may be increased, e.g., by inserting a post-transcriptional regulatory element in 3’ (i.e., downstream) of the sequence of interest. [0101] Examples of post-transcriptional regulatory element include, but are not limited to, the post-transcriptional regulatory element of Woodchuck hepatitis virus (WPRE) and the post-transcriptional regulatory element of hepatitis B virus (HPRE).

[0102] As demonstrated in the Examples, the duration of expression of the sequence of interest in a cell, in particular in the presence of a post-transcriptional regulatory element, may be at most 7 days (Fig. IB).

[0103] In some embodiments, the RNA or DNA molecule encodes for an mRNA molecule.

[0104] In some embodiments, the mRNA molecule is comprised within a non-viral vector. The present invention encompasses thus a non-viral vector comprising the RNA or DNA molecule described herein.

[0105] In some embodiments, the mRNA molecule may further comprise:

Upstream of the RNA Booster sequence: a 5’-UTR sequence comprising a cap structure, and downstream of the sequence of interest, a 3’-UTR and a 3’-polyA.

[0106] In some embodiments, the mRNA molecule may further comprise:

- upstream of a 5’-UTR sequence comprising a cap structure, of the sequence of interest, a 3’-UTR and a 3’-polyA; and

- downstream the RNA Booster sequence.

[0107] In some embodiments, the mRNA molecule is encoded by a DNA molecule.

[0108] In some embodiments, the DNA molecule is comprised within a non-viral vector selected in the group of: plasmid, cosmid, phage, Bacterial Artificial Chromosome (BAC), Yeast Artificial Chromosome, liposomes, exosomes, lipid nanoparticles, polypeptide nanoparticles, stable nucleic acid lipid particle (SNALP), and cationic lipoplexes.

[0109] In some embodiments, the cap structure is a 5 ’-terminal m ⁷ G(5’)ppp(5’)G. In some embodiments, the cap structure may be an analog of m ⁷ G(5’)ppp(5’)G, such as, e.g., 3’-O-Me-m ⁷ G(5’)ppp(5’)G (called anti-reverse cap analog or ARCA), m ⁷ G(5’)ppp(5’)A, G(5’)ppp(5’)G or G(5’)ppp(5’)A.

[0110] In some embodiments, the non-viral vector l) has efficient encapsulation and protection on mRNAs from nuclease-based degradation upon administration to a subject; 2) prolongs the mRNAs half-life by preventing rapid clearance by a subject’s kidney and phagocytosis by the subject’s liver or spleen upon administration; 3) enhances targeted tissue/organ penetration and accumulation; 4) facilitates targeted cell internalization;

5) avoids lysosomal degradation during intracellular trafficking pathway; and/or

6) enhances the release of mRNAs in a subject’s cell cytoplasm to exert gene effects.

[0111] Typical examples of non-viral RNA vectors include, but are not limited to, liposomes, exosomes, lipid nanoparticles, polypeptide nanoparticles, stable nucleic acid lipid particle (SNALP), and cationic lipoplexes.

[0112] Alternatively, the RNA or DNA molecule may be packaged into or otherwise comprised within a viral vector. The present invention encompasses thus a viral vector comprising the RNA or DNA molecule described herein.

[0113] In some embodiments, the RNA or DNA molecule is packaged into or otherwise comprised within an DNA virus vector, i.e., an engineered viral particle derived from an DNA virus.

[0114] In some embodiments, the RNA or DNA molecule is packaged into or otherwise comprised within an RNA virus vector, i.e., an engineered viral particle derived from an RNA virus. By “engineered viral particle”, it is implied that (i) at least one exogenous nucleic acid sequence is introduced into the genome of the RNA virus, and/or (ii) at least one endogenous gene from the RNA virus is be mutated or deleted, either partially or totally.

[0115] RNA viruses are viruses that have ribonucleic acid as their genetic material. RNA viruses can be classified into four groups according to the Baltimore classification system:

Group III (double-stranded RNA viruses), including the following families: Birnaviridae , Chrysoviridae, Cystoviridae, Hypoviridae, Partitiviridae, Reoviridae, Totiviridae and Endornavirus . Exemplary genera of double- stranded RNA viruses that can infect humans include, without limitation, Rotavirus and Coltivirus.

Group IV (positive-sense single-stranded RNA viruses), including the following families: Arteriviridae, Coronaviridae , Roniviridae, Astroviridae , Barnaviridae , Bromoviridae, Caliciviridae , Closteroviridae, Comoviridae, Dicistroviridae , Flaviviridae , Flexiviridae, Hepeviridae, Eeviviridae, Euteoviridae , Marnaviridae, Narnaviridae, Nodaviridae, Picornaviridae, Potyviridae, Sequiviridae, Tetraviridae, Togaviridae, Tombusviridae, Tymoviridae, Virgaviridae, Benyvirus, Cheravirus, Idaeovirus, Machlomovirus , Ourmiavirus, Sadwavirus Sobemovirus and Umbravirus. Exemplary species of positive-sense single-stranded RNA viruses that can infect humans include, without limitation, hepatitis C virus, yellow fever virus, West Nile virus, dengue virus, Zika virus, Chikungunya virus, rubella virus, MERS, SARS, and SARS-CoV-2.

Group V (negative- sense single- stranded RNA viruses), including the following families: Qinviridae, Aspiviridae, Chuviridae, Bornaviridae, Filoviridae, Mymonaviridae , Nyamiviridae, Paramyxoviridae, Pneumoviridae , Rhabdoviridae, Sunviridae, Yueviridae, Arenaviridae , Cruliviridae, Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae , Tospoviridae, Amnoonviridae and Orthomyxoviridae . Exemplary species of negative- sense single-stranded RNA viruses that can infect humans include, without limitation, Ebola virus, Lassa virus, Marburg virus, measles virus, rabies virus, mumps virus, Influenza viruses, and respiratory syncytial virus.

Group VI (negative-strand single-stranded RNA-reverse transcriptase viruses), including the following families: Metaviridae, Pseudoviridae and Retroviridae . Exemplary species of negative- strand single-stranded RNA-reverse transcriptase viruses that can infect humans include, without limitation, human immunodeficiency virus (HIV), and human T-cell leukemia-lymphoma virus (HTLV). [0116] In some embodiments, the RNA molecule is packaged into or otherwise comprised within an RNA virus vector derived from a Group III, Group IV, Group V or Group VI RNA virus.

[0117] In some preferred embodiments, the RNA molecule is packaged into or otherwise comprised within an RNA virus vector derived from a Group VI RNA virus.

[0118] In order to be packaged into or otherwise comprised within an RNA virus vector, the RNA molecule may comprise a packaging sequence.

[0119] Viral packaging sequences include bacteriophage packaging sequences (such as, e.g., DNA phage packaging sequences and RNA phage packaging sequences) and eukaryotic virus packaging sequences (such as, e.g., DNA virus packaging sequences and RNA virus packaging sequences).

[0120] When of viral origin, the packaging sequence may be:

(i) a wild-type packaging sequence from an RNA virus; or preferably

(ii) a wild-type packaging sequence from an RNA virus of the same group as the one of the RNA virus into which the RNA molecule is to be packaged into, or more preferably

(iii) a wild-type packaging sequence from an RNA virus of the same family as the one of the RNA virus into which the RNA molecule is to be packaged into, or even more preferably

(iv) a wild-type packaging sequence from an RNA virus of the same genus as the one of the RNA virus into which the RNA molecule is to be packaged into, or yet even more preferably

(v) the wild-type packaging sequence from the RNA virus into which the RNA molecule is to be packaged into.

[0121] In some embodiments, the packaging sequence may be a modified packaging sequence which shares more than 70 %, 75 %, 80 %, 85 %, 90 %, 95 % of sequence identity or even more with the wild-type packaging sequence of (i), (ii), (iii), (iv) or (v) above, while retaining its ability to trigger the process of packaging inside a viral capsid. [0122] Examples of packaging sequences include, but are not limited to, the psi ( ) packaging signal of HIV or SIV; the core encapsidation signal from Gammaretrovirus; the epsilon (s) encapsidation signal from HBV, and the encapsidation signal from BLV.

[0123] In some preferred embodiments, the RNA molecule is packaged into or otherwise comprised within an RNA virus vector derived from a Group VI RNA virus of the Retroviridae family. The present invention encompasses thus a retroviral vector comprising the RNA molecule described herein.

[0124] Retroviruses are enveloped viruses from the Retroviridae family. They package two identical single-stranded ribonucleic acid (RNA) molecules of typically 7 to 10 kb in length, forming their genome. The genome of retroviruses typically comprises gag, pol and env genes flanked by two long terminal repeat (LTRs) sequences. Each of these genes encodes for numerous peptides, which are initially expressed in the form of a single precursor polypeptide. The gag gene encodes for the internal structure proteins (matrix, capsid and nucleocapsid); the pol gene encodes for retroviral enzymes reverse transcriptase, integrase and protease; and the env gene encodes for viral envelope glycoprotein. The genome of retroviruses can further contain cA-acting elements, e.g., elements responsible for exporting out of the nucleus the unspliced viral genomic RNA which will be packaged, such as a Rev-response element (RRE) sequence. The 5’ and 3’ LTRs serve to promote transcription and also serve as a poly adenylation sequence of the viral RNAs. Sequences necessary for the initiation of reverse transcription of the genome and for the encapsidation of viral RNA in particles (psi [ ] packaging element) are typically adjacent to the 5’ LTR. If the sequences necessary for encapsidation are absent from the viral genome, the genomic RNA will not be actively packaged; it can, however, be passively packaged although with a lower yield. The genome of more complex retroviruses may comprise additional genes encoding accessory and/or regulatory proteins such as src, sag, tax, vif, vpr, vpx, vpu, nef, tat, rev, tmx, tas and/or bet. For example, the HIV-1 genome contains 7 accessory genes: vif, vpr, vpx, vpu, nef, tat and rev.

[0125] The Retroviridae family is subdivided into two subfamilies: Orthoretrovirinae and Spumaretrovirinae. In some embodiments, the retroviral vector is (or is derived from) an Orthoretrovirinae or a Spumaretrovirinae. In some preferred embodiments, the retroviral vector is (or is derived from) an Orthoretrovirinae .

[0126] The Orthoretrovirinae subfamily is subdivided into six genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, and Lentivirus.

[0127] Exemplary species of Alpharetrovirus include, but are not limited to, avian sarcoma leukosis virus (ASLV), Rous sarcoma virus (RSV), and avian myeloblastosis virus (AMV).

[0128] Exemplary species of Betaretrovirus include, but are not limited to, mouse mammary tumor virus (MMTV), Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor viruses (ENTV; including ENTV-1 and ENTV-2), simian retroviruses (SRV; including SRV-1 and SRV-2), and Mason-Pfizer monkey virus (M-PMV; formerly known as SRV-3).

[0129] Exemplary species of Deltaretrovirus include, but are not limited to, human T-lymphotropic viruses (HTLV; including HTLV-1, HTLV-2, HTLV-3 and HTLV-4), simian T-lymphotropic viruses (STLV; including STLV-1, STLV-2, STLV-3, and STLV-4), and bovine leukemia virus (BLV).

[0130] Exemplary species of Epsilonretrovirus include, but are not limited to, Walleye dermal sarcoma virus (WDSV), and Walleye epidermal hyperplasia viruses (WEHV; including WEHV-1 and WEHV-2).

[0131] Exemplary species of Gammaretrovirus include, but are not limited to, murine leukemia viruses (MLV), Abelson murine leukemia virus (AMLV), Friend virus (FV), feline leukemia virus (FeLV), koala retrovirus (KoRV), xenotropic murine leukemia virus-related virus (XMRV), chick syncytial virus (CSV), murine sarcoma viruses (MSV; including Finkel-Biskis-Jinkins murine sarcoma virus, Harvey murine sarcoma virus, Kirsten murine sarcoma virus and Moloney murine sarcoma virus), feline sarcoma viruses (FSV; including Gardner- Arnstein feline sarcoma virus, Hardy-Zuckerman feline sarcoma virus and Snyder- Theilen feline sarcoma virus), Gibbon ape leukemia virus (GaLV), guinea pig type-C oncovirus, porcine type-C oncovirus, reticuloendotheliosis virus, Trager duck spleen necrosis virus, viper retrovirus, and Woolly monkey sarcoma virus.

[0132] Exemplary species of Lentivirus include, but are not limited to, human immunodeficiency viruses (HIV; including HIV-1 and HIV-2), simian immunodeficiency viruses (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), puma lentivirus (PLV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus, and Jembrana disease virus.

[0133] In some embodiments, the retroviral vector is (or is derived from) an Alpharetrovirus, a Betaretrovirus, a Deltaretrovirus, an Epsilonretrovirus, a Gammaretrovirus or a Lentivirus.

[0134] In some preferred embodiments, the retroviral vector is (or is derived from) a Lentivirus. In some preferred embodiments, the retroviral vector is (or is derived from) a Lentivirus selected from the group comprising or consisting of human immunodeficiency viruses (HIV; including HIV-1 and HIV-2), simian immunodeficiency viruses (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), puma lentivirus (PLV), equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), Visna-maedi virus (VMV), and Jembrana disease virus (JDV).

[0135] In some preferred embodiments, the retroviral vector is (or is derived from) a human immunodeficiency virus, such as HIV-1 and HIV-2; more preferably HIV-1.

[0136] In some embodiments, a retroviral vector as described herein packages two RNA molecules, each comprising an RNA Booster sequence and a sequence of interest, as described above. According to this embodiment, the two RNA molecules may comprise the same sequence of interest, or different sequences of interest.

[0137] Also contemplated herein is therefore a retroviral vector comprising a recombinant ribonucleic genome, itself comprising, from 5’ to 3 ’or from 3’ to 5’: an RNA Booster sequence, and a sequence of interest. [0138] In some embodiments, the retroviral vector comprises a recombinant ribonucleic genome, itself comprising, from 5’ to 3 ’or from 3’ to 5’: a packaging sequence, an RNA Booster sequence, and a sequence of interest.

[0139] In some embodiments, the retroviral vector comprises a recombinant ribonucleic genome, itself comprising, from 5’ to 3’: a 5’ LTR, a packaging sequence, an RNA Booster sequence, a sequence of interest, and a 3’ LTR.

[0140] In some embodiments, the retroviral vector comprises a recombinant ribonucleic genome, itself comprising, from 5’ to 3’: a 5’ LTR, a packaging sequence, a Rev-response element, an RNA Booster sequence, a sequence of interest, and a 3’ LTR.

[0141] In some embodiments, the retroviral vector comprises a recombinant ribonucleic genome, itself comprising, from 5’ to 3’: a 5’ LTR, a packaging sequence, a Rev-response element, an RNA Booster sequence, a sequence of interest, a post-transcriptional regulatory element, and a 3’ LTR. [0142] In some embodiments, the retroviral vector is reverse transcriptase-defective.

[0143] In other words, the gene encoding the reverse transcriptase may be mutated or deleted, so that the reverse transcription process is altered and cannot be completed, cannot give rise to a full-length retroviral double-stranded DNA intermediate molecule upon infection of a target cell, and consequently, cannot generate a proviral vector genome. This inability, in the context of the invention, can result either from (i) an absence of the reverse transcriptase gene in the retroviral vector, or (ii) at least one mutation in the reverse transcriptase gene in the retroviral vector.

[0144] Examples of mutations in the reverse transcriptase gene to abolish reverse transcription activity are known in the art. Reference is made, inter alia, to WO 2013/060819 A2, from page 15 line 31 to page 42 line 14, which selected passage is hereby incorporated by reference. For instance, a HIV-1 reverse transcriptase with SEQ ID NO: 4 may comprise a D110E substitution resulting in an abolished reverse transcriptase activity.

[0145] In some embodiments, the retroviral vector might further be integrase-defective.

[0146] In other words, the gene encoding the integrase might be mutated or deleted, so that the integration process is altered and cannot be completed, i.e., a proviral vector genome cannot be integrated into the genome of a target cell. This inability can result either from (i) an absence of the integrase gene in the retroviral vector, or (ii) at least one mutation in the integrase gene in the retroviral vector.

[0147] In some embodiments, the RNA or DNA molecule, in particular when packaged into or otherwise comprised within an RNA virus vector, may further comprise one or several elements, in particular one or several of long terminal repeats (LTR), including a 5’ LTR and a 3’ LTR; a Rev -response element (RRE) sequence; and a post-transcriptional regulation element sequence.

[0148] In some embodiments, the RNA or DNA molecule comprises a 5’ LTR, located in 5’ of the packaging sequence (in other words, before the packaging sequence). [0149] In some embodiments, the RNA or DNA molecule comprises a Rev-response element (RRE) sequence, located in 3’ of the packaging sequence but in 5’ of the RNA Booster sequence (in other words, between the packaging sequence and the RNA Booster sequence).

[0150] In some embodiments, the RNA or DNA molecule comprises a post-transcriptional regulation element sequence, located in 3’ of the sequence of interest (in other words, after the sequence of interest).

[0151] In some embodiments, the RNA or DNA molecule comprises a 3’ LTR, located in 3’ of the sequence of interest (in other words, after the sequence of interest). When a post-transcriptional regulation element sequence is present, the 3’ LTR is located in 3’ of this post-transcriptional regulation element sequence.

[0152] In some embodiments, the RNA or DNA molecule may thus comprise, from 5’ to 3’: optionally, a 5’ LTR, optionally, a packaging sequence, optionally, a Rev-response element, compulsorily, an RNA Booster sequence, compulsorily, a sequence of interest, optionally, a post-transcriptional regulatory element, and optionally, a 3’ LTR.

[0153] In some embodiments, the RNA or DNA molecule may thus comprise, from 5’ to 3’: optionally, a 5’ LTR, compulsorily, a packaging sequence, optionally, a Rev-response element, compulsorily, an RNA Booster sequence, compulsorily, a sequence of interest, optionally, a post-transcriptional regulatory element, and optionally, a 3’ LTR. [0154] In some embodiments, the RNA or DNA molecule may thus comprise, from 5’ to 3’: optionally, a 5’ LTR, optionally, a packaging sequence, optionally, a Rev-response element, compulsorily, a sequence of interest, optionally, a post-transcriptional regulatory element, optionally, a 3’ LTR, and compulsorily, an RNA Booster sequence.

[0155] In some embodiments, the RNA or DNA molecule may thus comprise, from 5’ to 3’: optionally, a 5’ LTR, compulsorily, a packaging sequence, optionally, a Rev-response element, compulsorily, a sequence of interest, optionally, a post-transcriptional regulatory element, optionally, a 3’ LTR, and compulsorily, an RNA Booster sequence.

[0156] A second object of the invention is a composition comprising, consisting of, or consisting essentially of, the RNA or DNA molecule described above. In particular, an object of the invention is a composition comprising, consisting of, or consisting essentially of, the non- viral vector described above, or the viral vector, in particular the retroviral vector, described above.

[0157] In some embodiment, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable excipient or carrier.

[0158] Pharmaceutically acceptable excipients or carriers that may be used in the composition or pharmaceutical composition 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, poly acrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0159] A third object of the invention relates to the various uses and applications of the RNA or DNA molecule, in particular of the non-viral vector or viral vector, in particular the retroviral vector, or of the composition, as described above.

[0160] These uses and applications are particularly suitable in prokaryotes, whether in vitro, ex vivo or in vivo.

[0161] These uses and applications are particularly suitable in eukaryotes, whether in vitro, ex vivo or in vivo. In some embodiments, the eukaryote is an animal, preferably a mammal, even more preferably a human.

[0162] Encompassed in these uses and applications is a method, in particular an in vitro or ex vivo method, of transiently expressing at least one sequence of interest in a cell, which method comprises contacting or otherwise transfecting or transducing the cell with the RNA or DNA molecule, or with the non-viral vector or viral vector, in particular the retroviral vector, as described above.

[0163] In some embodiments, RNA or DNA molecule is under control of promoter noninducible.

[0164] In some embodiments, RNA or DNA molecule is under control of promoter inducible.

[0165] In some embodiments, RNA or DNA molecule is under control of strong promoter.

[0166] Also encompassed in these uses and applications is the RNA or DNA molecule, or the non-viral vector or viral vector, in particular the retroviral vector, or of the composition, for use in therapy, as a drug or as a medicament. [0167] Also encompassed in these uses and applications is the RNA or DNA molecule, or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, for use in preventing or treating a disease in a subject in need thereof; or a method of preventing or treating a disease, comprising administering to a subject in need thereof the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition.

[0168] In some embodiments, the disease is selected from the group consisting of cancer, infectious diseases, neuromuscular diseases, ocular diseases, blood diseases, cardiovascular diseases, skin diseases, and neurodegenerative diseases.

[0169] In some embodiments, the disease is cancer.

[0170] Examples of cancers include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 02 “Neoplasms”.

[0171] Further examples of cancers include, but are not limited to, recurrent, metastatic or multi-drug resistant cancer.

[0172] Further examples of cancers include, but are not limited to, adenofibroma, adenoma, agnogenic myeloid metaplasia, AIDS-related malignancies, ameloblastoma, anal cancer, angiofollicular mediastinal lymph node hyperplasia, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angiomatosis, anhidrotic ectodermal dysplasia, anterofacial dysplasia, apocrine metaplasia, apudoma, asphyxiating thoracic dysplasia, astrocytoma (including, e.g., cerebellar astrocytoma and cerebral astrocytoma), atriodigital dysplasia, atypical melanocytic hyperplasia, atypical metaplasia, autoparenchymatous metaplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, bile duct cancer (including, e.g., extrahepatic bile duct cancer), bladder cancer, bone cancer, brain tumor (including, e.g., brain stem glioma, cerebellar astrocytoma glioma, malignant glioma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, ependymoma, medulloblastoma, gestational trophoblastic tumor glioma, and paraganglioma), branchionia, female breast cancer, male breast cancer, bronchial adenomas/carcinoids, bronchopulmonary dysplasia, cancer growths of epithelial cells, pre-cancerous growths of epithelial cells, metastatic growths of epithelial cells, carcinoid heart disease, carcinoid tumor (including, e.g., gastrointestinal carcinoid tumor), carcinoma (including, e.g., carcinoma of unknown primary origin, adrenocortical carcinoma, islet cells carcinoma, adeno carcinoma, adeoncortical carcinoma, basal cell carcinoma, basosquamous carcinoma, bronchiolar carcinoma, Brown-Pearce carcinoma, cystadenocarcinoma, ductal carcinoma, hepatocarcinoma, Krebs carcinoma, papillary carcinoma, oat cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, squamous cell carcinoma, transitional cell carcinoma, Walker carcinoma, Merkel cell carcinoma, and skin carcinoma), cementoma, cementum hyperplasia, cerebral dysplasia, cervical cancer, cervical dysplasia, cholangioma, cholesteatoma, chondroblastoma, chondroectodermal dysplasia, chordoma, choristoma, chrondroma, cleidocranial dysplasia, colon cancer, colorectal cancer, local metastasized colorectal cancer, congenital adrenal hyperplasia, congenital ectodermal dysplasia, congenital sebaceous hyperplasia, connective tissue metaplasia, craniocarpotarsal dysplasia, craniodiaphysial dysplasia, craniometaphysial dysplasia, craniopharyngioma, cylindroma, cystadenoma, cystic hyperplasia (including, e.g., cystic hyperplasia of the breast), cystosarcoma phyllodes, dentin dysplasia, denture hyperplasia, diaphysial dysplasia, ductal hyperplasia, dysgenninoma, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctate, ectodermal dysplasia, Ehrlich tumor, enamel dysplasia, encephaloophthalmic dysplasia, endometrial cancer (including, e.g., ependymoma and endometrial hyperplasia), ependymoma, epithelial cancer, epithelial dysplasia, epithelial metaplasia, esophageal cancer, Ewing’s family of tumors (including, e.g., Ewing’s sarcoma), extrahepatic bile duct cancer, eye cancer (including, e.g., intraocular melanoma and retinoblastoma), faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibroma, fibromuscular dysplasia, fibromuscular hyperplasia, fibrous dysplasia of bone, florid osseous dysplasia, focal epithelial hyperplasia, gall bladder cancer, ganglioneuroma, gastric cancer (including, e.g., stomach cancer), gastrointestinal carcinoid tumor, gastrointestinal tract cancer, gastrointestinal tumors, Gaucher’s disease, germ cell tumors (including, e.g., extracranial germ cell tumors, extragonadal germ cell tumors, and ovarian germ cell tumors), giant cell tumor, gingival hyperplasia, glioblastoma, glomangioma, granulosa cell tumor, gynandroblastoma, hamartoma, head and neck cancer, hemangioendothelioma, hemangioma, hemangiopericytoma, hepatocellular cancer, hepatoma, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, histiocytonia, histiocytosis, hypergammaglobulinemia, hypohidrotic ectodermal dysplasia, hypopharyngeal cancer, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intestinal cancers, intestinal metaplasia, intestinal polyps, intraocular melanoma, intravascular papillary endothelial hyperplasia, kidney cancer, laryngeal cancer, leiomyoma, leukemia (including, e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, acute hairy cell leukemia, acute B-cell leukemia, acute T-cell leukemia, acute HTLV leukemia, chronic lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelogenous leukemia, chronic hairy cell leukemia, chronic B-cell leukemia, chronic T-cell leukemia, and chronic HTLV leukemia), Leydig cell tumor, lip and oral cavity cancer, lipoma, liver cancer, lung cancer (including, e.g., small cell lung cancer and non-small cell lung cancer), lymphangiomyoma, lymphaugioma, lymphoma (including, e.g., AIDS-related lymphoma, central nervous system lymphoma, primary central nervous system lymphoma, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma during pregnancy, non-Hodgkin’s lymphoma during pregnancy, mast cell lymphoma, B-cell lymphoma, adenolymphoma, Burkitt’s lymphoma, cutaneous T-cell lymphoma, large cell lymphoma, and small cell lymphoma), lymphopenic thymic dysplasia, lymphoproliferative disorders, macroglobulinemia (including, e.g., Waldenstrom’s macroglobulinemia), malignant carcinoid syndrome, malignant mesothelioma, malignant thymoma, mammary dysplasia, mandibulofacial dysplasia, medulloblastoma, meningioma, mesenchymoma, mesonephroma, mesothelioma (including, e.g., malignant mesothelioma), metaphysial dysplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, metastatic squamous neck cancer (including, e.g., metastatic squamous neck cancer with occult primary), Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple endocrine neoplasia syndrome, multiple epiphysial dysplasia, multiple myeloma/plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myeloid metaplasia, myeloproliferative disorders, chronic myeloproliferative disorders, myoblastoma, myoma, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, prostatic neoplasm, colon neoplasm, abdomen neoplasm, bone neoplasm, breast neoplasm, digestive system neoplasm, liver neoplasm, pancreas neoplasm, peritoneum neoplasm, endocrine glands neoplasm (including, e.g., adrenal neoplasm, parathyroid neoplasm, pituitary neoplasm, testicles neoplasm, ovary neoplasm, thymus neoplasm, and thyroid neoplasm), eye neoplasm, head and neck neoplasm, nervous system neoplasm (including, e.g., central nervous system neoplasm and peripheral nervous system neoplasm), lymphatic system neoplasm, pelvic neoplasm, skin neoplasm, soft tissue neoplasm, spleen neoplasm, thoracic neoplasm, urogenital tract neoplasm, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis, neuroma, nodular hyperplasia of prostate, nodular regenerative hyperplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, odontoma, opthalmomandibulomelic dysplasia, oropharyngeal cancer, osteoma, ovarian cancer (including, e.g., ovarian epithelial cancer and ovarian low malignant potential tumor), pancreatic cancer (including, e.g., islet cell pancreatic cancer and exocrine pancreatic cancer), papilloma, paraganglioma, nonchromaffin paraganglioma, paranasal sinus and nasal cavity cancer, paraproteinemias, parathyroid cancer, periapical cemental dysplasia, pheochromocytoma (including, e.g., penile cancer), pineal and supratentorial primitive neuroectodermal tumors, pinealoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, plasmacytoma, pleuropulmonary blastoma, polyostotic fibrous dysplasia, polyps, pregnancy cancer, pre-neoplastic disorders (including, e.g., benign dysproliferative disorders such as benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, esophageal dysplasia, leukoplakia, keratoses, Bowen’s disease, Farmer’s skin, solar cheilitis, and solar keratosis), primary hepatocellular cancer, primary liver cancer, primary myeloid metaplasia, prostate cancer, pseudoachondroplastic spondyloepiphysial dysplasia, pseudoepitheliomatous hyperplasia, purpura, rectal cancer, renal cancer (including, e.g., kidney cancer, renal pelvis, ureter cancer, transitional cell cancer of the renal pelvis and ureter), reticuloendotheliosis, retinal dysplasia, retinoblastoma, salivary gland cancer, sarcomas (including, e.g., uterine sarcoma, soft tissue sarcoma, carcinosarcoma, chondrosarcoma, fibrosarcoma, hemangiosarcoma, Kaposi’s sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, rhabdosarcoma, sarcoidosis sarcoma, osteosarcoma, Ewing sarcoma, malignant fibrous histiocytoma of bone, and clear cell sarcoma of tendon sheaths), sclerosing angioma, secondary myeloid metaplasia, senile sebaceous hyperplasia, septooptic dysplasia, Sertoli cell tumor, Sezary syndrome, skin cancer (including, e.g., melanoma skin cancer and non-melanoma skin cancer), small intestine cancer, spondyloepiphysial dysplasia, squamous metaplasia (including, e.g., squamous metaplasia of amnion), stomach cancer, supratentorial primitive neuroectodermal and pineal tumors, supratentorial primitive neuroectodermal tumors, symptomatic myeloid metaplasia, teratoma, testicular cancer, theca cell tumor, thymoma (including, e.g., malignant thymoma), thyroid cancer, trophoblastic tumors (including, e.g., gestational trophoblastic tumors), ureter cancer, urethral cancer, uterine cancer, vaginal cancer, ventriculoradial dysplasia, verrucous hyperplasia, vulvar cancer, Waldenstrom’s macroglobulinemia, and Wilms’ tumor.

[0173] In some embodiments, the disease is an infectious disease.

[0174] Examples of infectious diseases include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 01 “Certain infectious or parasitic diseases”.

[0175] Further examples of infectious diseases include, but are not limited to, bacterial infections, viral infections, fungal infections, parasitic infections, ectoparasitic infections, and the like.

[0176] In some embodiments, the disease is a neuromuscular disease.

[0177] Examples of neuromuscular diseases include, but are not limited to, acid maltase deficiency, amyotrophic lateral sclerosis, Andersen-Tawil syndrome, Becker muscular dystrophy, Becker myotonia congenita, Bethlem myopathy, bulbospinal muscular atrophy, carnitine deficiency, carnitine palmityl transferase deficiency, central core disease, centronuclear myopathy, Charcot-Marie-Tooth disease, congenital muscular dystrophy, congenital myasthenic syndromes, congenital myotonic dystrophy, Cori disease, Debrancher enzyme deficiency, Dejerine- Sottas disease, dermatomyositis, distal muscular dystrophy, Duchenne muscular dystrophy, dystrophia myotonica, Emery- Dreifuss muscular dystrophy, endocrine myopathies, Eulenberg disease, facioscapulohumeral muscular dystrophy, tibial distal myopathy, Friedreich's ataxia, Fukuyuma congenital muscular dystrophy, glycogenosis type 2, glycogenosis type 3, glycogenosis type 5, glycogenosis type 7, glycogenosis type 9, glycogenosis type 10, glycogenosis type 11, Gowers-Laing distal myopathy, hereditary inclusion-body myositis, hypothyroid myopathy, inclusion-body myositis, inherited myopathies, integrin-deficient congenital muscular dystrophy, spinal-bulbar muscular atrophy, spinal muscular atrophy, lactate dehydrogenase deficiency, Lambert-Eaton myasthenic syndrome, McArdei disease, merosin-deficient congenital muscular dystrophy, metabolic diseases of muscle, mitochondrial myopathy, Miyoshi distal myopathy, motor neuron disease, muscle-eye-brain disease, myasthenia gravis, myoadenylate deaminase deficiency, myofibrillar myopathy, myophosphorylase deficiency, myotonia congenital, myotonic muscular dystrophy, myotubular myopathy, nemaline myopathy, Nonaka distal myopathy, oculopharyngeal muscular dystrophy, paramyotonia congenital, Pearson syndrome, periodic paralysis, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, polymyositis, Pompe disease, progressive external ophthalmoplegia, spinal muscular atrophy, Ullrich congenital muscular dystrophy, Weiander distal myopathy, and ZASP- related myopathy.

[0178] In some embodiments, the disease is an ocular disease.

[0179] Examples of ocular diseases include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 09 “Diseases of the visual system”.

[0180] Further examples of ocular diseases include, but are not limited to, age-related macular degeneration (AMD, e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, diabetic macular edema (DME, e.g., focal, non-center DME and diffuse, center-involved DME), retinopathy, diabetic retinopathy (DR, e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR), other ischemia-related retinopathies, ROP, retinal vein occlusion (RVO, e.g., central (CRVO) and branched (BRVO) forms), choroidal neovascularization (CNV, e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal/choroidal neovascularization, pathologic myopia, von Hippel- Lindau disease, histoplasmosis of the eye, familial exudative vitreoretinopathy (FEVR), Coats’ disease, Nome disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g.. allergic) conjunctivitis), Leber congenital amaurosis, uveitis (e.g., infectious or non- infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, and Sjogren’s disease.

[0181] In some embodiments, the disease is a blood disease.

[0182] Examples of blood diseases include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 03 “Diseases of the blood or blood-forming organs”.

[0183] Further examples of blood diseases include, but are not limited to, acute myeloid leukemia, acute promyelocytic leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndromes, anemia, methaemoglobinaemia, hemophilia, non-thrombocytopenic purpura, thrombocytosis, and thrombocytopenia.

[0184] In some embodiments, the disease is a cardiovascular disease.

[0185] Examples of cardiovascular diseases include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 11 “Diseases of the circulatory system”.

[0186] Further examples of cardiovascular diseases include, but are not limited to, aneurysm, angina, arrhythmia, atherosclerosis, cardiomyopathy, stroke, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease coronary, artery disease dilated, cardiomyopathy, diastolic dysfunction, endocarditis, hypertension, hypertrophic cardiomyopathy, mitral valve prolapse, myocardial infarction, and venous thromboembolism. [0187] In some embodiments, the disease is a skin disease.

[0188] Examples of skin diseases include those listed in the 11 ^th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD), under chapter 14 “Diseases of the skin”.

[0189] Further examples of skin diseases include, but are not limited to, atopic dermatitis, hand dermatitis, contact dermatitis, allergic contact dermatitis, irritant contact dermatitis, neurodermatitis, perioral dermatitis, stasis dermatitis, dyshidrotic eczema, xerotic dermatitis, nummalar dermatitis, seborrheic dermatitis, eyelid dermatitis, diaper dermatitis, dermatomyositis, lichen planus, lichen sclerosis, alopecia areata, vitiligo, rosacea, epidermolysis bullosa, keratosis pilaris, pityriasis alba, pemphigus, vulvovaginitis, acne, chronic spontaneous urticaria, chronic idiopathic urticaria, chronic physical urticaria, Vogt-Koyanagi-Harada disease, Sutton nevus, post inflammatory hypopigmentation, senile leukoderma, chemical/drug-induced leukoderma, cutaneous lupus erythematosus, discoid lupus, palmoplantar pustulosis, pemphigoid, sweet's syndrome, hidradenitis suppurativa, psoriasis, plaque psoriasis, pustular psoriasis, nail psoriasis, flexural psoriasis, guttate psoriasis, psoriatic arthritis, erythrodermic psoriasis, or inverse psoriasis.

[0190] In some embodiments, the disease is a neurodegenerative disease.

[0191] Examples of neurodegenerative diseases include, but are not limited to, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, spinocerebellar ataxia (SCA) type 1, SCA type 2, SCA type 6, SCA type 7, SCA type 17, Machado -Joseph disease/SCA type 3 (MJD/SCA3), Huntington’s disease, dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), prion disease, and motor neuron disease.

[0192] Also encompassed in these uses and applications is the RNA molecule or DNA molecule or the non- viral vector or viral vector, in particular the retroviral vector, or the composition, for use in skin and/or skin appendages (such as hair, nails or skin glands) treatment; or a method of treating skin and/or skin appendages (such as hair, nails or skin glands) in a subject in need thereof against, comprising administering to the subject in need thereof the RNA molecule or DNA molecule or the non- viral vector or viral vector, in particular the retroviral vector, or the composition.

[0193] In some embodiments, these uses and methods are preferably non-therapeutic uses and methods, a cosmetic use or method.

[0194] In some embodiments, these non-therapeutic uses and methods are applicable to substantially healthy subject.

[0195] In some embodiments, a “substantially healthy subject” is a subject who has not been previously diagnosed or identified as suffering from skin and/or hair and/or nails diseases, a “substantially healthy subject” is a subject who has not been previously diagnosed or identified as suffering from skin and/or hair and/or nails diseases. In one embodiment, a “substantially healthy subject” is a subject who does not suffer from any known disease, disorder or condition. In one embodiment, a “substantially healthy subject” is a subject who is not seeking medical attention.

[0196] Examples of treatments of skin and/or skin appendages, in particular of non-therapeutic treatments, include for instance any or several of induction of hair growth, prevention of hair loss, induction of hair removal, hair coloring or bleaching, prevention of hair graying, promotion of hair thickening, promotion of or prevention of hair curling, promotion of skin healing, promotion of skin repairing, promoting skin appearance, prevention of wrinkle formation, improvement of skin elasticity, induction of skin tone homogenization, and reduction of sebum secretion.

[0197] In some embodiments, the non-therapeutic treatment corresponds to promoting skin healing by improving the appearance of the skin.

[0198] As exemplified herein, inducing hair growth and/or promoting skin healing can be achieved using the RNA or DNA molecule, in particular the retroviral vector, of the invention, in particular wherein the sequence of interest encodes the glia-activating factor (aka FGF9). [0199] However, the skilled artisan will readily recognize that other sequences of interest can be used for cosmetic purposes, including, for instance, sequences coding for growth factors.

[0200] In some embodiments, other sequences of interest that can be used for cosmetic purposes include, but are not limited to, sequences encoding the hepatocyte growth factor (HGF), the platelet-derived growth factor (PDGF), the fibroblast growth factor 5 (FGF5) (including FGF5-short [FGF5s] and FGF5-long [FGF51]), the fibroblast growth factor 7 (FGF7), the fibroblast growth factor 10 (FGF10), the transforming growth factor pi (TGFpi), the transforming growth factor a (TGFa), the keratinocyte growth factor (KGF), the insulin-like growth factor 1 (IGF-1), the insulin-like growth factor 2 (IGF-2), the insulin-like growth factor-binding protein 5 (IGFBP5), the vascular endothelial growth factor (VEGF), the acidic fibroblast growth factor (aFGF), the basic fibroblast growth factor (bFGF), the epidermal growth factor (EGF), the collagen genes (COL1A1, COL1A2, etc....), the L-dopachrome tautomerase (TRP2), noggin (NOG), protaetiamycine, thioredoxin (TRX), superoxide dismutase (SOD1), the stem cell factor (SCF), and the human growth hormone (hGH).

[0201] In some embodiment, the RNA molecule, the non- viral vector or viral vector, in particular the retroviral vector, or the composition is in non-therapeutic concentration, in particular in a concentration low enough not to induce a therapeutic effect. Therefore, the non-therapeutic concentration is depending of the RNA molecule and more specifically, on the sequence of interest. The skilled artisan is familiar with the non-therapeutic concentrations.

[0202] In some embodiments, the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically, in particular cutaneously or transdermally.

[0203] In some embodiments, the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically after collagen induction therapy (CIT). [0204] In some embodiments, the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is used as an epidermal and/or dermal stimulating agent, preferably as an epidermal agent.

[0205] As used herein, the term “collagen induction therapy”, also known as “microneedling”, “dermarolling”, or “skin needling”, refers to a cosmetic procedure which involves repeatedly puncturing the skin of a subject with microneedles (z.e., needles having a length typically ranging from about 100 pm to 1 000 pm).

[0206] Also encompassed herein is therefore a kit comprising a microneedling device and the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention.

[0207] Alternatively, the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically though application onto the skin of a transdermal patch, in particular of a microneedle transdermal patch, comprising said RNA molecule or DNA molecule or non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention.

[0208] Also encompassed herein is therefore a transdermal patch, in particular a microneedle transdermal patch, comprising the RNA molecule or DNA molecule the non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention.

[0209] In some embodiments, these uses and methods are preferably therapeutic uses and/or methods.

[0210] In some embodiments, these non-therapeutic uses and methods are applicable to substantially sick subject.

[0211] In some embodiments, a “substantially sick subject” is a subject who has been previously diagnosed or identified as suffering from skin and/or hair and/or nails diseases, a “substantially sick subject” is a subject who has been previously diagnosed or identified as suffering from skin and/or hair and/or nails diseases. In one embodiment, a “substantially sick subject” is a subject who does suffer from any known disease, disorder or condition. In one embodiment, a “substantially sick subject” is a subject who is seeking medical attention.

[0212] Examples of therapies of skin and/or skin appendages, in particular of therapeutic treatments, include for instance any or several of induction of promotion of skin and/or hair and/or nail healing.

[0213] As exemplified herein, promoting skin and/or hair healing can be achieved using the RNA molecule, in particular the retroviral vector, of the invention.

[0214] However, the skilled artisan will readily recognize that other sequences of interest can be used for therapeutic purposes, as promoting skin and/or hair healing including, for instance, sequences coding for growth factors.

[0215] In some embodiments, other sequences of interest that can be used for cosmetic purposes include, but are not limited to, sequences encoding the hepatocyte growth factor (HGF), the platelet-derived growth factor (PDGF), the fibroblast growth factor 5 (FGF5) (including FGF5-short [FGF5s] and FGF5-long [FGF51]), the fibroblast growth factor 7 (FGF 7), the fibroblast growth factor 10 (FGF10), the transforming growth factor pi (TGFpi), the transforming growth factor a (TGFa), the keratinocyte growth factor (KGF), the insulin-like growth factor 1 (IGF-1), the insulin-like growth factor 2 (IGF-2), the insulin-like growth factor-binding protein 5 (IGFBP5), the vascular endothelial growth factor (VEGF), the acidic fibroblast growth factor (aFGF), the basic fibroblast growth factor (bFGF), the epidermal growth factor (EGF), the collagen genes (COE1A1, COE1A2, etc....), the L-dopachrome tautomerase (TRP2), noggin (NOG), protaetiamycine, thioredoxin (TRX), superoxide dismutase (SOD1), the stem cell factor (SCF), and the human growth hormone (hGH).

[0216] In some embodiment, the RNA molecule, the non- viral vector or viral vector, in particular the retroviral vector, or the composition is in therapeutic concentration, in particular in a concentration high enough to induce a therapeutic effect. Therefore, the therapeutic concentration is depending of the RNA molecule and more specifically, on the sequence of interest. The skilled artisan is familiar with the therapeutic concentrations. [0217] In some embodiments, the RNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically, in particular cutaneously, transdermally or subdermally, and prefereably transdermally or subdermally, more preferably subdermally.

[0218] In some embodiments, administration to the subject may be carried out by systemic injection. Examples of systemic injections include, but are not limited to, intravenous (iv), subcutaneous (sc), intramuscular (im), intradermal (id), intraperitoneal (ip), and intranasal (in) injection.

[0219] Injections can be performed subcutaneously, intramuscularly, intranasally or intraperitoneally, with various combinations for the prime and boost injection.

[0220] In some embodiments, the RNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically after collagen induction therapy (CIT).

[0221] In some embodiments, the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is used as an epidermal and/or dermal stimulating agent, preferably as an dermal agent.

[0222] As used herein, the term “collagen induction therapy”, also known as “microneedling”, “dermarolling”, or “skin needling”, refers to a cosmetic procedure which involves repeatedly puncturing the skin of a subject with microneedles (z.e., needles having a length typically ranging from about 100 pm to 1 000 pm).

[0223] Also encompassed herein is therefore a kit comprising a microneedling device and the RNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention.

[0224] Alternatively, the RNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is to be administered topically though application onto the skin of a transdermal patch, in particular of a microneedle transdermal patch, comprising said RNA molecule, non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention. [0225] Also encompassed herein is therefore a transdermal patch, in particular a microneedle transdermal patch, comprising the RNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition of the invention.

[0226] Also encompassed in these uses and applications is the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, for use in vaccinating a subject in need thereof against an infectious disease or against cancer; or a method of vaccinating a subject in need thereof against an infectious disease or against cancer, comprising administering to the subject in need thereof the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition.

[0227] As exemplified herein, vaccinating a subject can be efficiently achieved using the RNA molecule, in particular the retroviral vector, of the invention.

[0228] As will be readily understood by the skilled artisan, the sequence of interest in this instance should encode an antigen against which an immunization is desired.

[0229] Examples of antigens include, without limitation, pathogen-related antigens (such as, e.g., antigens of microorganisms or parasites, such as viruses, fungi or bacteria, or archaea, or immunogenic molecules derived from them), self-antigens (such as, e.g., cellular antigens including cells containing normal transplantation antigens and/or tumor-related antigens, RR-Rh antigens, and antigens characteristic of, or specific to particular cells or tissues or body fluids), and allergen-related antigens (such as, e.g., those associated with environmental allergens, including grasses, pollens, molds, dust, insects, dander, venoms, and the like; occupational allergens, including latex, dander, urethanes, epoxy resins, and the like; food, including shellfish, peanuts, eggs, milk products, and the like; and drugs, including antibiotics, anesthetics, and the like).

[0230] Suitable examples of pathogen-related antigens include, but are not limited to, antigens derived from vaccinia, avipox virus, turkey influenza virus, bovine leukemia virus, feline leukemia virus, avian influenza, chicken pneumovirosis virus, canine parvovirus, equine influenza, FHV, Newcastle disease virus (NDV), Chicken/Pennsylvania/1/83 influenza virus, infectious bronchitis virus, Dengue virus, measles virus, Rubella virus, pseudorabies, Epstein-Barr virus, HIV, SIV, EHV, BHV, HCMV, MERS, SARS, SARS-CoV-2, Hantaan, C. tetani, mumps, Morbillivirus, Herpes Simplex virus type 1, Herpes Simplex virus type 2, Human cytomegalovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, respiratory syncytial virus, human papilloma virus, Influenza virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium, Toxoplasma, Cryptococcus, Streptococcus, Staphylococcus, Haemophilus, Diptheria, Tetanus, Pertussis, Escherichia, Candida, Aspergillus, Entamoeba, Giardia, Trypanosoma, Leishmania, and Malaria.

[0231] Suitable examples of self-antigens include, but are not limited to, lupus autoantigen, Smith, Ro, La, Ul-RNP, fibrillin, nuclear antigens, histones, glycoprotein gp70, ribosomal proteins, pyruvate dehydrogenase, dehydrolipoamide acetyltransferase (PCD-E2), hair follicle antigens, human tropomyosin isoform 5 (hTM5), proinsulin, insulin, IA2, GAD65, collagen type II, human cartilage gp 39 (HCgp39), gpl30-RAPS, dnaJpl, citrullinated proteins and peptides (including citrullinated type II collagen, citrullinated vimentin and citrullinated fibrinogen), myelin basic protein, proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), thyroid stimulating factor receptor (TSH-R), acetylcholine receptor (AchR), gliadin, PLP, glucose-6-phosphate isomerase, thyroglobulin, various tRNA synthetases, proteinase-3, and myeloperoxidase, and the like, including fragments thereof.

[0232] Suitable examples of tumor-related antigens include, but are not limited to, MART-l/Melan-A, gplOO, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-CO17-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-CI, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family (e.g. MUC1, MUC16, etc.), HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, pl20ctn, gpl00.sup.Pmelll7, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, pl5, gp75, GM2 and GD2 gangliosides, Smad family of cancer antigens brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2 and viral antigens such as the HPV-16 and HPV-18 E6 and E7 antigens and the EBV-encoded nuclear antigen (EBNA)-l, and the like, including fragments thereof. Further examples of tumor-related antigens are described in, e.g., Li et al., 2004. Cancer Immunol Immunother. 53(3): 139-43; Novellino et al., 2005. Cancer Immunol Immunother. 54(3):187-20; which are herein incorporated by reference in their entirety.

[0233] In some embodiments, the RNA molecule, DNA molecule, the non-viral vector or viral vector, in particular the retroviral vector, or the composition, can be administered to a subject in need thereof once, twice, or more.

[0234] When administered twice or more, the first administration is typically referred to as “priming step”, and the subsequent administration(s) are referred to as “boosting step”. In some embodiments, the boosting step can be carried out once, twice, three times, four times or more.

[0235] In some embodiments, the period of time between the priming step and the boosting step and/or between each iteration of the “boosting step” ranges from about 1 day to about 6 months, preferably from about 1 week to about 3 months, more preferably from about 2 weeks to about 1 month.

[0236] In one embodiment, the period of time between the priming step and the boosting step and/or between each iteration of the “boosting step” is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. [0237] In some embodiments, administration to the subject may be carried out by systemic injection. Examples of systemic injections include, but are not limited to, intravenous (iv), subcutaneous (sc), intramuscular (im), intradermal (id), intraperitoneal (ip), and intranasal (in) injection.

[0238] In case of prime/boosting administration, each injection may be carried out via the same route, or by different route. By way of non-limiting example, the priming step can be carried out by intramuscular injection and the boosting step can be carried out by intranasal injection; or the priming step can be carried out by intraperitoneal injection and boosting step by intranasal injection. Alternatively, both the priming and boosting steps can be carried out by intraperitoneal injection.

[0239] Also encompassed in these uses and applications is the RNA molecule or DNA molecule or the non- viral vector or viral vector, in particular the retroviral vector, or the composition, for use in genome engineering; or a method of genome engineering, comprising administering to a subject in need thereof the RNA molecule or DNA molecule or the non- viral vector or viral vector, in particular the retroviral vector, or the composition.

[0240] Such genome engineering applications are useful, in particular in the field of bioproduction, or in methods of therapeutic treatment such as cell therapy or gene therapy.

[0241] These uses and methods may be in vivo or in vitro.

[0242] They may also be ex vivo, wherein the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition, is contacted ex vivo with a cell or a population of cells from a subject, and optionally the cell or population of cells is then administered back to said subject.

[0243] As will be readily understood by the skilled artisan, the sequence of interest in this instance should encode a genome editor.

[0244] Examples of genome editors include, but are not limited to, CRISPR-associated proteins (Cas), zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALEN), and site-specific recombinases (such as, e.g., a Cre recombinase or flippase [Flp]).

[0245] Examples of CRISPR-associated proteins include, without limitation, class 2 Cas proteins such as, e.g., Cas9, Casl2 (including Casl2a (or Cpfl), Cas 12b (or C2cl), Casl2c (or C2c3), Casl2d (or CasY), Casl2e (or CasX), Casl2f (or Casl4 or C2cl0), Cas 12g, Casl2h, Casl2i, and Cas 12k (or C2c5)), and Cas 13 (including Cas 13a (or C2c2), Casl3b, Casl3c, and Casl3d). The nucleic acid coding for CRISPR-associated proteins can be fused with at least another nucleic acid sequence, such as prime editing guide RNA (pegRNA) and/or base editing RNA.

[0246] In some embodiments, genome editors may require at least one RNA or DNA molecule, in particular in the case of CRISPR-associated proteins, which RNA or DNA molecule is capable of interacting with the genome editor and targeting it to a locus of interest in a cell’s genome. Said RNA molecule may be known in the art as guide RNA (gRNA) and/or prime editing guide RNA (pegRNA) and/or base editing RNA. Alternatively, two RNA molecules (a CRISPR RNA named crRNA and a trans-activating CRISPR RNA named tracrRNA) may be used in lieu of a single gRNA.

[0247] In these embodiments, the uses and methods may thus comprise contacting the cell or administering to the subject such gRNA, or a mix of crRNA and tracrRNA, before, concomitantly with or after, the RNA molecule or DNA molecule or the non- viral vector or viral vector, in particular the retroviral vector, or the composition.

[0248] In some embodiments, genome editors may further require at least one exogenous nucleic acid, in particular at least one exogenous DNA, which is to be inserted in the genome of a target cell.

[0249] This exogenous nucleic acid may comprise, for instance, the sequence of a gene of interest to be inserted in the genome of a target cell. This gene of interest may be, for instance, a functional version of a malfunctioning endogenous gene, or alternatively, a malfunctional version of a normally functioning endogenous gene. It can also be any other gene which expression is desired in a cell, depending on the purpose. [0250] In some embodiments, the method of genome engineering may be particularly suitable for the transgenesis of an organism.

[0251] The organism may be a plant or an animal. In some embodiments, the animal is not a human.

[0252] The skilled artisan is familiar with means and methods for transgenesis.

[0253] In some embodiments, the method comprises the steps of transfecting or transducing an animal cell with the RNA molecule or DNA molecule or the non-viral vector or viral vector, in particular the retroviral vector, or the composition; collecting the animal cell’s nucleus; inserting the animal cell’s nucleus into an unfertilized oocyte; allowing the oocyte to develop, thereby obtaining a transgenic animal, preferably wherein the transgenic animal is not a human.

[0254] In particular, the sequence of interest of the RNA or DNA molecule is a genome editor, and the nucleic acid sequence encoding the transgene of interest is brought to the cell in addition to the RNA or DNA molecule of the invention, for stable integration into the genome of said cell and development of a transgenic organism.

[0255] In some embodiments, the method of genome engineering may be particularly suitable for bioproduction (or recombinant production), in particular for the generation of stable expression systems.

[0256] Such method is applicable, for instance, to the production of recombinant proteins of interest in any suitable expression system, including without limitation, bacterial cells, yeast cells, insect cells, mammalian cells, and human cells.

[0257] The skilled artisan is familiar with means and methods for recombinant protein production.

[0258] In some embodiments, the method comprises the steps of transfecting or transducing a cell or a population of cells with the RNA molecule or the DNA molecule or the RNA molecule vectorized or DNA molecule vectorized in a non- viral vector or in a viral vector, in particular the retroviral vector, or the composition; culturing the cell or population of cells for a period of time sufficient to allow the production by said cell or population of cells of a protein of interest encoded by the RNA or DNA molecule; recovering the protein of interest; optionally, purifying the protein of interest.

[0259] In particular, the sequence of interest of the RNA or DNA molecule is a genome editor, and the nucleic acid sequence encoding the protein of interest is brought to the cell or population of cells in addition to the RNA or DNA molecule of the invention, for stable integration into the genome of said cell or population of cells and recombinant production of the protein of interest.

[0260] A fourth object of the invention is a nucleic acid system comprising at least one nucleic acid sequence comprising the RNA Booster.

[0261] Another object of the invention is a kit comprising said nucleic acid system or RNA or DNA molecule; a cell or cell population comprising said nucleic acid system or RNA or DNA molecule; and/or a method of producing said nucleic acid system or the RNA molecule or the DNA molecule as described herein.

[0262] The invention can be a nucleic acid system; a kit comprising said nucleic acid system; a cell or cell population comprising said nucleic acid system; and a method of producing an RNA virus vector, in particular a retroviral vector, as described above, using said nucleic acid system or said cell or cell population.

[0263] According to the invention, the nucleic acid system comprises at least one nucleic acid sequence encoding a recombinant expression cassette comprising:

- optionally, a promoter, and/or

- an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, and/or - a multiple cloning site (MCS) or a sequence of interest, and/or

- optionally, an origin of replication (ORI), and/or

- optionally, a selectable marker.

[0264] In some embodiments, the nucleic acid system is in DNA form.

[0265] The skilled artisan will readily understand that the first nucleic acid system can be provided in a single linear nucleic acid or plasmid.

[0266] The purpose of the selectable marker is to allow for the selection or identification of cells or organisms that have taken up the gene of interest. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is selected in the group of: ampicillin, kanamycin, neomycin, tetracycline, bleomycin, chloramphenicol, streptomycin resistance gene.

[0267] In some embodiments, the origin of replication is selected in the group of: oriC, pl5A, ColEl, SV40.

[0268] In some embodiments, the nucleic acid system comprises:

(i) At least one first nucleic acid sequence encoding an RNA virus genome, in particular a retrovirus genome comprising at least a viral, preferably retroviral, gag and pol sequence; optionally wherein the pol sequence encodes a defective reverse-transcriptase (RT) or wherein the pol sequence lacks a RT gene;

(ii) At least one second nucleic acid sequence encoding an envelope glycoprotein; and

(iii) At least one third nucleic acid sequence encoding a recombinant expression cassette comprising: optionally, a promoter, optionally, a 5’ long terminal repeat (LTR), optionally, a packaging sequence, optionally, a Rev-response element, an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, optionally, a multiple cloning site (MCS), optionally, a sequence of interest, optionally, an origin of replication (ORI), optionally a selectable marker, optionally, a post-transcriptional regulatory element, and optionally, a 3’ LTR; wherein the first and second nucleic acid sequences lack a functional packaging sequence.

[0269] According to the invention, the nucleic acid system comprises:

(iv) At least one first nucleic acid sequence encoding an RNA virus genome, in particular a retrovirus genome comprising at least a viral, preferably retroviral, gag and pol sequence; optionally wherein the pol sequence encodes a defective reverse-transcriptase (RT) or wherein the pol sequence lacks a RT gene;

(v) At least one second nucleic acid sequence encoding an envelope glycoprotein; and

(vi) At least one third nucleic acid sequence encoding a recombinant expression cassette comprising: optionally, a 5’ long terminal repeat (LTR), a packaging sequence, optionally, a Rev-response element, an RNA Booster sequence characterized by a sequence mmsknkkkm or its reverse sequence mkkknksmm, optionally, a multiple cloning site (MCS), optionally, a sequence of interest, optionally, a post-transcriptional regulatory element, and optionally, a 3’ LTR; wherein the first and second nucleic acid sequences lack a functional packaging sequence.

[0270] Each nucleic acid sequence (i), (ii) and (iii) defined above is a DNA nucleic acid sequence. Hence, the RNA Booster sequence in DNA form will comprise thymine in lieu o/uracyl defined above for the RNA molecule. Exemplary RNA Booster sequences in the third nucleic acid sequence include thus, but are not limited to: a deoxyribonucleic acid sequence mmskngkkm or its reverse sequence mkkgnksmm, preferably mmskngkgm or its reverse sequence mgkgnksmm, more preferably cmskhgkgm or its reverse mgkghksmc, even more cmskwgkgm or its reverse sequence mgkgwksmc, yet even more preferably ccsuwgggm or its reverse sequence mgggwuscc, wherein:

■ “m” indicates an adenine (a) or cytosine (c);

■ “s” indicates a guanine (g) or a cytosine (c);

■ “k” indicates a guanine (g) or a thymine (t);

■ “h” indicates an adenine (a) or a cytosine (c) or a thymine (t);

■ “w” indicates an adenine (a) or a thymine (t);

■ “n” indicates any nucleotide; and in particular:

RNA Booster 1: cccgtgtgc, or its reverse sequence RNA Booster 10: cgtgtgccc

RNA Booster 2: aagtttggc, or its reverse sequence RNA Booster 11: cggtttgaa RNA Booster 3: ccctaggta, or its reverse sequence RNA Booster 12: atggatccc RNA Booster 4: ccgtggtgc, or its reverse sequence RNA Booster 13: cgtggtgcc RNA Booster 5: aactggggc, or its reverse sequence RNA Booster 14: cggggtcaa RNA Booster 6: ccctcgggc, or its reverse sequence RNA Booster 15: cgggctccc RNA Booster 7: cacgtgtgc, or its reverse sequence RNA Booster 16: cgtgtgcac RNA Booster 8: cccttgggc, or its reverse sequence RNA Booster 17: cgggttccc RNA Booster 9: ccgtaggga, or its reverse sequence RNA Booster 18: agggatgcc.

[0271] In some embodiment, the at least one first and the at least one second nucleic acid sequences are trans-complementation sequences, i.e., sequences encoding trans-complementation proteins or peptides which are intended to be part of the RNA viral vector, preferably the retroviral vector, as proteins or peptides, but not as nucleic acid information in its ribonucleic genome. These trans-complementation proteins or peptides are thus typically expressed in trans within a cell or population of cell during the production of the RNA viral vector, preferably the retroviral vector. In order to remain trans-complementary, trans-complementation sequences should be present in nucleic acid sequences lacking a functional packaging sequence.

[0272] In some embodiments, each at least one nucleic acid sequence (i), (ii) and (iii) defined above may, independently from each other, be a linear nucleic acid or a plasmid. [0273] The skilled artisan will readily understand that the first nucleic acid sequence encoding the RNA virus genome, preferably the retrovirus genome, can be provided in a single linear nucleic acid or plasmid, or in two or more linear nucleic acids or plasmids.

[0274] In some embodiments, the at least one second nucleic acid sequence encodes an envelope glycoprotein, such as an envelope glycoprotein from (or derived from) an enveloped virus, possibly from a Retroviridae or from any other enveloped virus, such as, e.g., from a Rhabdoviridae, or a cellular glycoprotein or a synthetic glycoprotein.

[0275] In some preferred embodiments, the at least one second nucleic acid sequence encodes an envelope glycoprotein from Indiana vesiculovirus (formerly known as vesicular stomatitis virus or VSV).

[0276] In some embodiments, the at least one third nucleic acid sequence does not comprise a sequence of interest. If so, it is desirable however that the at least one third nucleic acid sequence comprises at least one restriction site (and preferably at least two different restriction sites), or any other means suitable for introducing a gene of interest downstream the RNA Booster sequence.

[0277] According to the invention, the method of producing an RNA viral vector, preferably a retroviral vector, comprises the steps of:

1) transfecting at least one cell or cell population with at least one RNA or DNA molecule as described above, or at least one nucleic acid system as described above;

2) culturing the cell or cell population of a period of time sufficient for the production of the RNA viral vector, preferably the retroviral vector; and

3) recovering, and optionally purifying, the RNA viral vector, preferably the retroviral vector.

[0278] According to the invention, the method of producing a RNA viral vector, preferably a retroviral vector alternatively comprises the steps of:

1) providing at least one cell or cell population as described above;

2) culturing the cell or cell population of a period of time sufficient for the production of the RNA viral vector, preferably the retroviral vector; and 3) recovering, and optionally purifying, the RNA viral vector, preferably the retroviral vector.

[0279] According to the invention, the kit comprising at least one nucleic acid system as described above may comprise at least one first, second and third nucleic acid sequences in separate vials or containers.

[0280] Alternatively, two of the first, second and third nucleic acid sequences can be mixed in a single vial or container; in this case, the first and second nucleic acid sequences can preferably be mixed in a single vial or container, and the third nucleic acid sequence can be provided in a separate vial or container.

[0281] In some embodiments, the kit may further comprise instructions for use, in particular, instructions for performing the method of producing a RNA viral vector, preferably a retroviral vector, as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0282] Figures 1A-B show the expression of GFP in 293T cells transduced with an RNA vector derived from lentivirus expressing GFP and comprising one of RNA Booster 1 to RNA Booster 9, or without RNA Booster (0 RNA Booster). The RNA Booster being located about 500 nt upstream of GFP.

Figure 1A is a histogram showing the relative efficacy of GFP expression in 293T cells. The relative expression based on the level of expression after transduction with an RNA vector derived from lentivirus without RNA Booster is shown.

Figure IB is a histogram showing the transientness of GFP expression in 293T cells. Results are shown as a percentage of 293T cells expressing GFP at day 3 (D3) and day 7 (D7) post-transduction.

[0283] Figure 2 is a histogram showing the percentage of GFP-positive human PBMC after transduction with an integrating lentiviral vector expressing GFP [Integrating LV-GFP], or a RNA vector derived from lentivirus with a mutated reverse transcriptase (D110E substitution) expressing GFP and comprising RNA Booster 8 at two different doses, as indicated [LV-RNA Booster 8/GFP], versus negative control without any transduction [0 vector]. The RNA Booster being located about 500 nt upstream of GFP.

[0284] Figure 3 is a histogram showing the percentage of GFP-positive human dendritic cells after transduction with an integrating lentiviral vector expressing GFP [Integrating LV-GFP], a non-integrating lentiviral vector expressing GFP [Non-integrating LV-GFP], or a RNA vector derived from lentivirus with a mutated reverse transcriptase (D110E substitution) expressing GFP and comprising RNA Booster 8 [LV-RAA Booster 8/GFP], versus negative control without any transduction [0 vector]. The RNA Booster being located about 500 nt upstream of GFP.

[0285] Figure 4 is an histogram showing the percentage of GFP-positive human hematopoietic stem cells after transduction with an integrating lentiviral vector expressing GFP [Integrating LV-GFP], or a non-integrating lentiviral vector expressing GFP [Non-integrating LV-GFP], or a RNA vector derived from lentivirus with a mutated reverse transcriptase (D110E substitution) expressing GFP and comprising RNA Booster 8 at two different doses (Multiplicity Of Infection (MOI)), as indicated [LV-RAA Booster 8/GFP], versus negative control without any transduction [0 vector]. The RNA Booster being located about 500 nt upstream of GFP.

[0286] Figure 5 is a histogram showing the results of a cell proliferation assay of human hair follicle dermal papilla cells transduced or not with different doses (as indicated) of a RNA vector derived from lentivirus with a mutated reverse transcriptase (D110E substitution) expressing FGF9 and comprising RNA Booster 8 [LV-RAA Booster 8/FGF9], and cultured in presence or absence of cortisol and/or VEGF (as indicated). The RNA Booster being located about 500 nt upstream of FGF9.

[0287] Figure 6 is a histogram showing the quantification of immunoglobulin G (IgG) against ovalbumin (OVA) in C57BL/6J mice after prime/boost immunization with unadjuvanted ovalbumin [Pos Unadj OVA], adjuvanted OVA [Pos Adj OVA (Alun)], or with a RNA vector derived from lentivirus with a mutated reverse transcriptase (D110E substitution) expressing ovalbumin and comprising RNA Booster 8 [LV-RNA-OVA], or with a non-integrating lentiviral vector expressing ovalbumin, which does not comprise RNA Booster [LV-DNA-OVA], versus negative control without any transduction [Neg C]. The RNA Booster being located about 500 nt upstream of the gene of interest. Prime and boost injections were performed by various routes as indicated [prime/boost]: subcutaneously [SC], intramuscularly [IM], intranasally [IN], intraperitoneally [IP].

[0288] Figure ? is a histogram showing the percentage of GFP-positive HeLa cells constitutively expressing GFP after a co-transduction with a RNA vector derived from lentivirus with a mutated reverse transcriptase (DI 10E substitution) expressing Cas9 and comprising RNA Booster 8 [LV-RAA Booster 8/Cas9] and a non-integrating lentiviral vector expressing a guide RNA targeting the GFP [Non-integrating LV-gRNA], versus a co-transduction with a non-integrating lentiviral vector expressing Cas9 [Non-integrating LV-Cas9] and a non-integrating lentiviral vector expressing a guide RNA targeting GFP [Non-integrating LV-gRNA], versus negative control without any transduction [0 vector]. The RNA Booster being located about 500 nt upstream of Cas9.

[0289] Figure 8 is a histogram showing the expression of GFP in 293T cells transduced with an RNA vector derived from lentivirus expressing GFP, and comprising the RNA Booster 9 forward or its reverse sequence RNA Booster 9 reverse corresponding to RNA Booster 18, versus negative control corresponding to an RNA vector without RNA Booster (0 RNA Booster). The RNA Booster being located about 2000 nt upstream (5’) of GFP.

[0290] Figure 9 is a histogram showing the expression of GFP in 293T cells transduced with an RNA vector derived from lentivirus expressing GFP, and comprising the RNA Booster 9 forward or its reverse sequence RNA Booster 9 reverse corresponding to RNA Booster 18, versus a negative control corresponding to an RNA vector without RNA Booster (0 RNA Booster). The RNA Booster being located about 500 nt downstream (3’) of GFP.

[0291] Figure 10 shows the skin healing by the human epidermal keratinocyte migration kinetic.

Figure 10A shows a model of skin healing without treatment (control), with EGF at 10 ng/ml in the medium of culture or transduced with a RNA vector derived from lentivirus expressing FGF7, and comprising the RNA Booster 8. The RNA Booster being located about 500 nt upstream of FGF7.

Figure 10B is a histogram showing the human epidermal keratinocytes migration kinetic representing by the percentage of healing without treatment (control), with EGF at 10 ng/ml in the medium of culture or transduced with a RNA vector derived from lentivirus expressing FGF7, and comprising the RNA Booster 8. The RNA Booster being located about 500 nt upstream of FGF7.

EXAMPLES

[0292] The present invention is further illustrated by the following examples.

Materials and Methods

Plasmids

[0293] The envelope trans-complementation plasmid encodes the vesicular stomatitis virus envelope glycoprotein (VSV-G) with SEQ ID NO: 1, under control of a cytomegalovirus-immediate early (CMV-IE) promoter.

[0294] The capsid trans-complementation plasmid encodes a functional integrase (with SEQ ID NO: 2) and a functional reverse transcriptase (with SEQ ID NO: 4) of HIV-1; or a mutant integrase with abolished integrase activity (SEQ ID NO: 2 comprising a D64V substitution, as set forth in SEQ ID NO: 3); or a mutant reverse transcriptase with abolished reverse transcriptase activity (SEQ ID NO: 4 comprising a D110E substitution, as set forth in SEQ ID NO: 5).

[0295] The vector plasmid encodes a recombinant expression cassette comprising a 5’ LTR (with SEQ ID NO: 6) and a 3’ LTR (with SEQ ID NO: 7) flanking a transgene (z.e., a sequence of interest), either GFP including a tobacco extension signal sequence (with SEQ ID NO: 8, encoding SEQ ID NO: 9), human fibroblast growth factor 9 (FGF9) (with SEQ ID NO: 10 encoding SEQ ID NO: 11), human fibroblast growth factor 7 (FGF7) (with SEQ ID NO: 19 encoding SEQ ID NO: 20), ovalbumin (with SEQ ID NO: 12 encoding SEQ ID NO: 13), or Cas9 (with SEQ ID NO: 14 encoding SEQ ID NO: 15), with or without a RNA Booster sequence in 5’ or in 3’ of the transgene (RNA Booster 1 to RNA Booster 18, according in part to Table 1) inserted in a Sall restriction site. Regulatory sequences such as retroviral psi packaging signal (with

SEQ ID NO: 16), Rev-response element (RRE, with SEQ ID NO: 17) and WHV post-transcriptional regulatory element (WPRE, with SEQ ID NO: 18), were also included.

[0296] Table 1 : RNA Booster sequences Lentiviral vector production

[0297] Lentiviral vectors were generated by the transient transfection of 293T cells by using the calcium phosphate precipitation method. Briefly, cells were co-transfected with the VSV-G trans-complementation plasmid, the capsid trans-complementation plasmid and a vector plasmid. Supernatant was collected 48 hours after transfection, treated with DNasel and filtered. Viral particles were then concentrated by ultracentrifugation and resuspended in 0.1 M PBS. The genome of particles was quantified for each stock by RT-qPCR to determine a titer of gRNA by pL. Cell culture

[0298] 293T cells were grown in Dulbecco’s modified medium supplemented with antibiotics (lOO U/mL penicillin and 100 mg/mL streptomycin) and 10 % heat-inactivated fetal calf serum. The cells were plated and cultured in a humidified incubator at 37°C in a 5 % CO2 and 90 % air atmosphere.

[0299] Human PBMC were grown in RPMI-160 + L-G1U medium supplemented with 1 % HEPES 5 M, 0.1 % P-mercaptoethanol 55 mM, antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and 10 % heat- inactivated fetal calf serum. The cells were plated and cultured in a humidified incubator at 37 °C in a 5 % CO2 and 90 % air atmosphere.

[0300] Human dendritic cells were grown in RPMI-160 + L-G1U medium supplemented with 1 % HEPES 5 M, 100 ng/mL GM-CSF, 50 ng/mL IL-4, antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and 10 % heat-inactivated fetal calf serum. The cells were plated and cultured in a humidified incubator at 37°C in a 5 % CO2 and 90 % air atmosphere.

[0301] Human hematopoietic stem cells (HSC) were grown in StemSpan™ SFEM medium supplemented with the StemSpan™ CD34+ Expansion Supplement (lOx). The cells were plated and cultured in a humidified incubator at 37°C in a 5 % CO2 and 90 % air atmosphere.

[0302] Human hair follicle dermal papilla cells were grown in HFDPC Basal culture medium with HFDPC supplement mix. The cells were plated and cultured in a humidified incubator at 37°C in a 5 % CO2 and 90 % air atmosphere.

[0303] GFP ⁺ HeLa cells were generated with an integrating lentiviral vector expressing GFP. A clonal population with one integration and a stable expression of GFP was selected for the experiments. GFP ⁺ HeLa cells were grown in Dulbecco’s modified medium supplemented with antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) and 10 % heat- inactivated fetal calf serum. The cells were plated and cultured in a humidified incubator at 37°C in a 5 % CO2 and 90 % air atmosphere. Transduction

[0304] 293T cells were contacted and transduced with an RNA vector derived from lentivirus (with a mutated D110E reverse transcriptase) expressing GFP with one of RNA Booster 1 to 18, or without RNA Booster. During the transduction, the cells were incubated for 6 hours. GFP expression was measured 72 hours after transduction.

[0305] PBMC were contacted and transduced with an RNA vector derived from lentivirus (with a mutated D110E reverse transcriptase) expressing GFP with RNA Booster 8, or with an integrating lentiviral vector expressing GFP (without RNA Booster). GFP expression was measured by FACS 96 hours after transduction.

[0306] Human CD14 ⁺ monocytes were differentiated into dendritic cells during 4 days, before transduction, with GM-CSF (lOO ng/mE) and IE-4 (50 ng/mL). Differentiation was checked by cytometry with an anti-CDla antibody. Dendritic cells were then contacted and transduced with an RNA vector derived from lentivirus (with a mutated DI 10E reverse transcriptase) expressing GFP with RNA Booster 8, or with an integrating lentiviral vector expressing GFP (without RNA Booster), or with a non-integrating lentiviral vector expressing GFP (without RNA Booster). GFP expression was measured by FACS 96 hours after transduction.

[0307] Human hematopoietic stem cells (HSC) were contacted and transduced with an RNA vector derived from lentivirus (with a mutated D110E reverse transcriptase) expressing GFP with RNA Booster 8, or with an integrating lentiviral vector expressing GFP (without RNA Booster), or with a non-integrating lentiviral vector expressing GFP (without RNA Booster). GFP expression was measured by FACS 96 hours after transduction.

[0308] Human hair follicle dermal papilla cells were contacted and transduced with an RNA vector derived from lentivirus (with a mutated D110E reverse transcriptase) expressing FGF9 or FGF7 with RNA Booster 8. After transduction, the cells were treated with 300 nM of cortisol (which has a negative effect on proliferation). A control with or without VEGF (which inhibits the cortisol effect) was performed. Cell proliferation was measured through BrdU (5-bromo-2’-deoxyuridine) incorporation. [0309] GFP ⁺ HeLa cells were contacted and co-transduced with an RNA vector derived from lentivirus (with a mutated D110E reverse transcriptase) expressing Cas9 with RNA Booster 8 and a non-integrating lentiviral vector expressing a guide RNA targeting the GFP (without RNA Booster), or co-transduced with a non-integrating lentiviral vector expressing Cas9 (without RNA Booster) and a non-integrating lentiviral vector expressing a guide RNA targeting the GFP (without RNA Booster). GFP expression was measured by FACS 96 hours after.

Flow cytometry analysis

[0310] GFP expression was analyzed by flow cytometry to determine the percentage of GFP-positive cells. Transduced cells were harvested, trypsinized, and fixed with 1 % formaldehyde before analysis.

Mouse immunization

[0311] C57BL/6J mice were immunized with an RNA vector derived from lentivirus (with a mutated DI 10E reverse transcriptase) expressing ovalbumin with RNA Booster 8, or with a non-integrating lentiviral vector expressing ovalbumin (without RNA Booster). A boost injection with the same vector was administered 28 days after the prime injection. Injections were performed subcutaneously, intramuscularly, intranasally or intraperitoneally, with various combinations for the prime and boost injection. Immunization was measured through the dosage of ovalbumin- specific immunoglobulin G (IgG) 14 days after prime injection and again 14 days after boost injection.

[0312] Three controls were also carried out: a negative control, in which mice were administered saline; and two positive controls, in which mice were administered unadjuvanted ovalbumin or ovalbumin adjuvanted with alum.

Ovalbumin-specific IgG dosage

[0313] About 0.15 mL of blood were taken from each animal into dry capillaries from the mandibular vessels under isoflurane gas anesthesia. The blood samples were kept at room temperature for at least 30 minutes and serum was prepared within 60 minutes of sampling by centrifugation for 10 minutes at 1500 g at 4°C ± 2°C). Serum was frozen within 120 minutes post-sampling and stored at -70°C.

[0314] Ovalbumin-specific IgG were measured from thawed blood samples using the “Mouse anti-OVA IgG antibody assay kit” (Chondrex, Inc.; Ref. 3011).

Human epidermal keratinocytes migration assay

Culture and treatment

[0315] The keratinocytes have been seeded in culture medium in 24-well plates previously coated with a collagen I solution. After 24 hours of incubation, the medium will be replaced by test medium then a mechanical scraping has been carried out and the cells have been characterized with calcein-AM. After 30 minutes of incubation, images have been taken (TO) then the medium has been replaced by test medium containing or not (control) the test vector or the reference (EGF at 10 ng/ml). For the test vector, 15 pl of vector and 200 pl of test medium have been added and incubated for 5 hours then medium has been added (qsp 600 pl). The cells have been incubated until the next morning and again labeled with calcein-AM (30 minutes incubation) to produce the 24- hour time images. The cells have been incubated again for another 24 to 48 hours and then again labeled with calcein-AM and photographed according to a similar protocol.

Migration analyze

[0316] The cell migration area has been monitored with a high-resolution imaging system, INCell Analyzer™2200 automated microscope (GE Healthcare) and the artificial wound surface has been analyzed with Image J software. Representative images will be inserted in the report and all images will be provided via a secure sharing site.

[0317] The surface of the artificial wound (area without cells) has been measured after 24, 48 and 72 hours of culture and compared to the initial surface measured at TO. The effect of the compounds on the migration has been compared to the untreated control.

Data processing [0318] The raw data has been transferred and processed using Microsoft Excel software. Intergroup comparisons have been made using the unpaired two-tailed Student's t test. Statistical analyzes can be interpreted if n>5; however, for n<5, the calculated data is only provided as an indication.

Results

GFP in vitro expression in 293T cells (Figures 1A-B)

[0319] GFP expression from an RNA vector derived from lentivirus expressing GFP (with one of RNA Booster 1 to RNA Booster 9, or without RNA Booster) was compared at a same Multiplicity Of Infection (MOI).

[0320] As shown in Figure 1A, the presence of any of RNA Booster 1-9 in 5’ of the transgene of interest in the RNA vector derived from lentivirus improved the efficacy of GFP expression in transduced 293T cells, as compared to a lentiviral vector without RNA Booster. The RNA Booster sequences were shown to enhance GFP expression by at least a factor 2 (for RNA Booster 1) and increasingly, up to a factor 12 (for RNA Booster 9).

[0321] As seen in Figure IB, GFP was well expressed in 293T cells at day 3 posttransduction, but not at day 7 post-transduction anymore, confirming the transientness of this expression system.

[0322] These data demonstrate show that presence of a RNA Booster as defined herein, in 5’ of a transgene of interest, is able to improve the efficacy of transient expression of this transgene of interest in 293T cells.

GFP in vitro expression in human PBMC (Figure 2)

[0323] Figure 2 shows that the RNA vector derived from lentivirus expressing GFP, in presence of RNA Booster 8, and both at 10 pF and 15 pF doses, enabled to transduce human PBMC with high efficacy and induced an increase of the GFP expression by a factor 2.7-2.9, as compared to the integrating vector without RNA Booster. [0324] These data demonstrate that presence of a RNA Booster as defined herein, in 5’ of a transgene of interest, is able to improve the efficacy of expression of this transgene of interest in human PBMC.

[0325] This suggests that an RNA vector derived from lentivirus comprising such RNA Booster could be good candidates to induce transient expression of a gene of interest in PBMC, which cells are of interest in a wide range of immunotherapy strategies and gene therapy.

GFP in vitro expression in human dendritic cells (Figure 3)

[0326] Figure 3 shows that the RNA vector derived from lentivirus expressing GFP, in presence of RNA Booster 8, enabled to transduce human dendritic cells with high efficacy and induced an increase of the GFP expression 5.75 to 22 times higher than when using an integrative lentiviral vector or a non-integrative lentiviral vector in absence of RNA Booster, respectively.

[0327] This suggests that RNA vectors derived from lentivirus comprising such RNA Booster could be good candidates to induce transient expression of a gene of interest in primary cells such as dendritic cells.

GFP in vitro expression in human hematopoietic stem cells (Figure 4)

[0328] Figure 4 shows that the RNA vector derived from lentivirus expressing GFP, in presence of RNA Booster 8, enabled to transduce human hematopoietic stem cells with a higher efficacy than DNA integrating lentiviral vectors at a MOI 10 to 20 times lower, and induced an increase of the GFP expression around 35-42 times higher than when using a non-integrating lentiviral vector in absence of RNA Booster.

[0329] These data suggest that RNA vectors derived from lentivirus comprising such RNA Booster could be good candidates to induce transient expression of a gene of interest in human HSC, which cells are of interest in a wide range of immunotherapy strategies and gene therapy.

In vitro proliferation assay of human hair follicle dermal papilla cells (Figure 5) [0330] Figure 5 shows that cortisol decreased cell proliferation, as compared to the non-treated condition (without cortisol, VEGF or vector), while adding VEGF and cortisol restored cell proliferation (with cortisol and VEGF, without vector). The RNA vector derived from lentivirus expressing FGF9, in presence of RNA Booster 8, enabled human hair follicle dermal papilla cells treated with cortisol to be transduced and to restore cell proliferation at all doses (2 pL), to levels similar to the non-treated control (without cortisol, VEGF or vector).

[0331] These data suggest that these RNA vectors derived from lentivirus comprising such RNA Booster could be useful for treating a variety of diseases, for example promoting skin healing, or in non-therapeutic indications, for example in dermatology or cosmetology, to induce hair growth.

In vivo vaccination assay (Figure 6)

[0332] Figure 6 shows that an RNA vector derived from lentivirus expressing ovalbumin, in presence of RNA Booster 8, induced high levels of OVA- specific IgG when administered intramuscularly/intranasally, intraperitoneally/intraperitoneally, or intraperitoneally/intranasally (prime/boost - 5 x 10 ⁸ transducing units (TU) per injection). In particular, this RNA vector derived from lentivirus induced higher levels of OVA-specific IgG as compared to a non-integrative lentiviral vector expressing ovalbumin administered by the same routes.

[0333] Interestingly, very high doses of OVA-specific IgG were obtained after an intramuscular prime injection of the RNA vector derived from lentivirus (and to a lesser extent, after an intraperitoneal prime injection), suggesting that a boost injection could not even be needed.

[0334] These data demonstrate that RNA vectors derived from lentivirus comprising such RNA Booster can improve immunization in mice, and suggest that these RNA vectors derived from lentivirus could be useful for vaccination.

Cas9 in vitro expression in HeLa cells constitutively expressing GFP (Figure 7)

[0335] Figure 7 shows that an RNA vector derived from lentivirus expressing Cas9, in presence of RNA Booster 8, combined with a guide RNA targeting GFP brought to the cell using a non-integrating lentiviral vector, induces the knock-out of the GFP gene in HeLa cells with an efficacy close than 100 %.

[0336] Although non-integrating lentiviral vectors expressing Cas9 and a gRNA yield similar results, using these vectors in ex vivo or in vivo therapy is not desirable since DNA molecules have a non-zero probability of recombination with another DNA molecule, such as with a DNA genome. This would induce adverse effects. Moreover, non-integrating lentiviral vectors have been shown in the art to exhibit a residual level of integration of 0.1-0.5 %. Conversely, RNA vectors derived from lentivirus do not show any risk of reverse transcriptase activity leakage, which ensures thus 100 % of information transfer in the form of RNA, which cannot recombine with DNA.

[0337] The data shows the RNA vector comprising the RNA Booster performs at least as well as a DNA vector.

[0338] These data suggest that RNA vectors derived from lentivirus comprising such RNA Booster could be good candidates to induce transient expression of a genome editor, and could thus be useful for genome engineering in the field of bioproduction, cell therapy, gene therapy and transgenesis.

GFP in vitro expression in 293T cells (Figure 8 et 9)

[0339] GFP expression from an RNA vector derived from lentivirus expressing GFP with the forward and reverse sequence of the RNA Booster 9 at 2 different positions than initially, or without RNA Booster) was compared at a same Multiplicity Of Infection (MOI).

[0340] As shown in Figure 8, the presence of the forward and reverse RNA Booster 9 in 5’ and remote side of 2kb of the transgene of interest in the RNA vector derived from lentivirus improved the efficacy of GFP expression in transduced 293T cells, as compared to a lentiviral vector without RNA Booster. The RNA Booster sequences were shown to enhance GFP expression by a factor 2,5 for forward RNA Booster 9 and by a factor 2,2 for reverse RNA Booster 9. [0341] As shown in Figure 9, the presence of the forward and reverse RNA Booster 9 in 3’ of the transgene of interest in the RNA vector derived from lentivirus improved the efficacy of GFP expression in transduced 293T cells, as compared to a lentiviral vector without RNA Booster. The RNA Booster sequences were shown to enhance GFP expression by a factor 6.1 for forward RNA Booster 9 and by a factor 6.9 for reverse RNA Booster 9.

Human epidermal keratinocytes migration (Figure 10A et B)

[0342] As shown in Figure 10, the reduction in scrape over time is condition-dependent. The lentivirus-derived RNA vector expressing FGF7, in the presence of RNA Booster 8, transduced human epidermal keratinocyte cells and stimulated their migration. With this construction, the percentage of healing reaches 100% in 72h. These data suggest that lentivirus-derived RNA vectors comprising such RNA boosters could be good candidates for inducing transient FGF7 expression for skin repair.

LIST OF SEQUENCE;

List of sequences used in the present invention (from 5’ to 3’):

List of the sequences with SEQ ID:

SEQ ID NO 1:

MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLI GTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFTPSV EQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAVIVQVTPHHVLVDEYTGE WVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELS SLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSY LAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDD WAPYEDVEIGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQD AASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGI HLCIKLKHTKKRQIYTDIEMNRLGK

SEQ ID NO 2:

FLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHG

QVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAG RWPVKTVHTDNGS NETS TTVKA AC WWAGIKQEFGIPYNPQSQG VIES MNKELK KIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQ KQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKII RDYGKQMAGDDCVASRQDED

SEQ ID NO 3:

FLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVASCDKCQLKGEAMHG

QVDCSPGIWQLVCTHLEGKVILVAVHVASGYIEAEVIPAETGQETAYFLLKLAG RWPVKTVHTDNGS NETS TTVKA AC WWAGIKQEFGIPYNPQSQG VIES MNKELK KIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQ KQITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKII RDYGKQMAGDDCVASRQDED

SEQ ID NO 4:

PISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPY NTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVL DVGDAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCS MTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTP DKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQ

IYAGIKVRQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSK DLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHTNDVKQLTEAVQKI ATESIVIWGKTPKFKLPIQKETWEAW

SEQ ID NO 5:

PISPIETVPVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPY NTPVFAIKKKDSTKWRKLVDFRELNKRTQDFWEVQLGIPHPAGLKQKKSVTVL EVGDAYFSVPLDKDFRKYTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCS MTKILEPFRKQNPDIVIYQYMDDLYVGSDLEIGQHRTKIEELRQHLLRWGFTTP DKKHQKEPPFLWMGYELHPDKWTVQPIVLPEKDSWTVNDIQKLVGKLNWASQ IYAGIKVRQLCKLLRGTKALTEVVPLTEEAELELAENREILKEPVHGVYYDPSK DLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHTNDVKQLTEAVQKI ATESIVIWGKTPKFKLPIQKETWEAW

SEQ ID NO 6: tggaagggctaattcactcccaacgaagacaagatatccttgatctgtggatctaccaca cacaaggctacttccctgattagca gaactacacaccagggccagggatcagatatccactgacctttggatggtgctacaagct agtaccagttgagccagagaagt tagaagaagccaacaaaggagagaacaccagcttgttacaacctgtgagcctgcatggga tggatgacccggagagagaa gtgttagagtggaggtttgacagccgcctagcatttcatcacggtggcccgagagctgca tccggagtacttcaagaactgctg atatcgagcttgctacaagggactttccgctgggggactttccagggaggcgtggcctgg gcgggactggggagtggcgag ccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagac cagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagt gtgtgcccgtctgttgtgtgactctg gtaactagagatccctcagacccttttagtcagtgtggaaaatctctagca

SEQ ID NO 7: tggaagggctaattcactcccaacgaagacaagatcgtcgagagatgctgcatataagca gctgctttttgcttgtactgggtct ctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt aagcctcaataaagcttgccttgag tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagac ccttttagtcagtgtggaaaatctcta gca

SEQ ID NO 8: atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggac ggcgacgtaaacggccacaa gttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagtt catctgcaccaccggcaagct gcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccg ctaccccgaccacatgaagca gcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttctt caaggacgacggcaactacaag acccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggc atcgacttcaaggaggacg gcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatgg ccgacaagcagaagaacggca tcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgacc actaccagcagaacaccccc atcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctg agcaaagaccccaacgagaa gcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatgga cgagctgtacaag SEQ ID NO 9:

MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKL PVPWPTEVTTETYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNY KTRAEVKFEGDTEVNRIEEKGIDFKEDGNIEGHKEEYNYNSHNVYIMADKQKN GIKVNFKIRHNIEDGSVQEADHYQQNTPIGDGPVEEPDNHYESTQSAESKDPNE KRDHMVEEEFVTAAGITEGMDEEYK

SEQ ID NO 10: atggcacctctcggtgaagtcggaaactacttcggagtccaggatgccgtgcccttcggc aacgtgcctgtgctgccagtgga ttctccagtgctgctgtctgatcacctgggtcaaagcgaggctggaggcctgcccagagg tcctgcagtgactgatctggatca cctgaagggcattctcagacgcaggcaactgtactgcagaaccggattccatctcgaaat ctttccaaatggcaccattcaagg aaccagaaaggatcactctcgctttggcatcctggagttcatttccatcgctgttggcct cgtctctatcagaggcgtggacagc ggtctgtacctcggaatgaacgagaagggtgagctgtatggttccgagaagctcacacaa gaatgcgtgtttcgcgaacagttt gaagagaattggtacaacacctacagctccaatctgtacaagcatgtggatacaggtagg agatactatgtcgcactgaacaa agatggcactccacgcgagggtacacgcactaagaggcatcagaagttcacacatttcct gccaaggccagtggaccctgac aaggtgcctgagctctacaaggacatcctcagccagtcttga

SEQ ID NO 11:

MAPLGEVGNYFGVQDAVPFGNVPVLPVDSPVLLSDHLGQSEAGGLPRGPAVTD LDHLKGILRRRQLYCRTGFHLEIFPNGTIQGTRKDHSRFGILEFISIAVGLVSIRGV DSGLYLGMNEKGELYGSEKLTQECVFREQFEENWYNTYSSNLYKHVDTGRRY YVALNKDGTPREGTRTKRHQKFTHFLPRPVDPDKVPELYKDILSQS

SEQ ID NO 12: atgggtagcattggagcagccagcatggagttctgctttgacgtcttcaaggaactcaaa gtgcatcatgcaaacgagaacatc ttctactgtcccatcgcaatcatgagcgctctcgctatggtttacctgggagccaaagac agcaccaggacccagatcaacaa ggtggtccgcttcgataagctgcctggtttcggagacagcatcgaggcacagtgcggaac ctccgtgaacgttcattcttctct gagggacattctgaatcagatcacaaagcctaacgacgtgtatagcttctctctggcctc caggctgtacgctgaggaacgcta ccctatcctccctgaatacctgcagtgtgtcaaagagctgtatagaggtggactggaacc tatcaacttccaaactgcagcaga ccaggcacgcgagctgatcaattcttgggtggagtctcaaactaacggaatcatcaggaa cgtgctccagccctccagcgtg gacagccagactgcaatggtgctggtcaacgccattgtcttcaagggactctgggagaag actttcaaggacgaggacacac aggctatgcctttcagagttactgagcaggagagcaagcctgtccagatgatgtaccaga tcggtctgttcagagtggcatctat ggccagcgagaagatgaagatcctggagctgcccttcgcaagcggaactatgtctatgct cgtgctgctgccagatgaggttt ctggactggaacaactggagtccatcatcaactttgagaagctgacagagtggacaagct ctaacgtcatggaagagagaaa gatcaaggtgtacctcccaaggatgaagatggaagagaagtacaacctgactagcgtgct gatggcaatgggaatcacagat gtctttagctcttctgctaacctgtctggcatctcctccgcagagtccctgaagatttcc caggctgtccacgctgctcatgccga aatcaatgaagcaggtagggaagtggtcggatctgcagaggctggtgttgatgctgccag cgtctccgaagagtttagagctg accatccctttctgttctgtatcaaacatatcgccacaaatgcagtcctgttcttcggaa gatgtgtgtctcct

SEQ ID NO 13:

MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGAKDSTRTQI NKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAE ERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQP SSVDSQTAMVLVNAIVFKGLWEKTFKDEDTQAMPFRVTEQESKPVQMMYQIG LFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSS NVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKIS

QAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLF FGRCVSP

SEQ ID NO 14: atggacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtg atcaccgacgagtacaaggtg cccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctg atcggcgccctgctgttcgac agcggagaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccaga cggaagaaccggatctgc tatctgcaagagattttcagcaacgagatggccaaggtggacgacagcttcttccacaga ctggaagagtccttcctggtggaa gaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctac cacgagaagtaccccaccat ctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatcta tctggccctggcccacatgatc aagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggac aagctgttcatccagctggtgc agacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaagg ccatcctgtctgccagactga gcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcc tgttcggcaacctgattgccc tgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaac tgcagctgagcaaggacacct acgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttc tggccgccaagaacctgtccg acgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctga gcgcctctatgatcaagagata cgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctga gaagtacaaagaaatcttcttc gaccagagcaagaacggctacgccggctacatcgatggcggagccagccaggaagagttc tacaagttcatcaagcccatc ctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctg cggaagcagcggaccttcga caacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggca ggaagatttttacccattcctg aaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggc cctctggccaggggaaacagc agattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaa gtggtggacaagggcgccag cgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggt gctgcccaagcacagcctgct gtacgagtacttcaccgtgtacaacgagctgaccaaagtgaaatacgtgaccgagggaat gagaaagcccgccttcctgagc ggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtg aagcagctgaaagaggacta cttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaa cgcctccctgggcacataccacg acctgctgaagattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattc tggaagatatcgtgctgaccctg acactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttc gacgacaaagtgatgaagcagc tgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatcc gggacaagcagtccggcaa gacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgat ccacgacgacagcctgacctttaa agaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgc caatctggccggatcccccg ccattaagaagggcatcctgcagacagtgaagattgtggacgagctcgtgaaagtgatgg gccacaagcccgagaacatcg tgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgaga gaatgaagcggatcgaag agggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagc tgcagaacgagaagctgtac ctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccgg ctgtccgactacgatgtggacc acattgtgccccagtccttcatcaaggacgactccatcgataacaaagtgctgactcgga gcgacaagaaccggggcaagag cgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcgccagctgct gaatgccaagctgattaccca gaggaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggc cggcttcattaagcggcagc tggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaaca ctaagtacgacgagaacgac aaactgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgacttcaga aaggatttccagttttacaaagtg cgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaacc gccctgatcaaaaagtaccct aagctggaaagcgagttcgtgtacggcgattacaaggtgtacgacgtgcggaagatgatc gccaagagcgagcaggaaatc ggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgag atcacactggccaacggcgagatc agaaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggc cgggattttgccacagtgcg gaaagtgctgtccatgccccaagtgaatatcgtgaaaaagaccgaggtgcagaccggcgg cttcagcaaagagtctatcctg cccaagaggaactccgacaagctgatcgccagaaagaaggattgggaccctaagaagtac ggcggctttgacagccccac cgtggcctactctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaa gagtgtgaaagagctgctggg gatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaa gggctacaaagaagtgaaaaag gacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagcgg atgctggcttctgccggcgaact gcagaagggaaacgagctggccctgccctccaaatatgtgaacttcctgtacctggccag ccactatgagaagctgaagggc tcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggac gagatcatcgagcagattagcg agttctccaagcgcgtgatcctggccgatgccaacctggacaaggtgctgagcgcctaca acaagcaccgggataagccca tcagagagcaggccgagaatatcatccacctgtttaccctgaccaacctgggagcccctg ccgccttcaagtactttgacacca ccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccacc agagcatcaccggcctgtac gagacacggatcgacctgtctcagctgggaggcgaccccaagaaaaagcgcaaagtgctc gagtga

SEQ ID NO 15:

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD

SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE

EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMI

KFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS

KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTY

DDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYD

EHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILE

KMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKD

NREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGE

QKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDL

LKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK

RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKE DIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYY LQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSD

NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL

VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKV

REINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ

EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFAT

VRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFD

SPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYE KLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSIT

GEYETRIDESQEGGDPKKKRKVEE

SEQ ID NO 16: tcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggt gagtacgccaaaaattttga ctagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggag

SEQ ID NO 17: tccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacgg tacaggccagacaattattgtct ggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttg caactcacagtctggggcatcaa gcagctccaggcaagaatcctggctgtggaaagatacct

SEQ ID NO 18: ccgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatg ttgctccttttacgctatgtggatacgct gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttg tataaatcctggttgctgtctctttatgaggag ttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaaccccc actggttggggcattgccaccacc tgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatc gccgcctgccttgcccgctgctggac aggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctt tccatggctgctcgcctgtgttgcc acctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccg gctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgg gccgcctccccgcatcggg

SEQ ID NO 19: atgcacaaatggattctgacttggattctgcccaccctgctctatcgctcctgcttccac atcatctgtctcgtcggtacaatcagc ctggcttgtaacgacatgacaccagaacagatggctaccaacgtcaactgctccagccct gagagacacactcgctcttacga ctacatggaaggaggcgacattcgcgttagaaggctcttctgtcgcactcaatggtatct gaggattgacaagagaggcaagg tgaaaggcacccaggaaatgaagaacaactacaacatcatggaaatccgcactgtcgctg tcggtatcgttgccatcaaaggt gtcgaatccgagttctacctggctatgaacaaggagggcaagctgtacgcaaagaaggag tgcaacgaggattgcaacttca aggaactcattctggagaaccactacaacacttatgcttccgctaagtggactcacaacg gtggtgaaatgttcgtcgcactga accagaagggcatccctgttagaggtaagaagacaaagaaggagcagaagaccgcacact tcctgcctatggcaatcacat aa

SEQ ID NO 20: MHKWIETWIEPTEEYRSCFHIICEVGTISEACNDMTPEQMATNVNCSSPERHTRS

YDYMEGGDIRVRRLFCRTQWYLRIDKRGKVKGTQEMKNNYNIMEIRTVAVGI

VAIKGVESEFYLAMNKEGKLYAKKECNEDCNFKELILENHYNTYASAKWTHN

GGEMFVAENQKGIPVRGKKTKKEQKTAHFEPMAIT

Table of sequences without SEQ ID: