Synthetic nucleic acids and their therapeutic use

Abstract

The present invention relates to a synthetic nucleic acid comprising the following elements in the 5'-3' direction. a) At least one 5'UTR untranslated region selected from the 5'UTR (SEQ ID NO: 4), synthetic region 5'NeoUTR3 (SEQ ID NO: 5), the 5'UTR (SEQ ID NO: 6), and synthetic region 5'UTR4 (SEQ ID NO: 7) of the human beta-globin gene, b) Open Reading Frame (ORF), and c) At least one 3'UTR untranslated region selected from the 3'UTR region of the rotavirus VP6 gene (SEQ ID NO: 1) and the 3'UTR region of the manganese superoxide dismutase (MnSOD) gene.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acids, particularly messenger RNA for therapeutic purposes.

Background Art

[0002] The concept of gene therapy emerged in the 1960s with the development of molecular biology. Gene therapy is a strategy consisting of delivering nucleic acids into the cells of an organism in order to treat diseases caused by mutants, defective alleles. This approach has evolved. In addition to strategies to restore defective gene activity, it now also relates to any in situ production of further activity from exogenous nucleic acids that is likely to have a therapeutic effect.

[0003] Nucleic acids used in the therapeutic field include deoxyribonucleic acid (DNA )(double-stranded and single-stranded) and ribonucleic acid (RNA) (single-stranded, containing uracil instead of thymine in DNA).

[0004] Due to its physicochemical and physiological properties, RNA is a particularly powerful molecular biology tool for targeting genes of interest and / or for the expression of exogenous proteins. Technical obstacles related to its instability and sensitivity to RNA-degrading proteins have been overcome, and today, numerous RNA-targeting and / or RNA-based therapeutic agents have been approved.

[0005] A review article (Zhu et al., 2022) presents different types of RNA used in the following research and human treatments: antisense oligonucleotides that specifically inhibit the translation of RNA into proteins; interfering RNAs that cause the degradation of RNA molecules; guide RNAs that direct endonucleases, known as the "molecular scissors" of the CRISPR-Cas system, to genes that need modification; aptamers that can interact with proteins; and messenger RNA (mRNA) that enables the in-situ translation of the genetic information it encodes into proteins.

[0006] For the therapeutic use of mRNA, it is crucial to optimize the stability of the mRNA molecule used to promote its accurate intracellular integration and translation. Adjustment parameters include the molecular chemical composition (modified nucleotides, addition of untranslated sequences), possible complexation with chemical agents, and excipients used in the administered formulation.

[0007] In 2020, the emergence of COVID-19, a disease associated with SARS-CoV-2 coronavirus infection, accelerated the development of mRNA-based vaccines. In particular, the BNT162b2 vaccine developed by Pfizer-BioNTech (Lamb, 2021) and Moderna's mRNA-1273 vaccine (Baden et al., 2021) demonstrated their clinical efficacy. Both vaccines utilize ionized lipid-based formulations incorporating synthetic mRNA molecules containing modified nucleosides that encode the pre-fusion form of the coronavirus spike protein.

[0008] mRNA vaccines are also being developed for cancer treatment, with more than 20 vaccine candidates currently in clinical trials for the treatment of various solid tumors. In most cases, these mRNA vaccines are administered concurrently with immunomodulatory factors or cytokine cocktails.

[0009] Therapeutic messenger RNA (mRNA) involves the production of sequence-optimized mRNA and appropriate This is an advanced technology that requires the complexation of the substance with chemical agents for intracellular integration.

[0010] The conventional process of mRNA translation in eukaryotic cells relies on a scanning mechanism by ribosomes from a 5' cap that scans mRNA up to the first start codon. Another process used by viruses involves a so-called IRES (internal ribosome entry site) sequence, which allows ribosomes to be recruited at the start codon independently of the presence of the cap and the scanning mechanism.

[0011] Synthetic mRNA used in therapy typically contains a poly(A) tail, which is a sequence located downstream (3') of the coding sequence that protects the RNA molecule from degradation by RNA enzymes.

[0012] In addition to these elements, therapeutic mRNA typically contains untranslated nucleotide sequences called Untranslated Regions (UTRs), located upstream (5'UTR) and / or downstream (3'UTR) of the coding sequence. The use of such 5' and / or 3' untranslated sequences to extend and / or increase mRNA protein translation in transformed cells has been proposed, in particular, in European Patent No. 3494982.

[0013] A review by Uchida et al. (2020) presents this strategy for increasing the translation efficiency and half-life of synthetic mRNA, consisting of including UTR sequences derived from genes whose mRNA is highly translated and stable, such as alphaglobin, betaglobin, albumin, complement factor 3, or cytochrome CYP2E1 genes.

[0014] These untranslated sequences include, in particular, the following: - 5'UTR and 3'UTR sequences of human betaglobin disclosed in the paper (Babendure et al., 2006), International Publication No. 2014186334, and European Patent No. 0737750. - The 5'UTR sequence of either the human alpha-globin HBA1 or HBA2 gene (the 5'UTR sequence is identical in both genes). -5'NeoUTR3 element, a synthetic element disclosed in the paper by Cao et al., 2021 and U.S. Patent Application Publication No. 2020 / 0066375. This element is selected from a bank of 12,000 synthetic UTRs after functional evaluation. -5'UTR4 element, a synthetic element described in the paper (Linares-Fernandez et al., 2021). -3'mtRNR1 AES sequence, one derived from human mitochondrial 12S rRNA (mtRNR1), the other from human AES / TLE5 gene (AES)(Von Niessen A et al., 2019) It consists of two segments. - 3'UTR sequence of the rotavirus VP6 virus gene, Yang et al. It was isolated and characterized by al., 2004. - The 3'UTR sequence of the gene encoding manganese superoxide dismutase (MnSOD) and its effect on mRNA translation have been disclosed in a paper by Chung et al. (1998).

[0015] These untranslated elements are particularly used in mRNA used in vaccine compositions. This is especially true in the case of vaccines developed against the SARS-CoV-2 coronavirus that causes the so-called COVID-19 disease.

[0016] In particular, B developed by Pfizer and BioNTech (Xia, 2021) The NT162b2 vaccine contains mRNA that includes an open reading frame (ORF) encoding a pre-fusion version of the SARS-CoV-2 spike protein, and this ORF is adjacent to the following elements: - The 5'UTR of the human alphaglobin gene, in which the Kozak consensus sequence has been slightly modified, and -3'UTR mtRNR1 AES.

[0017] This particular combination of 5'UTR and 3'UTR elements used in this vaccine is thought to be a reference combination that enables maximum translation of the mRNA constructed in this way.

[0018] Another reference construct is the association between the 5'UTR element of human betaglobin and the mtRNR1 AES 3'UTR element.

[0019] The inventors have tested other untranslated elements, particularly other 3'UTR elements, that provide a much higher level of translation than is observed for the 5'UTR combination of the human betaglobin gene and the 3'UTR mtRNR1 AES.

[0020] Furthermore, the inventors tested several types of heterogeneous poly(A)-tail sequences, particularly poly(AG) or poly(G), to increase the stability of the synthetic nucleic acids of the present invention. [Overview of the Initiative]

[0021] The present invention relates to a synthetic nucleic acid comprising the following elements in the 5'-3' direction. a) At least one 5'UTR untranslated element selected from the 5'UTR of the human betaglobin gene (SEQ ID NO: 4), synthetic element 5'NeoUTR3 (SEQ ID NO: 5), the 5'UTR of the human alphaglobin gene (SEQ ID NO: 6), and synthetic element 5'UTR4 (SEQ ID NO: 7), b) Open Reading Frame (ORF), and c) At least one 3'UTR untranslated element selected from the 3'UTR element of the rotavirus VP6 gene (SEQ ID NO: 1) and the 3'UTR element of the manganese superoxide dismutase (MnSOD) gene.

[0022] The present invention also relates to an expression vector containing the synthetic nucleic acid as described above.

[0023] The present invention also relates to a host cell containing the synthetic nucleic acid or expression vector as described above, excluding human embryonic stem cells.

[0024] Another object of the present invention is a pharmaceutical composition or vaccine composition containing the synthetic nucleic acid as described above in a suitable pharmaceutical carrier, and optionally one or more excipients and / or one or more adjuvants.

[0025] The present invention also relates to the synthetic nucleic acid as described above for use as a drug.

[0026] The present invention also relates to the synthetic nucleic acid as described above for use in various applications, such as the treatment or prevention of bone disorders.

[0027] Finally, the present invention also relates to the in vitro use of the synthetic nucleic acid as described above to increase and / or extend the translation of the target protein from this nucleic acid in a host cell incorporating the nucleic acid defined above. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] [Figure 1A]This paper describes the expression dynamics of mRNA encoding nanoluciferase with different UTR combinations introduced into three different cell types. The combinations tested are as follows: -5'β-3'MT=5'UTR=5'UTR and 3'UTR of human betaglobin mtRNR1 AES -5'β-3'Rota=5'UTR of human betaglobin and 3'UTR of rotavirus VP6 gene -5'β-3'MnSOD=5'UTR of human betaglobin and 3'UTR of rat MnSOD gene -5'NeoUTR3-3'MT=Synthetic elements 5'NeoUTR3 and 3'UTR mtRNR1 AES -5'NeoUTR3-3'Rota=Synthetic element 5'NeoUTR3 and 3'UTR of rotavirus VP6 gene -5'NeoUTR3-3'MnSOD=Synthetic element 5'NeoUTR3 and 3'UTR of rat MnSOD gene Expression dynamics in HeLa human cancer cells. [Figure 1B] This paper describes the expression dynamics of mRNA encoding nanoluciferase with different UTR combinations introduced into three different cell types. The combinations tested are as follows: -5'β-3'MT=5'UTR=5'UTR and 3'UTR of human betaglobin mtRNR1 AES -5'β-3'Rota=5'UTR of human betaglobin and 3'UTR of rotavirus VP6 gene -5'β-3'MnSOD=5'UTR of human betaglobin and 3'UTR of rat MnSOD gene -5'NeoUTR3-3'MT=Synthetic elements 5'NeoUTR3 and 3'UTR mtRNR1 AES -5'NeoUTR3-3'Rota=Synthetic element 5'NeoUTR3 and 3'UTR of rotavirus VP6 gene -5'NeoUTR3-3'MnSOD=Synthetic element 5'NeoUTR3 and 3'UTR of rat MnSOD gene Expression dynamics in DC2.4 mouse dendritic cells. [Figure 1C]This paper describes the expression dynamics of mRNA encoding nanoluciferase with different UTR combinations introduced into three different cell types. The combinations tested are as follows: -5'β-3'MT=5'UTR=5'UTR and 3'UTR of human betaglobin mtRNR1 AES -5'β-3'Rota=5'UTR of human betaglobin and 3'UTR of rotavirus VP6 gene -5'β-3'MnSOD=5'UTR of human betaglobin and 3'UTR of rat MnSOD gene -5'NeoUTR3-3'MT=Synthetic elements 5'NeoUTR3 and 3'UTR mtRNR1 AES -5'NeoUTR3-3'Rota=Synthetic element 5'NeoUTR3 and 3'UTR of rotavirus VP6 gene -5'NeoUTR3-3'MnSOD=Synthetic element 5'NeoUTR3 and 3'UTR of rat MnSOD gene Expression dynamics in C2C12 mouse myoblasts. [Figure 2A] This shows the relative expression over time of a nanoluciferase-encoding mRNA adjacent to a specific UTR sequence, relative to a "standard" mRNA containing the untranslated elements 5'UTRβglo and 3'UTR MT, in three cell types. Expression levels are normalized to the expression levels measured for standard mRNA. Time-course expression in HeLa human cancer cells. [Figure 2B] This shows the relative time course of mRNA encoding nanoluciferase adjacent to specific UTR sequences compared to "standard" mRNA containing untranslated elements 5'UTRβglo and 3'UTR MT in three cell types. Expression levels are normalized to the expression levels measured for standard mRNA. Time course of expression in DC2.4 mouse dendritic cells. [Figure 2C] This shows the relative time course of mRNA encoding nanoluciferase adjacent to specific UTR sequences compared to "standard" mRNA containing the untranslated elements 5'UTRβglo and 3'UTR MT in three cell types. Expression levels are normalized to the expression levels measured for standard mRNA. Time course of expression in C2C12 mouse myoblasts. [Figure 3]This demonstrates the stability of polyadenylated tails on various plasmids. [Figure 4] This shows the expression dynamics of various mRNAs in C2C12 cells. [Figure 5] This shows the total production of nanoluciferase by various mRNAs in C2C12 cells. [Figure 6] This shows the expression dynamics of various mRNAs in HeLa cells. [Figure 7] This shows the production of nanoluciferase by various mRNAs in HeLa cells. [Figure 8] This study demonstrates total nanoluciferase production by various mRNAs in HeLa cells, with the mRNAs capped with ARCA or Cleancap AG analogues. [Figure 9] This shows the expression dynamics of various mRNAs in DC2.4 cells. [Figure 10] This shows the total production of nanoluciferase by various mRNAs in DC2.4 cells. [Figure 11] This study demonstrates total nanoluciferase production by various mRNAs in DC2.4 cells, with the mRNAs capped with ARCA or Cleancap AG analogues. [Figure 12] This shows the expression of various mRNAs in MoDC 24 hours after transfection. [Figure 13] This paper compares the expression of various mRNAs in MoDC 24 hours after transfection using ARCA or Cleancap AG caps. [Figure 14] This shows the intracellular stability of mRNAs with different 3'UTR sequences. A) Intracellular stability in HeLA cells, B) Intracellular stability in DC2.4 cells. [Figure 15] This shows the immunogenicity of the 3'UTR sequence in DC2.4 cells as measured by RT-qPCR. [Figure 16]The expression dynamics (A) and total protein production (B) after transfection with mRNA containing 5'UTRα globin and 3'UTR AES-mtRNR1 combined with either an A110 tail or a Tail X tail in DC2.4 cells are shown. [Figure 17] This shows the expression dynamics (A) and total protein production (B) after transfection in HeLa cells using mRNA containing 5'UTRα globin and 3'UTR AES-mtRNR1 combined with either an A110 tail or a Tail X tail. [Figure 18] This shows the pharmacokinetics (A) and total protein production (B) after transfection in HeLa cells using mRNA without a 5'UTR and mRNA containing a 3'UTR, VP6, or MnSOD. The control is an mRNA construct containing a 3'UTR AES-MT. [Figure 19] The dynamics (A) and total protein production (B) after transfection with mRNA lacking a 5'UTR and mRNA containing a 3'UTR, VP6, or MnSOD in DC2.4 cells are shown. The control is an mRNA construct containing a 3'UTR AES-MT. [Modes for carrying out the invention]

[0029] Combinations of 5'UTR and 3'UTR sequences have been identified, enabling higher protein expression than "conventional" non-coding sequences. Such combinations of non-coding elements can increase the expression of therapeutic target proteins and thus improve the efficacy of treatment.

[0030] According to a first aspect, the present invention relates to a synthetic nucleic acid comprising the following elements in the 5'-3' direction. a) At least one 5'UTR untranslated element selected from the 5'UTR of the human beta-globin gene (SEQ ID NO: 4), synthetic element 5'NeoUTR3 (SEQ ID NO: 5), the 5'UTR of the human alpha-globin gene (SEQ ID NO: 6), and synthetic element 5'UTR4 (SEQ ID NO: 7), b) Open Reading Frame (ORF), and c) At least one 3'UTR untranslated element selected from the 3'UTR element of the rotavirus VP6 gene (SEQ ID NO: 1) and the 3'UTR element of the manganese superoxide dismutase (MnSOD) gene.

[0031] The following terms are defined to better illustrate the objectives of the present invention.

[0032] The terms “element” and “sequence” are used interchangeably to refer to the 5'UTR and 3'UTR elements. It will be obvious to those skilled in the art that these elements consist of nucleotides and thus constitute a nucleotide sequence.

[0033] For the purposes of this invention, the term "synthetic nucleic acid" refers to isolated double-stranded or single-stranded nucleic acids obtained by linking different synthetic or natural polynucleotides.

[0034] Preferably, it is a single-stranded nucleic acid, and more preferably messenger RNA (mRNA).

[0035] The term "5'UTR untranslated element" refers to a portion of synthetic nucleic acid located at the 5' (i.e., "upstream") end of the open reading frame that is not translated into protein. This element acts as a translation activator. 5'UTR elements are also known as regulatory elements and may include elements that control gene expression. These regulatory elements may, for example, be ribosome binding sites.

[0036] The terms “open reading phase” or “open reading frame” (ORF) refer to a sequence of several triplets of nucleotides that can be translated into a peptide or protein. An open reading frame preferably includes, at its 5' end, a start codon, which is a combination of three consecutive nucleotides that commonly encodes the amino acid methionine (ATG), and a subsequent region that is generally a multiple of three nucleotides in length. An ORF is preferably terminated by a stop codon (e.g., TAA, TAG, TGA). Generally, this is the only stop codon in an open reading frame.

[0037] The term "3'UTR untranslated element" refers to the portion of a synthetic nucleic acid molecule located in the 3' (i.e., "downstream") of the open reading phase that is not translated into a protein. Generally, the 3'UTR is the protein coding region (ORF) and the nucleus. It is a portion of mRNA located between the acid's poly(A) or poly(AG) tail sequence.

[0038] The 3'UTR element of the MnSOD gene can originate from any organism. (Chung As shown in et al., 1998, there is high sequence identity between the 3'UTR MnSOD elements of bovine, mouse, rat, and human genes (see Figure 3 and Table 2 in particular in this paper). Therefore, depending on the embodiment desired by those skilled in the art, they can select the origin of the 3'UTR MnSOD element.

[0039] In this application, the presented examples were performed using a 3'UTR element derived from a rat gene having the sequence shown in Sequence ID No. 2.

[0040] According to one embodiment of the present invention, the 3'UTR element of the MnSOD gene is derived from a rat gene (SEQ ID NO: 2) or a human gene.

[0041] According to another embodiment of the present invention, the synthetic nucleic acid according to the present invention is characterized by comprising at least one of the following 3'UTR sequences. - The 3'UTR of the rotavirus VP6 gene having the sequence shown in Sequence ID No. 1, and - 3'UTR of the MnSOD gene.

[0042] Furthermore, these 3'UTR elements are combined with 5'UTR elements derived from different organisms.

[0043] Therefore, the 3'UTR element of the rotavirus VP6 gene does not combine with the 5'UTR element derived from the rotavirus gene, and the 3'UTR element of the rat gene encoding MnSOD does not combine with the 5'UTR element derived from the rat gene.

[0044] The synthetic nucleic acid according to the present invention comprises at least one 5'UTR element and at least one 3'UTR element. Therefore, it is understood that several non-coding elements may be present upstream and downstream of the ORF. In particular, typically two identical or different 5'UTR elements are positioned upstream of the ORF in a "head-tail" configuration.

[0045] In certain embodiments, the present invention corresponds to specific combinations of 5'UTR sequences and 3'UTR sequences. As shown in the examples, these combinations enable high expression of synthetic mRNA containing these elements in three different cell types, as well as in vivo in mice.

[0046] One hypothesis is that this high expression is achieved by increased mRNA stability due to specific combinations of these 5'UTR and 3'UTR elements.

[0047] In particular, the synthetic nucleic acid according to the present invention is characterized in that the 5'UTR element is selected from the following. -The 5'UTR of the human betaglobin gene having the sequence shown in Sequence ID No. 4, -Synthetic element 5'NeoUTR3 having the sequence shown in Sequence ID 5, - The 5'UTR of the human alpha-globin gene having the sequence shown in Sequence ID No. 6, and -A synthetic element 5'UTR4 having the sequence shown in sequence number 7.

[0048] Therefore, the following eight combinations are covered by the present invention. -3'Rota (3'UTR of the rotavirus VP6 gene) and 5'β (5'UTR of the human β-globin gene). -3'Rota (3'UTR of the rotavirus VP6 gene) and 5'NeoUTR3 (synthetic element). -3'MnSOD (3'UTR of the MnSOD gene) and 5'β (5'UTR of the human β-globin gene). -3'MnSOD (3'UTR of the MnSOD gene) and 5'NeoUTR3 (synthetic element). -3'Rota (3'UTR of the rotavirus VP6 gene) and 5'α (5'UTR of the human α-globin gene). -3'MnSOD (3'UTR of the MnSOD gene) and 5'α (5'UTR of the human α-globin gene). -3'Rota (3'UTR and 5'UTR4 of the rotavirus VP6 gene). -3'MnSOD (3'UTR and 5'UTR4 of the MnSOD gene).

[0049] Among these combinations of untranslated elements, preferred combinations include the 5'β element, i.e., the following combinations: -3'Rota (3'UTR of the rotavirus VP6 gene) and 5'β (human β-globulin). (5'UTR of the Bin gene). -3'MnSOD (3'UTR of the MnSOD gene) and 5'β (5'UTR of the human β-globin gene).

[0050] In particular, the 3'UTR of the MnSOD gene originates from either a rat or human gene.

[0051] A highly preferred combination is one containing rat 5'β and 3'MnSOD elements, each showing the sequences of SEQ ID NO: 4 and SEQ ID NO: 2 around the ORF, respectively.

[0052] The untranslated elements used in the synthetic nucleic acids of the present invention are defined by their origin and their nucleotide sequences. However, it is understood that these sequences may differ slightly and, in particular, may be optimized by replacing a small percentage of nucleotides while remaining within the scope of the present invention.

[0053] Therefore, for the purposes of the present invention, the untranslated element is defined as follows: - The 5'UTR of the human betaglobin gene has at least 90% sequence identity with the sequence of Sequence ID No. 4. - The synthetic element 5'NeoUTR3 has at least 90% sequence identity with the sequence of sequence number 5, - The 5'UTR of the human alphaglobin gene has at least 90% sequence identity with sequence number 6. - The synthetic element 5'UTR4 has at least 90% sequence identity with the sequence of sequence number 7, - The 3'UTR element of the rotavirus VP6 gene has at least 90% sequence identity with the sequence of Sequence ID No. 1. - The 3'UTR element of the rat-derived manganese superoxide dismutase (MnSOD) gene has at least 90% sequence identity with the sequence of Sequence ID No. 2.

[0054] The synthetic nucleic acid according to the present invention is preferably a single-stranded nucleic acid, preferably ribonucleic acid (RNA), and most preferably messenger RNA (mRNA).

[0055] According to a preferred embodiment, the synthetic nucleic acid according to the present invention is also characterized by further comprising a cap at 5'.

[0056] A cap is an entity that "caps" the 5' end of mature mRNA, usually a modified nucleotide. Numerous examples of cap structures, such as ARCA, Cap0, Cap1, or Cap2 types, are known to those skilled in the art.

[0057] According to a preferred embodiment, the synthetic nucleic acid according to the present invention is characterized by further comprising a poly(A) tail or a poly(AG) tail at 3'. According to this embodiment, the nucleic acid is mRNA.

[0058] A poly(A)tail or 3'-poly(A)tail is an adenosine nucleotide sequence located at the 3' end of mRNA, containing up to approximately 400 adenosine nucleotides. A poly(AG)tail or 3'-poly(AG)tail is an adenosine and guanine nucleotide sequence containing up to approximately 400 nucleotides, composed of a mixture of adenosine and guanine in any ratio that a person skilled in the art could conceive. In particular, a poly(AG)tail contains at least one guanine.

[0059] In particular, poly(A) or poly(AG) tails contain 100 to 150 nucleotides. , in particular, it contains 120 nucleotides.

[0060] The synthetic nucleic acid according to the present invention includes an open reading phase (ORF) that encodes the target protein.

[0061] In the examples presented in the Experiments section, the target protein is nanoluciferase.

[0062] Those skilled in the art will know how to select the most appropriate target protein depending on its intended use.

[0063] In a preferred embodiment, the protein of interest is a protein from the bone morphogenetic protein (BMP) family. These proteins are growth factors involved in embryogenesis. In particular, BMP-2 is involved in bone and cartilage development. Therefore, in a particular embodiment of the present invention, ORF encodes a BMP protein, preferably BMP2.

[0064] Other non-limiting examples of therapeutic applications include tissue regeneration using mRNA encoding bone morphogenetic factors or angiogenic factors, anti-cancer vaccination using mRNA encoding tumor antigens or coagulation factors, or treatment of fibrotic conditions. It is also possible to cite the use of prescribed sequences to deliver healthy copies of proteins to treat rare diseases such as phenylketonuria.

[0065] A further object of the present invention is an expression vector containing the synthetic nucleic acid described above.

[0066] An "expression vector" is understood as a vector containing a nucleic acid molecule encoding the target protein and the elements necessary to enable its expression. In particular, the nucleic acid molecule encoding the target protein is operably ligated to an appropriate regulatory sequence, such as a promoter having constitutive or inductive activity.

[0067] A further object of the present invention is a host cell containing the above-mentioned synthetic nucleic acid or expression vector, excluding human embryonic stem cells.

[0068] Those skilled in the art are familiar with the many methods of introducing nucleic acids or vectors into cells, particularly transfection.

[0069] The host cells are selected based on the intended use, preferentially eukaryotic cells, and preferably mammalian cells other than human embryonic stem cells.

[0070] In the examples presented in the section on in vitro experiments, the host cells selected are as follows: -HeLa cells, which are human cancer cells, -Mouse dendritic cells, specifically DC2.4 cells, and - Mouse myoblast cells, C2C12 cells.

[0071] These cells, with their diverse origins and functions, demonstrate that the synthetic nucleic acids according to the present invention can be efficiently translated into any cell type.

[0072] The host cells may be cells isolated in in vitro culture, or cells isolated from living multicellular organisms, excluding human embryonic stem cells.

[0073] The host cells may include primary human cells such as monocytes derived from blood, human mesenchymal stem cells, or primary cells derived from surgical waste, excluding human embryonic stem cells.

[0074] According to one embodiment of the present invention, the host cells may be primary human cells derived from the patient, excluding human embryonic stem cells.

[0075] The present invention also relates to a pharmaceutical composition or vaccine composition comprising a synthetic nucleic acid according to the present invention, and optionally one or more excipients and / or one or more adjuvants, in a suitable pharmaceutical carrier.

[0076] "Appropriate pharmaceutical carrier" refers to any carrier that is acceptable for use in the target, preferably in humans.

[0077] Pharmaceutical compositions or vaccine compositions are formulated for oral, topical, or parenteral administration to a target.

[0078] Vaccine compositions are intended for use as vaccines, that is, to induce an immune response to a specific antigen in a target. Vaccination may be prophylactic or therapeutic.

[0079] The term "excipient" refers to a substance other than the active pharmaceutical ingredient (in this specification, synthetic nucleic acid or expression vector) that has been evaluated as non-toxic.

[0080] Numerous excipients and / or carriers, such as water, buffer water, physiological saline, glycine solution and its derivatives, as well as agents necessary to reproduce physiological conditions, such as pH buffers and adjusters, surfactants, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc., can be used, and this list is not exhaustive. Furthermore, the pharmaceutical composition can be sterilized by sterilization techniques well known to those skilled in the art.

[0081] An "adjuvant" refers to a substance that, when administered to a target (in the form of synthetic nucleic acid or expression vector), can induce and / or enhance an immune response to an antigen.

[0082] The present invention also relates to synthetic nucleic acids for use as pharmaceuticals. According to this embodiment, synthetic nucleic acids are administered to subjects, particularly humans, to treat symptoms or diseases.

[0083] More specifically, if the target protein encoded by the synthetic nucleic acid is a BMP protein, the present invention relates to such synthetic nucleic acids for use in the treatment or prevention of bone or cartilage disorders.

[0084] These conditions include osteoarthritis, rheumatoid arthritis, osteoporosis, osteomalacia, bone dystrophy, and bone cancers such as osteosarcoma.

[0085] Other medical conditions, such as hemophilia, lysosomal storage diseases, and cystic fibrosis, can be treated with such synthetic nucleic acids.

[0086] The target protein may also be an antigen. In this case, the present invention comprises a synthetic nucleic acid including an open reading phase that encodes the antigen. A vaccine composition containing the nucleic acid is used for vaccinating a subject at risk (human or animal).

[0087] The present invention also provides a host cell incorporating the nucleic acid defined above, from this nucleic acid. This relates to the in vitro use of synthetic nucleic acids as described above for increasing and / or extending the translation of a target protein. [Examples]

[0088] Materials and methods 1) Nucleotide sequences of the UTR and poly(A) elements used The sequences used are shown in Table 1.

[0089] [Table 1]

[0090] 2) Cloning of UTR sequences and polyadenylated sequences The UTR sequence was obtained from GENSCRIPT, and restriction sites were added to the 5' and 3' ends. These sequences were inserted into a plasmid containing the T7 promoter for in vitro mRNA production by conventional cloning.

[0091] The poly(A) (SEQ ID NOs. 8, 10, 11) and poly(AG) (SEQ ID NOs. 9) sequences were obtained from GENSCRIPT. They contain restriction sites at 5' and 3'. These sequences were inserted after the 3' UTR sequence by conventional cloning. These techniques are (Sa mbrook, Joseph.Molecular Cloning: a Laboratory Manual.Cold Spring Harbor,NY:Cold This is described in Spring Harbor Laboratory Press, 2001. The UTR and polyadenylated sequences were obtained from Genscript.

[0092] 3) Stability of polyadenylated sequences in bacterial transformants Colonies isolated from the Top 10 Escherichia coli strains (Thermo Fisher) transformed with plasmids containing a poly(A) tail (SEQ ID NO: 8), A120 tail (SEQ ID NO: 11), A110 tail (SEQ ID NO: 10), or AG tail (SEQ ID NO: 9) were subjected to 10 consecutive passages. Plasmid DNA from the 10th passage colony was isolated using the Nucleospin® plasmid kit (Macherey Nagel). The DNA was sequenced using Eurofins.

[0093] 4) In vitro transcription (IVT) For in vitro transcription, the plasmid was linearized with restriction enzymes that cleaved after the polyadenylated sequence. A High-Yield T7 RNA Synthesis Kit (Jena Bioscience) was used for IVT. The reaction conditions are shown in Table 3. IVT was performed using two cap analogs: ARCA (Anti Reverse Cap Analogue) or Cleancap® analogue (Trilink Biotechnologies). The IVT reaction mixture was incubated at 37°C for 2 hours.

[0094] [Table 2]

[0095] After the IVTG reaction, template DNA was removed using Turbo(trademark) DNAse (ThermoFisher) according to the supplier's protocol.

[0096] Next, linear plasmids were purified using kits (e.g., Gel & PCR Cleanup (Macherey Nagel), Monarch® RNA Cleanup Kit (New England Biolabs)). The linear fragments were then used for in vitro transcription using kits distributed by either NEW ENGLAND BIOLABS (hiScribe) or THERMOPISHER (mMessage mMachine). These kits also specify procedures for purifying and quality control of the resulting mRNA.

[0097] 5)Cell culture mRNA expression was evaluated in the following cell models. -HeLa human cancer cells, available from ATCC (https: / / www.atcc.org / products / ccl-2) -DC2.4 mouse dendritic cells, available from Merck (https: / / www.merckmillipore.com / FR / fr / product / DC2.4-Mo use-Dendritic-Cell-Line,MM_NF-SCC142), - Mouse myoblast cells, C2C12 cells, are available from ATCC (https: / / www.atcc.org / products / crl-1772), and -Monocyte-derived human dendritic cells (MoDCs) were obtained from blood samples of Etablissement Francais du Sang according to the method described below (Linares-Fernandez S et al., Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA, Mol Ther Nucleic Acids. 2021 Dec 3:26:945-956). After isolating monocytes, they were differentiated into MoDCs for 6 days using interleukin-4 (62.5 ng / mL) and granulocyte-macrophage colony-stimulating factor (75 ng / mL). The acquisition of MoDC CD45+ / CD14- / CD209+ moDCs was confirmed by flow cytometry.

[0098] 6) Cell culture The cells were cultured in the culture media recommended by the suppliers. These media and their additives were obtained from MERCK and LONZA.

[0099] HeLa cells were supplemented with 10% decomplemented fetal bovine serum and cultured in Minimum Eagle's Medium (MEM) containing 100 U / mL penicillin and 100 μg / mL streptomycin (Fischer Bioblock, Illkirch, France).

[0100] DC2.4 cells were supplemented with 10% decomplemented fetal bovine serum and cultured in RPMI 1640 medium (Roswell Park Memory Institute) containing 100 U / mL penicillin and 100 μg / mL streptomycin (Fischer Bioblock, Illkirch, France).

[0101] C2C12 cells were supplemented with 10% decomplemented fetal bovine serum and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 100 U / mL penicillin and 100 μg / mL streptomycin (Fischer Bioblock, Illkirch, France).

[0102] 7) Transfection, which involves introducing mRNA into cells. Cells were cultured in 96-well or 24-well plates at a cell density that would allow for 70–80% confluence at the time of transfection, 24 hours prior to transfection.

[0103] Transfection was performed using 200 ng of mRNA per well in a 96-well plate and 1 μg of mRNA per well in a 24-well plate.

[0104] The mRNA was complexed using the commercially available vector Lipofectamine messengerMAX®, distributed by ThermoFisher, according to the supplier's protocol (https: / / www.thermofisher.com / fr / fr / home / life-science / cell-culture / transfection / transfection-reagents / lipofectamine-messengermax-reagent.html).

[0105] 8) Measurement of nanoluciferase reporter gene expression The expression of the nanoluciferase reporter gene was measured using the Nano-Glo® Luciferase Assay System kit provided by PROMEGA (https: / / france.promega.com / products / luciferase-assays / reporter-assays / nano_glo-luciferase-assay-system / ?catNum=N1110).

[0106] Bioluminescence was measured using the IVIS Lumina LT bioimager (Perkin Elmer).

[0107] 9) Reverse transcription and quantitative PCR Total RNA was extracted using Trizol® (ThermoFisher) and converted to cDNA using the LunaScript® RT SuperMix kit (New England Biolabs). For qPCR, the Luna qPCR Master mix (New England Biolabs) and the LightCycler® 480 PCR system (Roche) were used.

[0108] 10) Cytotoxicity test Cell viability after transfection with different mRNAs formulated with Lipofectamine messengerMAX™ was evaluated using the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the procedure described below (Perche F et al., Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA, Nanomedicine. 2011 Aug;7(4):445-53.). Cell viability was normalized by the viability of untransfected cells.

[0109] Example 1. Translational dynamics of mRNA introduced into cells Different cell types were transfected with mRNA constructs encoding nanoluciferase reporter genes adjacent to different combinations of UTR untranslated elements.

[0110] The following cell models were used. -DC2.4 cells, mouse dendritic cells. - HeLa cells, human cancer cells. -C2C12 cells, mouse myoblasts.

[0111] The following were the UTR untranslated elements: - Three 3'UTR elements: 3'UTR mtRNR1-AES (sequence number 3), abbreviated as MT; 3'UTR MnSOD (sequence number 2), abbreviated as MnSOD; and 3'UTR derived from rotavirus (sequence number 1), abbreviated as Rota. -Two 5'UTR elements: the 5'UTR of beta-globin, abbreviated as 5'β (sequence number 4), and 5'NeoUTR3 (sequence number 5).

[0112] Six combinations were tested, and the results are shown in Figures 1A, 1B, and 1C.

[0113] In HeLa cells (Figure 1A), the two 3'UTR Rota and MnSOD sequences enable better expression than standard 3'UTR MTs.

[0114] Figure 1B shows that the 5'β-3'MnSOD combination provides better expression in DC2.4 cells than the 5'β-3'MT and 5'β-3'Rota combination.

[0115] No increase in expression was observed in C2C12 cells at 96 hours. Nevertheless, the results obtained at 6 hours favor the 5'β-3'MnSOD combination. [Figure 1C]

[0116] The 5'UTRNeo3 element appears to be less effective than 5'β for all three cell types.

[0117] Example 2. Translation of mRNA into protein The relative expression of mRNAs with different combinations of 3'UTR and 5'UTR elements was compared with that of "standard" mRNA containing 5'β and 3'MT elements.

[0118] The results obtained from HeLa cells are summarized in Table 2 below.

[0119] [Table 3]

[0120] 3'UTR MnSOD resulted in a 5-fold increase in expression in HeLa cells within 6 hours and a more than 4-fold increase within 24 hours. 3'UTR Rota resulted in nearly a 2-fold increase in mRNA expression compared to the standard combination (Figure 2A).

[0121] In DC2.4 cells, 3'UTR MnSOD increased nanoluciferase expression fivefold 6 hours after transfection and more than twice as much 24 hours after transfection in HeLa cells. The 3'UTR Rota element enabled only about a 1.5-fold increase in these DC2.4 cells 6 hours after transfection, and slightly less at 96 hours after transfection (Figure 2B).

[0122] No significant difference was measured in C2C12 cells 96 hours after transfection (Figure 2C). Nevertheless, a transient effect of increased translation was observed at 6 hours with the 3'Rota and 3'MnSOD elements. The inventors are testing several hypotheses to explain this phenomenon.

[0123] The 5'UTRNeo3 element appears to be less effective than the 5' element for all three cell types.

[0124] Example 3: Stability and Expression Stability of polyadenylated tails The inventors were the first to demonstrate that poly(A / G) tails are stable on template plasmids. (Figure 3). In fact, after 10 consecutive transplants, 100% of the bacterial clones had intact A / G tails. This percentage was 95% for clones with the A110 construct disclosed in U.S. Patent Application Publication 2020 / 0392518. The inventors confirmed the instability of adenosine-only tails. Only 48% of A120 clones had intact tails.

[0125] Expression dynamics of various mRNAs according to the present invention The 3'UTR sequences MnSOD (MnSOD) and VP6 (Rota), combined with 5'UTRα-globin (α), enabled better mRNA expression in C2C12 cells than the reference sequence AES-mtRNR1 (AES) (Figures 4 and 5). In C2C12 cells, the 3'UTR sequences MnSOD (MnSOD) and VP6 (Rota), when combined with UTR4 (4) or UTR3 (3), enabled expression equivalent to or better than that of the AES sequence.

[0126] In HeLa cells, the 3'UTR sequences MnSOD (MnSOD) and VP6 (Rota), when combined with the 5'UTR α-globin (α), UTR4 (4), or UTR3 (3), enabled higher expression than the AES sequence (Figures 6 and 7). The inventors demonstrated that this difference is consistent with ARCA or Cleancap AG caps (Figure 8).

[0127] In DC2.4 cells, the 3'UTR sequences MnSOD (MnSOD) and VP6 (Rota), when combined with 5'UTR α-globin (α) or UTR3 (3), enabled expression equivalent to that of the AES sequence (AES) (Figures 9 and 10). The combination of 5'UTR4 / 3'UTR MnSOD (4-NL-MnSOD) was ineffective in DC2.4 cells. The inventors demonstrated that this equivalence is valid for ARCA or Cleancap AG caps (Figure 11).

[0128] In human primary monocyte-derived dendritic cells (MoDCs), the 3'UTR sequences MnSOD (MnSOD) and VP6 (Rota), when combined with the 5'UTR α-globin (α), UTR4 (4), or UTR3 (3), enabled expression equivalent to that of the AES sequence (AES) (Figure 12).

[0129] The inventors have shown that in MoDC, the combination of 5'UTRα-globin-3'UTR AES (α-NL-AES) is equivalent to the combination of 5'UTRα-globin-3'UTR MnSOD (α-NL-MnSOD) or 5'UTRα-globin-3'UTR VP6 (α-NL-Rota), regardless of whether an ARCA cap or a Cleancap AG cap is used (Figure 13). ARCA cap analogs are described in (Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG. RNA 7:1486-1495). Clean Cap analogues are an invention of Trilink Technologies (https: / / www.trilinkbiotech.com / legal-notices).

[0130] Intracellular mRNA stability The 3'UTR sequence VP6 increased the intracellular stability of mRNA with 5'UTR3 relative to the reference 3'UTR in both HeLa cells (Figure 14A) and DC2.4 cells (Figure 14B).

[0131] Immunogenicity of the 3'UTR sequence The 3'UTR sequences MnSOD or VP6 did not result in greater immunogenicity than the reference 3'UTR sequence in DC2.4 cells (Figure 15).

[0132] Other expression dynamics mRNA containing 5'UTRα-globin and 3'UTR AES-mtRNR1 combined with an AG tail showed better expression in DC2.4 cells than this same combination with an A110 tail (Figure 16).

[0133] mRNA containing 5'UTRα-globin and 3'UTR AES-mtRNR1 combined with an AG tail resulted in better expression in HeLa cells than this same combination with an A110 tail (Figure 17).

[0134] The inventors also evaluated mRNA expression using various 3'UTR sequences that were not combined with a 5'UTR sequence. The 3'UTR sequences MnSOD and VP6 enabled expression in both HeLa cells (Figure 18) and DC2.4 cells (Figure 19) that was at least equivalent to that obtained using mRNA containing the 3'UTR sequence AES.

[0135] References European Patent No. 3494982 U.S. Patent Application Publication No. 20200066375 International Publication No. 2014186334 European Patent No. 0737750 U.S. Patent Application Publication No. 20200392518 Zhu Y,Zhu L,Wang X,Jin H.RNA-based therapeutics:an overview and prospectus.Cell Death Dis.2022 Jul 23;13(7):644. Lamb YN.BNT162b2 mRNA COVID-19 Vaccine:First Approval.Drugs.2021 Mar;81(4):495-501. Baden LR,El Sahly HM,Essink B,Kotloff K,Frey S,Novak R,Diemert D,Spector SA,Rouphael N,Creech CB,McGettigan J,Khetan S,Segall N,Solis J,Brosz A,Fierro C,Schwartz H,Neuzil K,Corey L,Gilbert P,Janes H,Follmann D,Marovich M,Mascola J,Polakowski L,Ledgerwood J,Graham BS,Bennett H,Pajon R,Knightly C,Leav B,Deng W,Zhou H,Han S,Ivarsson M,Miller J,Zaks T;COVE Study Group.Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine.N Engl J Med.2021 Feb 4;384(5):403-416。 Uchida S,Perche F,Pichon C,Cabral H.Nanomedicine-Based Approaches for mRNA Delivery.Mol Pharm.2020 Oct 5;17(10):3654-3684。 Babendure JR,Babendure JL,Ding JH,Tsien RY.Control of mammalian translation by mRNA structure near caps.RNA.2006 May;12(5):851-61。 Cao J,Novoa EM,Zhang Z,Chen WCW,Liu D,Choi GCG,Wong ASL,Wehrspaun C,Kellis M,Lu TK.High-throughput 5’UTR engineering for enhanced protein production in non-viral gene therapies.Nat Commun.2021 Jul 6;12(1):4138。 Linares-Fernandez S,Moreno J,Lambert E,Mercier-Gouy P,Vachez L,Verrier B,Exposito JY.Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA.Mol Ther Nucleic Acids.2021 Dec 3;26:945-956。 Orlandini von Niessen AG,Poleganov MA,Rechner C,Plaschke A,Kranz LM,Fesser S,Diken M,Lower M,Vallazza B,Beissert T,Bukur V,Kuhn AN,Tureci o,Sahin U.Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3’ UTRs Identified by Cellular Library Screening.Mol Ther.2019 Apr 10;27(4):824-836。 Yang AD,Barro M,Gorziglia MI,Patton JT.Translation enhancer in the 3’-untranslated region of rotavirus gene 6 mRNA promotes expression of the major capsid protein VP6.Arch Virol.2004 Feb;149(2):303-21。 Chung DJ,Wright AE,Clerch LB.The 3’untranslated region of manganese superoxide dismutase RNA contains a translational enhancer element.Biochemistry.1998 Nov 17;37(46):16298-306。 Xia X.Detailed Dissection and Critical Evaluation of the Pfizer / BioNTech and Moderna mRNA Vaccines.Vaccines(Basel).2021 Jul 3;9(7):734。 Sambrook,Joseph.Molecular Cloning:a Laboratory Manual.Cold Spring Harbor,N.Y.:Cold Spring Harbor Laboratory Press,2001。 Linares-Fernandez S et al,Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA,Mol Ther Nucleic Acids.2021 Dec 3:26:945-956。 Perche F et al,Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA,Nanomedicine.2011 Aug;7(4):445-53。 Stepinski J,Waddell C,Stolarski R,Darzynkiewicz E,Rhoads RE(2001)Synthesis and properties of mRNAs containing the novel「anti-reverse」cap analogs 7-methyl(3’-O-methyl)GpppG and 7-methyl(3’deoxy)GpppG.RNA 7:1486-1495

Claims

1. In the 5'-3' direction, the following elements: a) At least one 5'UTR untranslated element selected from the 5'UTR of the human beta-globin gene (SEQ ID NO: 4), synthetic element 5'NeoUTR3 (SEQ ID NO: 5), the 5'UTR of the human alpha-globin gene (SEQ ID NO: 6), and synthetic element 5'UTR4 (SEQ ID NO: 7), b) Open Reading Frame (ORF) and, c) A synthetic nucleic acid comprising at least one 3'UTR untranslated element selected from the 3'UTR element of the rotavirus VP6 gene (SEQ ID NO: 1) and the 3'UTR element of the manganese superoxide dismutase (MnSOD) gene.

2. The synthetic nucleic acid according to claim 1, characterized in that the 3'UTR element of the MnSOD gene is derived from a rat gene (SEQ ID NO: 2) or a human gene.

3. The synthetic nucleic acid according to any one of claim 1 or 2, characterized in that the nucleic acid is RNA, particularly messenger RNA.

4. The synthetic nucleic acid according to any one of claims 1 to 3, characterized in that the nucleic acid further comprises an ARCA, Cap0, Cap1, or Cap2 type cap at the 5' position.

5. The synthetic nucleic acid according to any one of claims 1 to 4, characterized in that the nucleic acid further comprises a poly(A) or poly(AG) tail, preferably a poly(AG) tail containing at least one G nucleotide base, at 3'.

6. The synthetic nucleic acid according to any one of claims 1 to 5, characterized in that the ORF encodes a target protein, i.e., a BMP protein, preferably BMP2.

7. An expression vector comprising a synthetic nucleic acid according to any one of claims 1 to 6.

8. A host cell comprising, excluding human embryonic stem cells, the synthetic nucleic acid described in any one of claims 1 to 6 or the expression vector described in claim 7.

9. A pharmaceutical composition or vaccine composition comprising a synthetic nucleic acid according to any one of claims 1 to 6 in a suitable pharmaceutical carrier, and optionally one or more excipients and / or one or more adjuvants.

10. A synthetic nucleic acid according to any one of claims 1 to 6 for use as a drug.

11. The synthetic nucleic acid according to claim 6, for use in the treatment or prevention of bone diseases.

12. In vitro use of the synthetic nucleic acid according to any one of claims 1 to 6 for increasing and / or extending the translation of a target protein from the nucleic acid in a host cell into which the nucleic acid according to claim 8 is incorporated.

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