Nucleic acid molecule

Abstract

The present disclosure relates to a nucleic acid molecule comprising a 5 '-UTR and / or 3'-UTR sequence that yields a high level of translation. Aspects of the disclosure further relate to nucleic acid molecules suitable for use as vaccines for the treatment and prevention of infectious diseases, including those caused by coronavirus, compositions comprising the nucleic acid molecules, and methods of treating or preventing infectious diseases.

Description

Technical Field

[0001] This disclosure relates to a nucleic acid molecule comprising 5'-UTR and / or 3'-UTR sequences that produce high levels of translation. Aspects of this disclosure further relate to nucleic acid molecules suitable for use as vaccines for the treatment and prevention of infectious diseases, including those caused by coronaviruses. Background Technology

[0002] Nucleic acid vectors have a variety of uses in the treatment and research of human diseases. In molecular biology, nucleic acid vectors can be used as a assay to introduce disease-related genes or proteins of interest into cell or animal models. Furthermore, numerous therapeutic settings exist where the expression of nucleic acid sequences is desirable, including the expression of functional proteins to treat diseases caused by the deficiency of said proteins or as nucleic acid-based vaccines. Nucleic acid vectors typically contain: a simple structure consisting of coding sequences that encode peptides or proteins of interest; 5' and 3' untranslated regions (UTRs) that contribute to the translation and stability of the vector; and polyadenylation signals that protect the vector from enzymatic degradation and play a crucial role in translation. The components of nucleic acid vectors can be modified to treat or mimic specific diseases.

[0003] The recent success of nucleic acid vaccines during the COVID-19 pandemic has rekindled interest in the field of nucleic acid-based therapies, particularly those for expressing heterologous proteins of interest. For example, interest has now shifted to pan-mRNA vaccines, in which antigens from multiple disease-associated pathogens are simultaneously administered to provide pan-immunity against a variety of seasonal pathogens (e.g., Clinical Trial NCT05596S734, which evaluates mRNA vaccine candidates against COVID-19 and influenza).

[0004] Improvements to nucleic acid vectors will greatly facilitate the development of pan-vaccines. For example, maximizing or improving the translation efficiency of a vector could achieve a high neutralizing response against antigens at lower mRNA dose levels. This would enable the simultaneous administration of multiple vectors encoding multiple antigens without exceeding the recommended mRNA dose for each vaccination. Furthermore, improved translation efficiency could reduce the costs associated with the production of mRNA therapeutics by achieving effective responses at lower dose levels. This advantage would apply to any nucleic acid-based expression-based biotherapy (e.g., antibodies expressed in vivo, wild-type proteins, cancer neoantigens, and conventional antipathogenic vaccines). Summary of the Invention

[0005] This disclosure relates to a nucleic acid molecule comprising at least one coding sequence side-joined and operably linked to a 5'-UTR and / or a 3'-UTR. The UTR is engineered to enhance the translation of a protein of interest encoded by the coding sequence. One or more coding sequences may encode one or more peptides or fragments thereof of an infectious agent (such as a receptor-binding domain or spike protein from one or more coronavirus variants). The molecule may also comprise coding sequences for polymerizing units, enabling the formation of a multimeric complex upon assembly.

[0006] Some examples of this disclosure are summarized below. This list is merely exemplary and not an exhaustive list of all examples provided in this disclosure.

[0007] 1. A nucleic acid molecule comprising a 5' untranslated region (5'-UTR), a coding sequence, and a 3' untranslated region (3'-UTR), wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein...

[0008] (i) The 5'-UTR contains a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human citrate synthase (CS).

[0009] (ii) The 5'-UTR contains a sequence of the 5'-UTR derived from the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains a sequence of the 3'-UTR derived from human chitinase-1 (CHIT1);

[0010] (iii) The 5'-UTR contains a sequence of the 5'-UTR derived from the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains a sequence of the 3'-UTR derived from human citrate synthase (CS);

[0011] (iv) The 5'-UTR contains a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1), wherein the coding sequence is not derived from human chitinase-1 (CHIT1).

[0012] (v) The 5'-UTR contains a sequence derived from the 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human citrate synthase (CS); or

[0013] (vi) The 5'-UTR contains a sequence derived from the 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1).

[0014] 2. The nucleic acid molecule according to Clause 1, wherein the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, and (i) the 5'-UTR contains the sequence of SEQ ID NO:1 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (ii) the 5'-UTR contains the sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (iii) the 5'-UTR contains the sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:11. (iv) The 5'-UTR contains the sequence of SEQ ID NO: 9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (v) The 5'-UTR contains the sequence of SEQ ID NO: 5 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO: 9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; or (vi) The 5'-UTR contains the sequence of SEQ ID NO: 5 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO: 9. The sequence of NO:11 or a sequence that has at least 80%, 85%, 90% or 95% identity with it.

[0015] 3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a ribonucleic acid molecule (RNA), and (i) the 5'-UTR contains the sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (ii) the 5'-UTR contains the sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (iii) the 5'-UTR contains the sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10. (iv) The 5'-UTR contains the sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (v) The 5'-UTR contains the sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; or (vi) The 5'-UTR contains the sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:10. The sequence of NO:12 or a sequence that has at least 80%, 85%, 90% or 95% identity with it.

[0016] 4. The nucleic acid molecule according to Clause 1 or Clause 3, wherein the 5'-UTR comprises or is composed of the sequence of SEQ ID NO:2, and the 3'-UTR comprises or is composed of the nucleic acid sequence of SEQ ID NO:10.

[0017] 5. The molecule according to any of the preceding clauses, wherein the coding sequence comprises a sequence encoding a therapeutic protein or peptide.

[0018] 6. The molecule according to Clause 5, wherein the therapeutic protein or peptide is a wild-type sequence of a human protein.

[0019] 7. The molecule according to Clause 5 or Clause 6, wherein the therapeutic protein or peptide is an antibody or an antigen-binding fragment thereof.

[0020] 8. The molecule according to Clause 5, wherein the therapeutic protein or peptide comprises a disease-associated antigen (DAA).

[0021] 9. The molecule according to Clause 8, wherein the disease-associated antigen is a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0022] 10. The molecule according to Clause 9, wherein the tumor-associated antigen is not expressed in normal tissues or is mutated in tumor cells.

[0023] 11. The molecule according to any one of clauses 1 and 3 to 10, wherein the nucleic acid molecule is mRNA.

[0024] 12. The molecule according to any one of clauses 8 to 11, wherein the coding sequence further encodes a multipolymerization unit (MU).

[0025] 13. The molecule according to Clause 12, wherein the polymerizing unit (MU) is ferritin.

[0026] 14. The molecule according to Clause 12 or Clause 13, wherein the coding sequence further encodes a linker—optionally a glycine-serine linker.

[0027] 15. The molecule according to Clause 14, wherein the linker is encoded between the DAA and the MU to encode a DAA-MU fusion protein.

[0028] 16. The molecule according to any one of clauses 9 to 15, wherein the viral antigen is a coronavirus antigen.

[0029] 17. The molecule according to Clause 16, wherein the coronavirus is selected from SARS-CoV-1 and / or SARS-CoV-2.

[0030] 18. The molecule according to Clause 16 or Clause 17, wherein the coronavirus antigen is a coronavirus spike (S) protein or an antigenic fragment thereof.

[0031] 19. The molecule according to Clause 18, wherein the S protein is stable in its pre-fusion conformation.

[0032] 20. The molecule according to Clause 18 or Clause 19, wherein the S protein comprises K986P and V987P mutations.

[0033] 21. The molecule as described in Clause 18, wherein the antigen fragment thereof is a receptor-binding domain (RBD).

[0034] 22. A composition comprising a first nucleic acid carrier comprising a molecule according to any one of claims 11 to 21, wherein the disease-associated antigen is a Wuhan variant spike (S) protein or a Delta variant spike (S) protein.

[0035] 23. The composition according to Clause 22, wherein the first nucleic acid vector comprises the antigen-linker-ferritin sequence shown in SEQ ID NO:23 or SEQ ID NO:25.

[0036] 24. The composition according to any one of Clauses 22 to 23, wherein the composition further comprises a second nucleic acid vector comprising a molecule according to any one of Clauses 11 to 21, wherein the disease-associated antigen is an Omicron variant spike (S) protein, optionally variant BA.2, BA.4 / 5, or XBB.1.5.

[0037] 25. The composition according to any one of clauses 22 to 24, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises a polyadenylated sequence comprising 60 to 100 adenine nucleotides.

[0038] 26. The composition according to any one of clauses 22 to 25, wherein the first nucleic acid carrier and / or the second nucleic acid carrier comprises N1-methylpseudouridine at 80% to 100% of the uridine sites.

[0039] 27. The composition according to any one of clauses 22 to 26, wherein the first nucleic acid vector and / or nucleic acid vector comprises a 5'-cap structure, optionally a cap 1 structure.

[0040] 28. The composition according to any one of clauses 22 to 27, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence shown in SEQ ID NO:19 and the 3'-UTR sequence shown in SEQ ID NO:21.

[0041] 29. The composition according to any one of claims 24 to 28, wherein the second nucleic acid vector comprises the antigen-linker-ferritin sequence shown in SEQ ID NO:27.

[0042] 30. The composition according to any one of claims 24 to 28, wherein the second nucleic acid vector comprises the antigen-linker-ferritin sequence shown in SEQ ID NO:42.

[0043] 31. A composition comprising a nucleic acid vector comprising a molecule according to any one of claims 11 to 21, wherein the disease-associated antigen is an Omicron variant spike (S) protein, optionally variant BA.2, BA.4 / 5, or XBB.1.5.

[0044] 32. The composition according to claim 31, wherein the first nucleic acid vector comprises the antigen-linker-ferritin sequence shown in SEQ ID NO:27 or SEQ ID NO:42.

[0045] 33. The composition according to any one of claims 31 to 32, wherein the nucleic acid vector comprises a polyadenylated sequence comprising 60 to 100 adenine nucleotides.

[0046] 34. The composition according to any one of clauses 31 to 33, wherein the nucleic acid vector comprises N1-methylpseudouridine at 80% to 100% of the uridine sites.

[0047] 35. The composition according to any one of clauses 31 to 34, wherein the nucleic acid vector comprises a 5'-cap structure, optionally a cap 1 structure.

[0048] 36. The composition according to any one of clauses 31 to 35, wherein the nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence shown in SEQ ID NO:19 and the 3'-UTR sequence shown in SEQ ID NO:21.

[0049] 37. The molecule or composition according to any one of clauses 11 to 36, wherein the nucleic acid carrier is formulated in lipid nanoparticles (LNPs).

[0050] 38. The composition or nucleic acid molecule according to any of the preceding clauses, wherein the composition or nucleic acid molecule is used in a pharmaceutical product.

[0051] 39. A vaccine comprising a nucleic acid molecule or composition according to any of the preceding clauses.

[0052] 40. The composition or vaccine according to any one of clauses 22 to 39, in a method of preventing and / or treating an infectious disease, optionally a disease caused by a coronavirus.

[0053] 41. A method for preventing and / or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of a composition or vaccine according to any one of clauses 22 to 39, wherein the infectious disease is optionally a disease caused by a coronavirus.

[0054] 42. A method of vaccinating a subject against an infectious disease, optionally a disease caused by a coronavirus, said method comprising administering an effective amount of a composition or vaccine according to any one of clauses 22 to 39.

[0055] 43. A nucleic acid vector comprising a 5' cap 1 structure, a 5'-UTR sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), a coding sequence encoding a Wuhan variant spike (S) protein fused with a ferritin sequence or a Delta variant spike (S) protein fused with a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylated sequence comprising 70 to 90 adenine nucleotides.

[0056] 44. A nucleic acid vector comprising a 5' cap 1 structure, a 5'-UTR sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), a coding sequence encoding the spike (S) protein of an Omicron variant BA.2, BA.4 / 5, or XBB.1.5 fused to a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylated sequence comprising 70 to 90 adenine nucleotides.

[0057] 45. A composition comprising the nucleic acid vector according to clause 43 and the nucleic acid vector according to clause 44.

[0058] 46. ​​A nucleic acid vector comprising the sequence shown in SEQ ID NO:36.

[0059] 47. A nucleic acid vector comprising the sequence shown in SEQ ID NO:37.

[0060] 48. A nucleic acid vector comprising the sequence shown in SEQ ID NO:38.

[0061] 49. A nucleic acid vector comprising the sequence shown in SEQ ID NO:43.

[0062] 50. A composition comprising a carrier according to clause 46 or clause 47 and a carrier according to clause 48 or clause 49.

[0063] 51. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:39.

[0064] 52. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:40.

[0065] 53. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:41.

[0066] 54. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:44.

[0067] 55. The nucleic acid vector according to any one of clauses 51 to 54, wherein the nucleic acid vector further comprises a 5'-UTR and a 3'-UTR as described in any one of clauses 1-4 or 28.

[0068] 56. The nucleic acid vector according to any one of clauses 51 to 55, wherein the nucleic acid vector is mRNA.

[0069] 57. The nucleic acid vector according to Clause 56, wherein the mRNA comprises any of the features described in Clauses 26 to 28.

[0070] 58. A composition comprising the carrier according to clause 51 and the carrier according to clause 52.

[0071] 59. A composition comprising a carrier according to clause 51 or clause 52 and a carrier according to clause 53 or clause 54. Attached Figure Description

[0072] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of this teaching in any way.

[0073] Figure 1This is a bar graph comparing the expression of eGFP-encoding mRNA (in A549 cells) with a constant 3'-UTR region (albumin) and candidate 5'-UTRs (UTRs -11, -37, -52, and -53 identified in the examples). Black bars represent vectors with a control 5'-UTR region (HSD17B4). The selected 5'-UTRs increased the fluorescence intensity of eGFP in A549 cells. Compared to the control 5'-UTR (HSD17B4), the candidate 5'-UTRs showed an ability to increase eGFP expression in A549 cells. The data represent three independent experiments with three to four technical replicates in each experiment.

[0074] Figure 2 This is a bar graph comparing the expression of eGFP-encoding mRNA (in HeLa cells) with a constant 3'-UTR region (albumin) and candidate 5'-UTRs (UTR-11, -37, -52, -53). Black bars represent vectors with control 3'-UTR (albumin) and control 5'-UTR (HSD17B4). Candidate 5'-UTRs showed an ability to increase eGFP expression in HeLa cells compared to control 5'-UTR (HSD17B4). Data represent 3 independent experiments with 3–4 technical replicates in each experiment.

[0075] Figure 3 This is a bar graph comparing the expression of eGFP mRNA (in A549 cells) with candidate 3'-UTR regions (UTR-3, UTR-4, and UTR-36 identified in the examples) and the control 5'-UTR region (HSD17B4). Black bars represent the control 3'-UTR (albumin) and 5'-UTR (HSD17B4). When compared to the control 3'-UTR, the candidate 3'-UTR increased eGFP mRNA expression in A549 cells. Data represent two independent experiments with three technical replicates in each experiment.

[0076] Figure 4 This is a bar graph comparing the expression of eGFP mRNA (in HeLa cells) with candidate 3'-UTRs (UTR-3, UTR-4, UTR-36) and control 5'-UTR (HSD17B4). Candidate 3'-UTRs increased eGFP mRNA expression in HeLa cells compared to the control 3'-UTR. Black bars represent the control 3'-UTR (albumin) and control 5'-UTR (HSD17B4). Data represent two independent experiments with three technical replicates in each experiment.

[0077] Figure 5This is a bar graph comparing the expression of eGFP mRNA (in A549 cells) with a combination of candidate 3'-UTRs (CHIT-1, CS) and 5'-UTRs (GOT1, PRKACB, CHIT1) with that of a control consisting of a control 3'-UTR (albumin) and a control 5'-UTR (HSD17B4). Black bars represent eGFP mRNA expression in the control. Combining candidate 3'-UTRs with candidate 5'-UTRs increases eGFP mRNA expression in A549 cells. Data represent three independent experiments with three technical replicates in each experiment.

[0078] Figure 6 This is a bar graph showing the expression of eGFP-encoded modified mRNAs (in A549 cells) with combinations of candidate 5' (GOT1, PRKACB, CHIT) and 3'-UTR (CHIT, CS) regions. Black bars represent the expression of eGFP-encoded modified mRNAs with control 5' (HSD17B4) and 3'-UTR (albumin) regions. The combination of candidate 5'-UTR and candidate 3'-UTR increased the expression of eGFP-encoded modified mRNAs compared to the expression of control 5' and 3'-UTR. Data represent three independent experiments with three technical replicates in each experiment.

[0079] Figure 7 Antibody expression in A549 cells with scFv-Fc-encoded mRNA having a combination of candidate or control 5'-UTR and 3'-UTR is shown. Black bars represent the expression of scFv-Fc-encoded mRNA with control 5'-UTR (HSD17B4) and control 3'-UTR (albumin). The combination of PRKACB(5'-UTR) / CHIT(3'-UTR) or CHIT(5'-UTR) / CS(3'-UTR) produces higher expression of scFv-Fc-encoded mRNA compared to the expression of control 5'-UTR and 3'-UTR regions. Data represent 3 independent experiments with 3 technical replicates in each experiment.

[0080] Figure 8Antibody expression in A549 cells is shown with scFv-Fc-encoded modified mRNA (in A549 cells) having a combination of candidate or control 5'-UTR and 3'-UTR. The modified mRNA contains modified uridine (5-methoxyuridine). Black bars represent the expression of scFv-Fc-encoded mRNA with control 5'-UTR (HSD17B4) and control 3'-UTR (albumin). Compared with the expression of control 5'-UTR and 3'-UTR regions, the combinations of PRKACB(5'-UTR) / CHIT(3'-UTR), PRKACB(5'-UTR) / CS(3'-UTR), CHIT(5'-UTR) / CHIT(3'-UTR), and CHIT(5'-UTR) / CS(3'-UTR) produced higher expression of scFv-Fc-encoded mRNA. Data represent 3 independent experiments with 3 technical replicates in each experiment.

[0081] Figure 9 shows that mRNAs with 5'-UTR CHIT1 and 3'-UTR CS UTR produced the highest levels of EGFP expression in both BHK-21 cells (Figure A) and HEK293 cells (Figure B) compared to two competing mRNA molecules. mRNA_AZ, mRNAcomp A, and mRNAcomp B all contained 100% pseudouridine (pseudo U). For comparative purposes, mRNA_AZ with 0% pseudouridine is included.

[0082] Figure 10 The improved panvariant immunogenicity of the mRNA molecule according to this disclosure in mice is shown, which encodes the Delta antigen fused to ferritin via a linker, compared to encoding the native spike.

[0083] Figure 11 The improved panvariant immunogenicity of the mRNA molecule according to this disclosure in non-human primates is shown, which encodes the Delta antigen fused to ferritin via a linker, compared to encoding the native spike. Detailed Implementation

[0084] All references mentioned are incorporated in full into this paper.

[0085] Benefiting from the teachings presented in the foregoing description and associated drawings, those skilled in the art to which this disclosure pertains will conceive of numerous modifications and other instances of the disclosure set forth herein. Therefore, it should be understood that this disclosure is not limited to the specific instances disclosed, and that modifications and other instances are intended to be included within the scope of the appended claims. Although specific terminology is used herein, it is used only in a general and descriptive sense and not for purposes of limitation.

[0086] Units, prefixes, and symbols may be represented in their International System of Units (SI) acceptable form. Unless otherwise specified, nucleic acids are written from left to right with a 5' to 3' orientation; amino acid sequences are written from left to right with the amino group to the carboxyl group, respectively. Numerical ranges include values ​​that define that range. Amino acids may be represented herein by commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be represented by commonly accepted single-letter codes. The terms defined below are defined more fully by referring to the entire specification.

[0087] Definition

[0088] The term “nucleic acid sequence” is intended to encompass polymers of DNA or RNA, namely polynucleotides, which may be single-stranded or double-stranded and may contain non-natural or altered nucleotides, such as modified uridines. As used herein, the terms “nucleic acid” and “polynucleotide” refer to polymeric forms of nucleotides of any length, ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and therefore include both double-stranded and single-stranded DNA as well as both double-stranded and single-stranded RNA. These terms include RNA or DNA analogs made from nucleotide analogs and modified polynucleotides (such as, but not limited to, methylated polynucleotides and / or capped polynucleotides) as equivalents. Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, although many other bonds (e.g., thiophosphates, boron phosphates, etc.) are known in the art.

[0089] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acids of any length. This polymer may be linear or branched, may include modified amino acids, and may be interrupted by non-amino acid components. These terms also cover polymers of naturally modified or intervened amino acids; such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeled component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art. It should be understood that because the polypeptides of this disclosure are antibody-based, in some respects, the polypeptide may exist as a single chain or an associated chain.

[0090] "Identity percentage" refers to the degree of identity between two sequences (e.g., nucleic acid sequences). The identity percentage can be determined by aligning two sequences, introducing gaps to maximize the identity between them. The identity percentage should generally be calculated between the same type of nucleic acid, i.e., for either a DNA or RNA sequence. Therefore, it should be understood that if a DNA sequence "corresponds" to an RNA sequence, or vice versa, in the first step, the RNA sequence is converted to the corresponding DNA sequence (particularly by replacing uracil (U) with thymidine (T) throughout the sequence), or vice versa, the DNA sequence is converted to the corresponding RNA sequence (particularly by replacing T with U throughout the sequence). Alignment can be performed using procedures known in the art. For the purposes of this document, nucleotide sequence alignment can be performed using the blastn (homology analysis) procedure with default parameter settings (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov). The identity between two sequences can be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region (including gaps). The identity score is counted only for perfect matches. Empty spaces at the end of the sequence are not included, while empty spaces inside are included in the length.

[0091] The “5'-untranslated region (5'-UTR)” has the commonly accepted meaning among those skilled in the art. It is a region of a nucleic acid molecule located at the 5' end of the coding sequence that is not translated into a protein. The 5'-UTR typically begins at the transcription start site and ends before the start codon of the coding sequence.

[0092] The “3'-untranslated region (3'-UTR)” has a commonly accepted meaning among those skilled in the art. It is a region of a nucleic acid molecule located at the 3' end of the coding sequence that is not translated into a protein. The 3'-UTR is typically the 3' end of the coding sequence. If the molecule contains a polyadenylation signal, the 3'-UTR is usually located between the coding sequence and the polyadenylation signal.

[0093] A “coding sequence” is a continuous extension of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). Coding sequences typically encode polypeptides. The coding sequences disclosed herein are operatively ligated to the 5' and 3' UTRs described herein.

[0094] "Merchant RNA (mRNA)" is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. Those skilled in the art will understand that, unless otherwise stated, the nucleic acid sequences described herein may be described as "T" in a representative DNA sequence, but where the sequence represents RNA (e.g., mRNA), "T" will be replaced by "U". Therefore, any DNA disclosed and identified herein by a specific sequence identifier also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" in the DNA sequence is replaced by "U".

[0095] A "nucleoside" is a compound containing a sugar molecule (such as a pentose or ribose) or a derivative thereof combined with an organic base (such as a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). Nucleic acids may contain one or more regions of linked nucleosides. Such regions may have variable backbone bonds. The bond can be a standard phosphodiester bond, in which case the nucleic acid will contain nucleotide regions ("nucleotide" refers to a nucleoside, including a phosphate group).

[0096] The “expression” of a nucleic acid sequence refers to one or more of the following events: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing RNA transcripts (e.g., by splicing, editing, 5' cap formation and / or 3' end processing); and (3) translating RNA into a polypeptide or protein.

[0097] The term "pharmaceutical composition" refers to a formulation in which the bioactivity of the active ingredient is effective and which contains no additional components that would have unacceptable toxicity to a subject to which the composition will be administered. The composition may be sterile.

[0098] As used herein, the terms “subject” and “patient” are used interchangeably. A subject can be an animal. In some respects, a subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate). In other respects, a subject is a human.

[0099] As used in this disclosure and claims, the singular forms “a,” “an,” and “the” include the plural forms, unless the context clearly specifies otherwise.

[0100] It should be understood that wherever the term "comprises" is used to describe aspects herein, other similar aspects described by "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprises," "containing," and "having" may mean "includes," etc.; "consisting essentially of" is open-ended and allows for the existence of more than those listed, provided that the essential or novel features of the listed ones are not altered by the existence of more than those listed, but excluding prior art aspects.

[0101] Unless otherwise specified or apparent from the context, the term “or” as used herein should be understood as inclusive. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include both “A and B”, “A or B”, and “A and B”. Similarly, the term “and / or” as used in phrases such as “A, B and / or C” is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0102] Any molecule, carrier, composition, use or method provided herein may be combined with one or more of any other molecule, carrier, composition, use or method provided herein.

[0103] Untranslated Region (UTR)

[0104] This disclosure provides a nucleic acid molecule comprising a 5' untranslated region (5'-UTR), a coding sequence, and a 3' untranslated region (3'-UTR), wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein...

[0105] (i) The 5'-UTR contains or is composed of a sequence derived from or composed of a 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains or is composed of a sequence derived from or composed of a 3'-UTR of human citrate synthase (CS).

[0106] (ii) The 5'-UTR contains or is composed of the 5'-UTR of the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains or is composed of the 3'-UTR of human chitinase-1 (CHIT1).

[0107] (iii) The 5'-UTR contains or is composed of the 5'-UTR of the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains or is composed of the 3'-UTR of human citrate synthase (CS).

[0108] (iv) The 5'-UTR contains or is composed of a sequence derived from or composed of a 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains or is composed of a sequence derived from or composed of a 3'-UTR of human chitinase-1 (CHIT1), wherein the coding sequence is not derived from human chitinase-1 (CHIT1).

[0109] (v) The 5'-UTR contains or is composed of a sequence derived from or consisting of the 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains or is composed of a sequence derived from or consisting of the 3'-UTR of human citrate synthase (CS); or

[0110] (vi) The 5'-UTR contains or is composed of a sequence derived from or composed of a 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains or is composed of a sequence derived from or composed of a 3'-UTR of human chitinase-1 (CHIT1).

[0111] The nucleic acid molecule disclosed herein is a nucleic acid vector.

[0112] The 5'-UTR and 3'-UTR sequences disclosed herein are particularly useful for increasing the translation level of coding sequences. Examples demonstrate that the combination of the 5'-UTR and 3'-UTR disclosed herein enhances the translation level of a variety of proteins of interest, including GFP and scFv-Fc (e.g., therapeutic proteins). Higher levels of translation are achieved compared to clinically validated UTR combinations derived from regulatory-approved mRNA vaccine products (e.g., see [link to relevant documentation]). Figure 10 Therefore, this disclosure provides nucleic acid molecules capable of enhancing the translation of proteins of interest, potentially paving the way for lower-dose regimens of mRNA vaccines as currently achievable in the art. This could enable the development of combination mRNA vaccines against multiple pathogens that can be administered as a single dose. Furthermore, the UTR combinations described herein can be preferentially used in conjunction with therapeutic proteins to be expressed in vivo, such as antibodies (or variants thereof) targeting intracellular targets. The UTRs disclosed herein can enhance the dynamic range of in vivo expression levels, thereby enabling the exploration of a wider dose range to identify the most effective dose.

[0113] The level of translation obtained using the UTR of this disclosure can be assessed by any appropriate assay available to a person skilled in the art, for example by measuring the concentration of the protein of interest encoded by the coding sequence, such as by detecting a labeled protein (such as GFP) or a therapeutic protein (such as scFv-Fc) or a vaccine antigen (such as the SARS-CoV-2 spike protein).

[0114] Compared to albumin-derived 5'-UTR sequences (optionally comprising or consisting of the sequence shown in SEQ ID NO:16 or the corresponding RNA sequence) and HSD17B4-derived 3'-UTR sequences (optionally comprising or consisting of the sequence shown in SEQ ID NO:17 or the corresponding RNA sequence), the 5'-UTR and 3'-UTR of this disclosure are capable of increasing the translation of coding sequences, optionally in HeLa cells or A549 cells.

[0115] In one instance, the 5'-UTR derived from CHIT1 does not contain a sequence containing ATG, and optionally, the 5'-UTR derived from CHIT1 does not contain a sequence consisting of ATGGGCTGCAGCCTGCCGCTGA (SEQ ID NO:35) or the corresponding RNA sequence.

[0116] This disclosure also provides a deoxyribonucleic acid (DNA) molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein...

[0117] (i) The 5'-UTR contains or consists of a sequence of SEQ ID NO:1 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it;

[0118] (ii) The 5'-UTR contains or is composed of a sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90% or 95% identity with it, and the 3'-UTR contains or is composed of a sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90% or 95% identity with it;

[0119] (iii) The 5'-UTR contains or is composed of a sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90% or 95% identity with it, and the 3'-UTR contains or is composed of a sequence of SEQ ID NO:9 or a sequence having at least 80%, 85%, 90% or 95% identity with it;

[0120] (iv) The 5'-UTR contains or consists of a sequence of SEQ ID NO:1 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it;

[0121] (v) The 5'-UTR contains or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 5, and the 3'-UTR contains or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 9; or

[0122] (vi) The 5'-UTR contains or consists of a sequence of SEQ ID NO:5 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it.

[0123] This disclosure also provides a ribonucleic acid (RNA) molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein...

[0124] (i) The 5'-UTR contains or is composed of a sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90% or 95% identity with it, and the 3'-UTR contains or is composed of a sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90% or 95% identity with it;

[0125] (ii) The 5'-UTR contains or consists of a sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it;

[0126] (iii) The 5'-UTR contains or consists of a sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it;

[0127] (iv) The 5'-UTR contains or consists of a sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it;

[0128] (v) The 5'-UTR contains or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 6, and the 3'-UTR contains or consists of a sequence having at least 80%, 85%, 90%, or 95% identity with SEQ ID NO: 10; or

[0129] (vi) The 5'-UTR contains or consists of a sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains or consists of a sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it.

[0130] In one example, the nucleic acid molecule of this disclosure comprises a 5'-UTR and a 3'-UTR, wherein the 5'-UTR comprises or consists of a sequence of SEQ ID NO:19 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR comprises or consists of a sequence of SEQ ID NO:21 or a sequence having at least 80%, 85%, 90%, or 95% identity with it. This disclosure also provides a nucleic acid molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR.

[0131] The 5'-UTR contains a sequence derived from the 5'-UTR of any one of human chitinase-1 (CHIT1), glucuronidase β (GUSB1), human protein kinase cAMP-activated catalytic subunit β (PRKACB), or aspartate aminotransferase 1 (GOT1), and / or the 3'-UTR contains a sequence derived from the 3'-UTR of any one of human chitinase-1 (CHIT1), pyruvate kinase L / R (PKLR), or human citrate synthase (CS).

[0132] The coding sequence is operatively linked to the 5'-UTR and 3'-UTR, and the coding sequence is not CHIT1, GUSB1, PRKACB, GOT1, PKLR or CS.

[0133] Examples show that, as evaluated according to this disclosure, these 5'-UTRs and / or these 3'UTRs can enhance the translation of encoded sequences.

[0134] This disclosure also provides a deoxyribonucleic acid (DNA) molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the 5'-UTR comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence having at least 80%, 85%, 90%, or 95% identity with such a sequence, and / or the 3'-UTR comprises a sequence selected from SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:12, or a sequence having at least 80%, 85%, 90%, or 95% identity with such a sequence.

[0135] This disclosure also provides a ribonucleic acid molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the 5'-UTR comprises a sequence selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8, or a sequence having at least 80%, 85%, 90%, or 95% identity with such a sequence, and / or the 3'-UTR comprises a sequence selected from SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14, or a sequence having at least 80%, 85%, 90%, or 95% identity with such a sequence.

[0136] In one instance, the 5'-UTR of this disclosure comprises a sequence having at least 80%, 85%, 90%, or 95% identity with a sequence selected from any one of SEQ ID NO:1 to 8, wherein when a constant 3'-UTR sequence is included in both molecules, the sequence is capable of increasing the translation of the coding sequence compared to a reference 5'-UTR—optionally HSD17B4 5'-UTR (SEQ ID NO:17), optionally in HeLa cells or A549 cells. A constant 3'-UTR sequence means the same 3'-UTR sequence paired with both the 5'-UTR of this disclosure and the reference 5'-UTR.

[0137] In one instance, the 3'-UTR of this disclosure comprises a sequence having at least 80%, 85%, 90%, or 95% identity with a sequence selected from any one of SEQ ID NO: 9 to 14, wherein when a constant 5'-UTR sequence is included in both molecules, the sequence is capable of increasing the translation of the coding sequence compared to a reference 3'-UTR—optionally the albumin 3'-UTR (SEQ ID NO: 16), optionally in HeLa cells or A549 cells. A constant 5'-UTR sequence means the same 5'-UTR sequence paired with both the 3'-UTR of this disclosure and the reference 3'-UTR.

[0138] In one example, the nucleic acid molecule of this disclosure comprises a 5'-UTR and a 3'-UTR, the 5'-UTR comprising a sequence or corresponding RNA sequence having at least 80%, 85%, 90%, or 95% identity with a sequence selected from SEQ ID NO:1, and the 3'-UTR comprising a sequence or corresponding RNA sequence having at least 80%, 85%, 90%, or 95% identity with a sequence selected from SEQ ID NO:9, wherein the 5'-UTR sequence and the 3'-UTR sequence are capable of increasing the translation of the coding sequence compared to translation of a nucleic acid molecule comprising a reference 5'-UTR (optionally HSD17B4 5'-UTR (SEQ ID NO:17, or corresponding RNA sequence)) and a reference 3'-UTR (optionally albumin 3'-UTR (SEQ ID NO:16, or corresponding RNA sequence)) operatively linked to the coding sequence, optionally in HeLa cells or A549 cells.

[0139] In one instance, the 5'-UTR consists of a sequence selected from any one of SEQ ID NO:1 to 8 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, wherein the sequence is capable of enhancing the translation of the encoded sequence, as assessed according to this disclosure, and / or the 3'-UTR consists of a sequence selected from any one of SEQ ID NO:9 to 14 or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with it, wherein the sequence is capable of enhancing the translation of the encoded sequence, as assessed according to this disclosure.

[0140] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the human chitinase-1 (CHIT1) 5'-UTR, optionally according to the sequence of SEQ ID NO:1 or SEQ ID NO:2, or a sequence having at least 80%, 85%, 90%, or 95% identity with it. Examples demonstrate that, as assessed according to this disclosure, the 5'-UTR is capable of enhancing the translation of the coding sequence.

[0141] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 3'-UTR comprises a sequence derived from the 3'UTR of human citrate synthase (CS), optionally according to the sequence of SEQ ID NO:9 or SEQ ID NO:10, or a sequence having at least 80%, 85%, 90%, or 95% identity with it. Examples demonstrate that, as assessed according to this disclosure, the 3'-UTR is capable of enhancing the translation of the coding sequence.

[0142] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to both the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the human chitinase 1 (CHIT1) 5'-UTR, and wherein the 3'-UTR comprises a sequence derived from the human citrate synthase (CS) 3'-UTR. Examples demonstrate that nucleic acid molecules containing these 5' and 3'-UTRs produce the highest translational levels of a variety of proteins of interest in multiple cell lines compared to clinically validated combinations of 5' and 3'-UTRs.

[0143] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the 5'UTR of the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, optionally according to the sequence of SEQ ID NO:3 or SEQ ID NO:4, or a sequence having at least 80%, 85%, 90%, or 95% identity with it, the sequence being capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0144] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 3'-UTR comprises a sequence derived from the human chitinase-1 (CHIT1) 3'-UTR, optionally according to the sequence of SEQ ID NO:11 or SEQ ID NO:12, or a sequence having at least 80%, 85%, 90%, or 95% identity with it, the sequence being capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0145] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the 5'-UTR of the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, which is capable of increasing the expression of the coding sequence, as assessed according to this disclosure, and wherein the 3'-UTR comprises a sequence derived from the 3'-UTR of chitinase-1 (CHIT1), optionally according to the sequence of SEQ ID NO:5, which is capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0146] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the 5'-UTR of aspartate aminotransferase 1 (GOT1), optionally according to the sequence of SEQ ID NO:5 or SEQ ID NO:6, or a sequence having at least 80%, 85%, 90%, or 95% identity with it, the sequence being capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0147] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from the 5'-UTR of glucuronidase β (GUSB1), optionally according to the sequence of SEQ ID NO:7 or SEQ ID NO:8, or a sequence having at least 80%, 85%, 90%, or 95% identity with it, the sequence being capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0148] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein the 3'-UTR comprises a sequence derived from a pyruvate kinase L / R (PKLR) sequence, optionally according to the sequence of SEQ ID NO:13 or SEQ ID NO:14, or a sequence having at least 80%, 85%, 90%, or 95% identity with it, the sequence being capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0149] Table 1 lists the UniProt (version 2022_04) codes for the genes mentioned in this disclosure:

[0150] Table 1

[0151] Gene Name UniProt Code Organism Full Protein Name CHIT1 Q13231·CHIT1_Human Human Chitinase 1 / Chitotriosidase-1 CS O75390·CISY_Human Human Citrate Synthase GUSB P08236·BGLR_Human Human β-Glucuronidase PKLR P30613·KPYR_Human Human Pyruvate Kinase L / R PRKACB P22694·KAPCB_Human Human Protein Kinase cAMP-Activated Catalytic Subunit β GOT1 P17174·AATC_Human Human Aspartate Aminotransferase 1

[0152] In one instance, the 5'-UTR and / or 3'-UTR sequences of this disclosure include sequences having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences disclosed herein, wherein these sequences are capable of increasing the expression of the coding sequence, as assessed according to this disclosure.

[0153] The nucleic acid molecules disclosed herein may be, for example, plasmids, episomes, granules, or phages. Suitable vectors and methods for their preparation are well known in the art (see, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994)).

[0154] In one instance, the nucleic acid molecule disclosed herein is a closed circular molecule or a linear molecule.

[0155] In various instances, nucleic acid molecules according to this disclosure contain a promoter, a side coding sequence, and a 5'-UTR and 3'-UTR of a polyadenylation signal in the 5' to 3' transcriptional direction.

[0156] In one instance, the nucleic acid molecule disclosed herein also includes a 5'-cap structure, optionally a cap 1 structure. Other suitable cap structures and methods for generating suitable cap structures are disclosed in the following references: WO2017 / 053297 and Tusup et al., Design of in vitro Transcribed mRNA Vectors for Research and Therapy, Chim Int J Chem. 2019; 73(5):391-394; both of which are incorporated herein by reference. 5'-capping of polynucleotides can be performed simultaneously during in vitro transcription using the following chemical RNA cap analogs to generate 5'-guanosine cap structures according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; 35G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA can be performed post-transcriptionally using a vaccinia virus capping enzyme to generate a "cap 0" structure: structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The cap 1 structure can be generated using both a vaccinia virus capping enzyme and a 2'-O methyltransferase to produce: m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure can be generated from the cap 1 structure, followed by 2'-O-methylation of the penultimate nucleotide at 5' using a 2'-O methyltransferase. The cap 3 structure can be generated from the cap 2 structure, followed by 2'-O-methylation of the fourth penultimate nucleotide at 5' using a 2'-O methyltransferase. The enzymes can be derived from recombinant sources. Other suitable means for generating appropriate cap structures are disclosed in WO2016 / 193226, which is incorporated herein by reference.

[0157] In one instance, the nucleic acid molecule of this disclosure contains a promoter that is any promoter of a DNA-dependent RNA polymerase. For example, T7 (optionally containing or consisting of the sequence TAATACGACTCACTATAAGG (SEQ ID NO:15)), T3, SP6, or Syn5 RNA polymerase.

[0158] In some instances, the nucleic acid molecules disclosed herein contain a polyadenylation signal (poly-A tail). A poly-A tail is a long sequence of adenine residues located at the 3' end of the molecule. The role of the poly-A tail is dual. The poly-A tail is essential for translation, in which poly(A)-binding protein (PABP) recruits translation factors to enhance translational levels. Furthermore, the poly-A tail increases the stability of the nucleic acid molecule by binding the poly(A) in mRNA to PABP and protecting it from exonuclease digestion. In mRNA, the poly-A tail is also known to play a crucial role in the transport of mRNA from the nucleus to the ribosome (Shlake, T. et al., RNA Biol., (2012), 9(11), 1319-1330). In one instance, the nucleic acid molecule disclosed herein contains a poly-A tail having approximately 50 to approximately 500 adenosine nucleotides. For example, the poly-A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosines. In some instances, the poly-A tail contains 50 to 250 adenosines. In some instances, the poly-A tail contains 60 to 100 adenosines. In some instances, the poly-A tail contains 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 adenosines. In some instances, the poly-A tail contains 77 adenosine molecules.

[0159] In one example of this disclosure, the nucleic acid molecule comprises a split poly(A) tail. The split poly(A) tail may comprise at least two adenosine-containing elements, optionally each having 30 to 60 adenosines, the two elements being separated by a spacer optionally having 1 to 25 nucleotides.

[0160] In one instance, the nucleic acid molecule disclosed herein is a ribonucleic acid (RNA) molecule.

[0161] In one instance, the ribonucleic acid (RNA) molecule is mRNA.

[0162] Leader Sequence

[0163] In some instances, the coding sequences disclosed herein include a leader sequence. The leader sequence may encode a signal peptide. In some instances, the signal peptide is fused to an expressed therapeutic protein. In such instances, the leader sequence and the gene of interest are located within the same open reading frame (ORF).

[0164] Signal peptides (comprising the N-terminal 15-60 amino acids of a protein) typically require transmembrane translocation in the secretory pathway and control the entry of most proteins into the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) and initiates the transport of the growing peptide chain therefore for processing. ER processing yields mature proteins, in which the signal peptide is usually cleaved by resident signal peptidases, at least for secretory proteins.

[0165] Signal peptides can be 15-60 amino acids in length. For example, signal peptides can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids in length. In some instances, the signal peptides have 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-5 The length can be 0, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids. In some instances, the signal peptide has the following sequence: MPLLLLLPLLWAGALA (SEQ ID NO:34).

[0166] Nucleosides and Nucleotides

[0167] In some instances, the nucleic acid molecules provided herein are unmodified and contain standard nucleotides adenine (A), thymine (T), or uracil (U), wherein the nucleic acid molecules are RNA, guanine (G), or cytosine (C).

[0168] In some instances, nucleic acid molecules contain modified nucleotides. Many modified nucleotides are known in the art, such as those disclosed in WO2007 / 024708, which is incorporated herein by reference. Modifications may include naturally occurring or non-naturally occurring modifications. Modifications may include those well-known in the art at the sugar, backbone, or nucleobase sites of nucleotides and / or nucleosides.

[0169] In some instances, the nucleic acid molecules described herein may contain natural (i.e., standard) nucleotides or nucleosides, non-natural or naturally occurring modified nucleotides or nucleosides, or any combination thereof.

[0170] In one instance, the nucleic acid molecule is RNA, which may contain standard A, G, and C nucleotides as well as modified U nucleotides.

[0171] In some instances, nucleic acid molecules containing modified nucleosides or nucleotides (e.g., "modified RNA nucleic acid molecules") exhibit reduced immunogenicity in cells or organisms compared to unmodified RNA nucleic acid molecules containing the same sequence.

[0172] In some instances, the modified nucleosides in the nucleic acid molecules (e.g., RNA nucleic acid molecules, such as mRNA) provided herein comprise N1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (Ψ). In some instances, the modified nucleotides in the nucleic acid molecules (e.g., RNA nucleic acid molecules, such as mRNA) comprise 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some instances, the RNA nucleic acid molecules comprise a combination of at least two (e.g., 2, 3, 4, or more) of any of the above-described modified nucleosides.

[0173] In some instances, the nucleic acid molecules described herein contain N1-methyl-pseudouridine (m1Ψ) at one or more or all uridine sites in the nucleic acid molecule.

[0174] In some instances, nucleic acid molecules contain 5-methoxy-uridine (mo5U) at one or all of the uridine sites in the nucleic acid molecule.

[0175] In some instances, nucleic acid molecules contain approximately 1% to approximately 100% modified nucleotides (relative to the total nucleotide content or relative to one or more types of nucleotides (i.e., any one or more of A, G, U, T, or C)). In some instances, nucleic acid molecules contain any intermediate percentage of modified nucleotides. For example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 2 0% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%. The remaining percentages consist of unmodified A, G, U, T, or C.

[0176] Nucleic acid molecules may contain at least 1% and at most 100% of modified nucleotides, or any intermediate percentage, such as at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, or at least 90% of modified nucleotides. For example, nucleic acids may contain modified pyrimidines, such as modified uracil or cytosine. In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the nucleic acid is replaced by modified uracil (e.g., 5-substituted uracil). The modified uracil may be replaced by a compound having a single unique structure, or by multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some instances, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the nucleic acid is replaced by a modified cytosine (e.g., 5-substituted cytosine). The modified cytosine may be replaced by a compound having a single unique structure or by multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0177] In some instances, the nucleic acid molecule is mRNA, in which uridine is replaced by a compound having a single, unique structure. In some instances, this single, unique structure is N1-methyl-pseudouridine. In some instances, the nucleic acid molecule contains at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% N1-methyl-pseudouridine.

[0178] In one instance, the mRNA contains modified nucleobases. In some instances, the modified nucleobases are modified adenine (A), cytosine (C), uracil (U), and guanine (G).

[0179] In one instance, the modified nucleobase is a modified U. In some instances, the modified U is 1-methylpseuuridine (m1Ψ) and pseudouridine (Ψ), as disclosed in WO2007 / 024708, which is incorporated herein by reference.

[0180] In one example, the nucleic acid molecule disclosed herein contains a UTR sequence that includes 5-methoxy-uridine (mo5U) at one or more or all uridine sites of the nucleic acid molecule. The molecule may contain at least a 25% ratio of modified uridine to unmodified uridine, including 25% to 50%, or at least 50%.

[0181] Examples show that modification of uridine in the 5'-UTR sequence of human CHIT1 and the 3'-UTR sequence of human citrate synthase (CS) leads to a particularly significant increase in translation.

[0182] In one example, the nucleic acid molecule of this disclosure comprises the following sequence, which contains N1-methyl-pseudouridine (m1Ψ) at one or all of the uridine positions of the nucleic acid molecule. The molecule may contain at least 75% modified uridine and unmodified uridine, including 100%.

[0183] Coding Sequence

[0184] In one instance, the nucleic acid molecule of this disclosure comprises a coding sequence encoding a therapeutic protein or peptide, optionally a wild-type sequence of a human protein or antibody or an antigen-binding fragment thereof.

[0185] Therefore, the nucleic acid molecules disclosed herein can be used in gene therapy.

[0186] In some instances, the encoded sequence is not CHIT1, GUSB1, PRKACB, GOT1, PKLR, or CS.

[0187] In one instance, the nucleic acid molecule of this disclosure contains a coding sequence encoding a disease-associated antigen (DAA). This molecule of the present disclosure is a vaccine vector.

[0188] Vaccine Vector

[0189] Nucleic acid vaccine vectors offer substantial advantages over conventional vaccination methods. In terms of safety, RNA-based vaccines are non-infectious, unlike live or attenuated live vaccines. Furthermore, RNA vaccines do not integrate into the genome, thus eliminating the risk of mutagenesis. Additionally, vaccines based on both DNA and RNA have proven highly effective against many infectious agents, including Zika, influenza, rabies, and SARS-CoV-2. Nucleic acid-based vaccines provide a cost-effective, rapid, and easily scalable alternative to conventional vaccination methods (Pardi, N. et al., Nature Reviews, (2018), 17, 261-279).

[0190] In one example of this disclosure, the disease-associated antigen may be a viral antigen, a bacterial antigen, or a tumor-associated antigen.

[0191] Once inside the cell, the coding sequence of a DNA molecule can be transcribed and translated, or the coding sequence of an RNA molecule can be translated to produce an antigen protein or fragment thereof.

[0192] When an antigen is produced, the host immune system's exposure to a protein or protein fragment may stimulate an immune response. This immune response can include stimulating B cells to produce antibodies and generating memory B cells, which are capable of producing antibodies against the antigen or fragments of a specific infectious agent. Following infection with the same infectious agent, the host immune system becomes sensitized to that antigen or antigenic protein fragment, thereby reducing the time span of the immune response against the infectious agent. This reduces or prevents the onset of post-infection symptoms.

[0193] In one example of this disclosure, the nucleic acid molecule contains a sequence encoding a nanoantigen particle. The antigen can be an antigen as described anywhere herein.

[0194] In one instance, the nucleic acid molecule contains a coding sequence that encodes a multimerizing unit. In one instance, the multimerizing unit is ferritin. The multimerizing unit can be a scaffold for nanoantigen particles. In some instances, the ferritin is Helicobacter pylori ferritin. In some instances, the nucleic acid molecule contains sequences that encode both an antigen protein and ferritin, which assemble to form nanoantigen particles.

[0195] In one example of this disclosure, the coding sequence further encodes a linker. The linker may encode between a DAA and a multimerizing unit (optionally a metro protein), such that the DAA is fused to the multimerizing unit in the coding molecule.

[0196] In one instance, the nucleic acid molecule contains RNA 5'-UTR and 3'-UTR sequences as well as an RNA coding sequence—optionally an mRNA sequence.

[0197] Coronavirus Vaccine

[0198] The recent Covid-19 pandemic has created an urgent need for improved vaccines targeting the coronavirus of concern. Several variants of SARS-CoV-2 have been identified to date, among which the Delta and Omicron variants are some of the most infectious. Currently approved vaccines for treating SARS-CoV-2 all provide stimulating immunity against the SARS-CoV-2 spike protein. Although numerous mutations in the receptor-binding domain of the spike protein have been identified in newer variants of SARS-CoV-2, this is thought to lead to increased vaccine resistance in these emerging variants (Zhao, J. et al., Environmental Research, (2022), 206(112240)). Studies estimate that current vaccines are approximately three to five times less effective against the Delta variant than against the Alpha variant of SARS-CoV-2 (Planas, D. et al., Nature, (2021), 596, 276-280).

[0199] Therefore, there is a continued need for improved vaccines overall, including those that can be used to prevent and treat coronaviruses.

[0200] Therefore, one example of this disclosure provides a nucleic acid molecule encoding a coronavirus (CoV) antigen as described herein. In one example, the nucleic acid molecule comprises RNA 5'-UTR and 3'-UTR sequences and an RNA coding sequence. In one example, the coronavirus antigen may be selected from SARS-CoV-1 and / or SARS-CoV-2. In one example, the nucleic acid molecule encodes a SARS-CoV-2 antigen selected from one or more variants of the following: Wuhan, Alpha, Beta, Delta, and Omicron, optionally BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, and XBB.1.5.

[0201] Coronavirus particles contain a large number of glycosylated spike (S) proteins that protrude from the surface of the virus particle. These S proteins form a trimer structure and mediate viral entry into host cells, making them a primary target for vaccine design.

[0202] The coronavirus spike protein is 1273 amino acids long and contains a signal peptide as well as S1 and S2 subunits. The S1 subunit contains a receptor-binding domain (RBD), which recognizes and binds to a specific host cell receptor—angiotensin-converting enzyme 2 (ACE2). The S2 subunit mediates viral cell membrane fusion.

[0203] Therefore, in one instance, the nucleic acid molecule contains a sequence encoding the S protein or an antigenic fragment thereof. After delivery to a host cell, the S protein is translated and processed within the host cell, resulting in the presentation of a trimerized S protein on the host cell surface.

[0204] In some instances, nucleic acid molecules contain sequences encoding CoV S protein and ferritin, which assemble to form nanoantigen particles.

[0205] The S protein can be stabilized in its pre-fusion conformation. Furthermore, the S protein can contain K986P and / or V987P mutations.

[0206] In one instance, the nucleic acid molecule of this disclosure encodes an antigen fragment, which is a receptor-binding domain (RBD). In one instance, the antigen fragment is the RBD of the SARS-CoV-2S protein. In one instance, the antigen fragment is the RBD of the SARS-CoV-1S protein.

[0207] In some instances, it may be advantageous to fuse fragments of antigenic proteins with ferritin in nanoparticles. In one instance, the fragment of the antigenic protein may be an RBD (reactive protein divider). The RBD in the nanoparticles may originate from antigens of the same infectious agent, i.e., monovalent, or the RBD in the nanoparticles may originate from antigens of more than one infectious agent, i.e., polyvalent.

[0208] Polymerization Unit

[0209] In some instances, the nucleic acid molecules provided herein encode fusion proteins comprising vaccine antigens linked to multimerization units. In some instances, such multimerization units confer desired properties to the antigens encoded by the nucleic acid molecules. For example, examples demonstrate that multimerization units improve the immunogenicity of antigens (e.g., the COVID spike protein) compared to the immunogenicity of the same antigen not expressed with multimerization units. Furthermore, the multimerization units provided herein improve panvariant responses against antigens. For example, nucleic acid molecules comprising coding sequences encoding COVID spike protein-multimerization unit fusion proteins, as provided herein, elicit a broader immune response against SARs-CoV-2 variants compared to the spike protein alone.

[0210] In some instances, the multimerizing unit is a protein capable of self-assembling into protein nanoparticles that are highly symmetrical, stable, and structurally organized, with a diameter of 10–150 nm, a highly suitable size range for optimal interaction with various cells of the immune system. In some instances, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some instances, the multimerizing unit is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of about 22 nm and lacks nucleic acids, thus being non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14(2016)58–68). In some instances, the multimerizing unit is hepatitis B core antigen (HBcAg) that self-assembles into particles with a diameter of 24–31 nm, which is similar to the viral core obtained from the liver of a person infected with HEY. HBcAg self-assembles into two different types of nanoparticles with diameters of 300 Å and 360 Å, corresponding to 180 or 240 protopolymers, respectively. In some instances, the antigen is fused with HBsAG or HBcAG to promote the self-assembly of nanoparticles displaying the antigen.

[0211] In some instances, the polymerizing units are selected from the following self-assembling proteins: ferritin, dioxetine synthase, and encapsuling.

[0212] Ferritin is a protein whose primary function is intracellular iron storage. Ferritin consists of 24 subunits, each composed of four α-helical bundles, which self-assemble into a quaternary structure with octahedral symmetry (Cho KJ et al. J Mol Biol. 2009; 390: 83-98). Several high-resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin consists of 24 identical protomeres, while in animals, ferritin light and heavy chains exist, which can assemble individually or in different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem. 2003; 8: 105-111; Lawson DM et al. Nature. 1991; 349: 541-544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Therefore, ferritin nanoparticles are well-suited for carrying and exposing antigens.

[0213] Dioxane-tetrahydropteridine synthase (LS) is also well-suited as a nanoparticle platform for antigen display. LS (responsible for the penultimate catalytic step in riboflavin biosynthesis) is an enzyme found in a variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber SEFlavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). LS monomers are 150 amino acids long and consist of β-sheets flanked by tandem α-helices. Many different quaternary structures of LS have been reported, illustrating its morphological diversity: from homopentamers to symmetrical assemblies of 12 pentamers forming a capsid with a diameter of 150 Å. LS cages with more than 100 subunits have even been described (Zhang X. et al. J Mol Biol. 2006; 362: 753-770).

[0214] Encapsulin (a novel protein cage-like nanoparticle isolated from the thermophilic bacterium *Thermophyton floccosum*) can also serve as a platform for antigen presentation on the surface of self-assembled nanoparticles. Encapsulin is assembled from 60 identical 31 kDa monomers with a thin, icosahedral T=1 symmetric cage structure, with an inner diameter of 20 nm and an outer diameter of 24 nm (Sutter M. et al., *Nat Struct Mol Biol.*, 2008, 15: 939-947). Although the exact function of encapsulin in *Thermophyton floccosum* remains unclear, its crystal structure has recently been resolved, and its function has been hypothesized as a cellular compartment encapsulating proteins involved in oxidative stress responses, such as DyP (dye decolorizing peroxidase) and Flp (ferritin-like protein) (Rahmanpour R. et al., *FEES J.*, 2013, 280: 2097-2104).

[0215] In some instances, the nucleic acid molecules provided herein contain coding sequences that encode coronavirus antigens (e.g., the SARS-CoV-2 spike (S) protein) fused to ferritin subunits.

[0216] Linker

[0217] In some instances, the nucleic acid molecules disclosed herein encode fusion proteins. In such instances, each domain of the fusion protein (e.g., antigen and polymerizing unit) can be separated by a coding sequence that encodes a linker sequence. The linker sequence can be self-cleaving. In other words, the linker can be a self-cleaving linker. In other instances, the linker can be a protease-sensitive linker. In some instances, the linker can be a glycine-serine linker.

[0218] In some instances, the self-cleaving linker is selected from F2A linkers, P2A linkers, T2A linkers, E2A linkers, and combinations thereof. This family of self-cleaving peptide linkers (referred to as 2A peptides) has been described in the art (see, for example, Kim, JH et al. (2011) PLoS ONE 6:e18556).

[0219] In some instances, the glycine-serine linker has the following amino acid sequence: GSGGSG (SEQ ID NO:28). In some instances, the glycine-serine linker is encoded by SEQ ID NO:29 or SEQ ID NO:30.

[0220] Those skilled in the art will understand that other linkers recognized in the art can be used for the constructs disclosed herein (e.g., those encoded by the nucleic acid molecules provided herein). Those skilled in the art will also understand that other polycistronic constructs (nucleic acid molecules encoding more than one antigen / peptide within the same molecule) can be used for the purposes provided herein.

[0221] Manufacture

[0222] The nucleic acid vaccine vector of this disclosure can be manufactured according to in vitro transcription. In vitro transcription of RNA is known in the art and described in International Publication WO2014 / 152027, which is incorporated herein by reference in its entirety. In some instances, the RNA of this disclosure is prepared according to any one or more methods described in WO2018 / 053209 and WO2019 / 036682, each of which is incorporated herein by reference. Generally, a DNA template is generated, typically as a linearized plasmid, followed by in vitro transcription to synthesize RNA, with or after capping.

[0223] The 5' cap can be added via a multi-step enzymatic reaction or via co-transcription. In co-transcriptional capping, a cap analog (such as...) is added... AG) is added directly to the in vitro transcription mixture. Alternatively, enzymatic capping using vaccinia virus capping enzyme is performed separately from in vitro transcription.

[0224] After purification, the mRNA product can be encapsulated in lipid nanoparticles (LNPs).

[0225] Composition

[0226] This disclosure also provides a pharmaceutical composition comprising a nucleic acid molecule or LNP as defined anywhere herein and a pharmaceutical carrier.

[0227] In one example, the pharmaceutical composition is a monovalent composition comprising a nucleic acid molecule encoding a first antigen or an immunogenic fragment or immunogenic variant thereof according to the present disclosure.

[0228] In one example, the pharmaceutical composition is a bivalent composition comprising another nucleic acid molecule encoding a second antigen or an immunogenic fragment or immunogenic variant thereof according to the present disclosure, wherein the second antigen is different from the first antigen.

[0229] This disclosure also provides a composition comprising a first nucleic acid molecule according to this disclosure, wherein the disease-associated antigen is a Delta variant S protein. In one example, the disease-associated antigen of the first nucleic acid molecule is a Wuhan variant S protein.

[0230] In one instance, the composition according to this disclosure may further comprise a second nucleic acid molecule that encodes an Omicron variant S protein—optionally variants BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, or XBB.1.5.

[0231] In one instance, the second nucleic acid molecule encodes the Omicron variant S protein BA.4 / 5.

[0232] In one instance, the second nucleic acid molecule encodes the Omicron variant S protein XBB.1.5.

[0233] In one example of this disclosure, the composition comprises:

[0234] 1) A first nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:19, and the 3'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding a Delta variant S protein, a linker, and ferritin, wherein the encoded protein is a Delta variant S protein-ferritin fusion protein, and

[0235] 2) A second nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:19, and the 3'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding an Omicron variant S protein, a linker, and ferritin, wherein the encoded protein is an Omicron variant S protein-ferritin fusion protein.

[0236] In one example of this disclosure, the composition comprises:

[0237] 1) A first nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising or consisting of the sequence of SEQ ID NO:19, and the 3'-UTR comprising or consisting of the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding a Delta variant S protein, a linker, and ferritin, wherein the encoded protein is a Delta variant S protein-ferritin fusion protein, and

[0238] 2) A second nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising or consisting of the sequence of SEQ ID NO:19, and the 3'-UTR comprising or consisting of the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding the Omicron variant S protein, a linker, and ferritin, wherein the encoded protein is an Omicron variant S protein-ferritin fusion protein.

[0239] In one example of this disclosure, the first nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:25, and / or the second nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:27.

[0240] In one example of this disclosure, the first nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:25, and / or the second nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:42.

[0241] In one example of this disclosure, the first nucleic acid molecule comprises the sequence shown in SEQ ID NO:37, and the second nucleic acid molecule comprises the sequence shown in SEQ ID NO:38. In another example, the first nucleic acid molecule consists of the sequence shown in SEQ ID NO:37, and the second nucleic acid molecule consists of the sequence shown in SEQ ID NO:38.

[0242] In one example of this disclosure, the first nucleic acid molecule comprises the sequence shown in SEQ ID NO:37, and the second nucleic acid molecule comprises the sequence shown in SEQ ID NO:43. In another example, the first nucleic acid molecule consists of the sequence shown in SEQ ID NO:37, and the second nucleic acid molecule consists of the sequence shown in SEQ ID NO:43.

[0243] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:41.

[0244] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:41.

[0245] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:44.

[0246] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:44.

[0247] In one example of this disclosure, the composition comprises:

[0248] 1) A first nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:19, and the 3'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding a Wuhan variant S protein, a linker, and ferritin, wherein the encoded protein is a Wuhan variant S protein-ferritin fusion protein, and

[0249] 2) A second nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:19, and the 3'-UTR comprising a sequence having at least 95% identity with the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding an Omicron variant S protein, a linker, and ferritin, wherein the encoded protein is an Omicron variant S protein-ferritin fusion protein.

[0250] In one example of this disclosure, the composition comprises:

[0251] 1) A first nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising or consisting of the sequence of SEQ ID NO:19, and the 3'-UTR comprising or consisting of the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding a Wuhan variant S protein, a linker, and ferritin, wherein the encoded protein is a Wuhan variant S protein-ferritin fusion protein, and

[0252] 2) A second nucleic acid molecule comprising a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising or consisting of the sequence of SEQ ID NO:19, and the 3'-UTR comprising or consisting of the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding the Omicron variant S protein, a linker, and ferritin, wherein the encoded protein is an Omicron variant S protein-ferritin fusion protein.

[0253] In one example of this disclosure, the first nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:23, and the second nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:27.

[0254] In one example of this disclosure, the first nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:23, and the second nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:42.

[0255] In one example of this disclosure, the first nucleic acid molecule comprises the sequence shown in SEQ ID NO:36, and the second nucleic acid molecule comprises the sequence shown in SEQ ID NO:38. In another example, the first nucleic acid molecule consists of the sequence shown in SEQ ID NO:36, and the second nucleic acid molecule consists of the sequence shown in SEQ ID NO:38.

[0256] In one example of this disclosure, the first nucleic acid molecule comprises the sequence shown in SEQ ID NO:36, and the second nucleic acid molecule comprises the sequence shown in SEQ ID NO:43. In another example, the first nucleic acid molecule consists of the sequence shown in SEQ ID NO:36, and the second nucleic acid molecule consists of the sequence shown in SEQ ID NO:43.

[0257] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:41.

[0258] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:41.

[0259] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:44.

[0260] In one instance, the first nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence encoding the polypeptide sequence shown in SEQ ID NO:44.

[0261] In one instance, the first and / or second nucleic acid molecules of any composition of this disclosure contain 80% to 100% N1-methyl-pseudouridine (m1Ψ) at the uridine position of the nucleic acid molecule.

[0262] In one instance, the first nucleic acid vector and / or the nucleic acid vector contains a 5'-cap structure, optionally a cap 1 structure.

[0263] In one instance, the first and / or second nucleic acid molecules of the composition contain a T7 promoter sequence, optionally corresponding to an RNA sequence shown in SEQ ID NO:15.

[0264] In one example, the first and / or second nucleic acid molecules of the composition encode a glycine-serine linker, optionally having the sequence of SEQ ID NO:28.

[0265] In one example, the first and / or second nucleic acid molecules of the composition contain a polyA tail having 70 to 90 adenosine nucleotides.

[0266] In one example, the first and / or second nucleic acid molecules of the composition contain a leader sequence. The leader sequence is cleaved from a mature expression antigen. In one example, the leader sequence encodes the following amino acid sequence: MPLLLLLPLLWAGALA (SEQ ID NO:34).

[0267] This disclosure also provides a monovalent composition comprising a nucleic acid molecule according to this disclosure, wherein the disease-associated antigen is an Omicron variant S protein, optionally variant BA.1, BA.2, BA.2.86, BA.3, BA.4 / 5, BQ.1, BQ.1.1, JN.1, XBB.1, or XBB.1.5.

[0268] In one instance, the nucleic acid molecule encodes the Omicron variant S protein BA.4 / 5.

[0269] In one instance, the nucleic acid molecule encodes the Omicron variant S protein XBB.1.5.

[0270] In one example of this disclosure, the composition comprises a nucleic acid molecule having a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR containing a sequence having at least 95% identity with the sequence of SEQ ID NO:19, and the 3'-UTR containing a sequence having at least 95% identity with the sequence of SEQ ID NO:21, wherein the coding sequence contains a sequence encoding an Omicron variant S protein, a linker, and ferritin, wherein the encoded protein is a Delta variant S protein-ferritin fusion protein.

[0271] In one example of this disclosure, the composition comprises a nucleic acid molecule having a 5'-UTR and a 3'-UTR operatively linked to a coding sequence, the 5'-UTR comprising or consisting of the sequence of SEQ ID NO:19, and the 3'-UTR comprising or consisting of the sequence of SEQ ID NO:21, wherein the coding sequence comprises a sequence encoding an Omicron S protein, a linker, and ferritin, wherein the encoded protein is a Delta variant S protein-ferritin fusion protein.

[0272] In one example of this disclosure, the nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:27.

[0273] In one example of this disclosure, the nucleic acid molecule contains a coding sequence as shown in SEQ ID NO:42.

[0274] In one example of this disclosure, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:38. In another example, the nucleic acid molecule consists of the sequence shown in SEQ ID NO:38.

[0275] In one example of this disclosure, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:43. In another example, the nucleic acid molecule consists of the sequence shown in SEQ ID NO:43.

[0276] In one instance, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence shown in SEQ ID NO:41.

[0277] In one instance, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence shown in SEQ ID NO:41.

[0278] In one instance, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence shown in SEQ ID NO:44.

[0279] In one instance, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence shown in SEQ ID NO:44.

[0280] In one example, the nucleic acid molecule of the composition contains 80% to 100% N1-methyl-pseudouridine (m1Ψ) at the uridine position of the nucleic acid molecule.

[0281] In one instance, the nucleic acid vector contains a 5'-cap structure, optionally a cap 1 structure.

[0282] In one instance, the nucleic acid molecule of the composition contains a T7 promoter sequence, optionally corresponding to an RNA sequence shown in SEQ ID NO:15.

[0283] In one example, the nucleic acid molecule of the composition encodes a glycine-serine linker and optionally has the sequence of SEQ ID NO:28.

[0284] In one example, the nucleic acid molecule of the composition contains a polyA tail having 70 to 90 adenosine nucleotides.

[0285] In one example, the nucleic acid molecule of the composition contains a leader sequence. The leader sequence is cleaved from a mature expression antigen. In one example, the leader sequence encodes the following amino acid sequence: MPLLLLLPLLWAGALA (SEQ ID NO:34).

[0286] In all instances of this disclosure, the sequence encoding the antigen can be further optimized via mutation to increase protein stability (such as the structure of the CoV spike protein or RBD), maximize protein translation, and reduce unwanted side effects.

[0287] The composition may contain an effective amount of nucleic acid molecules as defined herein. The effective amount of nucleic acid molecules to be used therapeutically will depend on, for example, the treatment objective, route of administration, and patient condition. In one instance, the effective amount of nucleic acid molecules within the pharmaceutical composition, as defined anywhere herein, is sufficient to effectively treat or prevent disease associated with coronavirus infection.

[0288] The composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition comprising a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. A pharmaceutically acceptable carrier may comprise one or more excipients. Pharmaceutically acceptable excipients are known and include carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. In the context of this disclosure, any suitable carrier may be used, and such carriers are well known in the art. The choice of carrier will be determined in part by the specific site on which the composition can be applied and the specific method of application. A physiologically acceptable excipient may be a pH-buffered aqueous solution. Examples of physiologically acceptable excipients include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol; salt-forming ions such as sodium; and / or nonionic surfactants such as TWEEN. TM Polyethylene glycol (PEG) and PLURONICS TM .

[0289] The composition may optionally be sterile. The composition may be frozen or lyophilized and reconstituted in a suitable sterile carrier prior to use. The composition may be prepared according to conventional techniques described in the following literature: e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0290] The composition can be administered intravenously. It can also be administered parenterally or subcutaneously.

[0291] Methods of administering a pharmaceutical composition as defined herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal administration (e.g., intranasal and oral routes). In specific examples, the pharmaceutical composition is administered intranasally, intramuscularly, intravenously, or subcutaneously. The composition may be administered via any convenient route, such as by infusion or bolus, absorption through the epithelial or mucosal lining of the skin (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Administration may be systemic or local. Each dose may or may not be administered via the same route of administration.

[0292] Delivery System

[0293] Various delivery systems are known and can be used to administer prophylactic or therapeutic agents (e.g., nucleic acid molecules disclosed herein), including but not limited to encapsulation in liposomes, microparticles, microcapsules, and constructing nucleotides as part of retroviruses or other vectors. Additionally, pulmonary administration can also be employed, for example, through inhalers or nebulizers, and formulations containing nebulizers.

[0294] This disclosure relates to nucleic acid molecules that are suitable for use as vaccine vectors.

[0295] Lipid nanoparticles (LNPs) can be used as a platform for vaccine delivery. LNPs can contain ionizable cationic lipids, cholesterol, phospholipids (such as distearate phosphatidylcholine), and polyethylene glycol (PEG)-lipids. The ionizable cationic lipids participate in nanoparticle packaging by interacting with negatively charged RNA molecules. After administration, LNPs are rapidly cleared from the injected tissue and are therefore less likely to cause inflammation and tissue damage.

[0296] Therefore, in one example of this disclosure, nucleic acid molecules as described anywhere herein are packaged into a delivery system. In one example, the delivery system is an LNP. Therefore, this disclosure also relates to LNPs containing nucleic acid molecules as described anywhere herein.

[0297] In one instance, an LNP comprises a nucleic acid molecule as described anywhere herein, wherein the nucleic acid molecule encodes an antigen protein. In another instance, an LNP comprises one or more nucleic acid molecules as described anywhere herein, wherein the molecule encodes a CoV S protein.

[0298] Use

[0299] This disclosure provides the nucleic acid molecules described herein for use in pharmaceuticals.

[0300] In one instance, the nucleic acid molecules encoding therapeutic proteins or peptides disclosed herein can be used to treat diseases or conditions characterized by the absence of said therapeutic proteins or peptides.

[0301] In one instance, the nucleic acid molecule encoding a disease-related antigen disclosed herein can be used as a nucleic acid vaccine vector.

[0302] After administration to a patient, the coding sequence is transcribed and translated in the case of a DNA sequence and translated in the case of an RNA sequence, into the antigen protein or antigen protein fragment it encodes. The production of these antigen proteins or antigen protein fragments stimulates an immune response, leading to the production of neutralizing antibodies. Upon infection with the corresponding infectious agent, the presence of neutralizing antibodies and memory B cells increases the speed of the immune response, thereby minimizing the severity and duration of symptom onset.

[0303] The vaccine vector disclosed herein can be used as a prophylactic therapy against target antigens that cause disease. In one example of this disclosure, the vaccine vector can be used to prevent CoV, particularly SARS-CoV-2.

[0304] The vaccine vector disclosed herein can also be used for treatment of infected subjects with target antigens. In one example of this disclosure, the vaccine vector can be used to treat CoV, particularly SARS-CoV-2.

[0305] This disclosure also provides a method for preventing or treating a disease or condition, the method comprising administering a nucleic acid molecule as described anywhere herein to a patient in need. This disclosure also relates to the use of the nucleic acid molecule as described anywhere herein in a method of preparing a medicament that can be used to prevent or treat a disease. In one instance, the disease is caused by CoV. In one instance, the disease is COVID-19.

[0306] Furthermore, this disclosure relates to a method for inducing an immune response in a subject, the method comprising administering to the subject a nucleic acid molecule, combination, composition, pharmaceutical composition, or formulation as described anywhere herein.

[0307] In one instance, the subject was a person.

[0308] Example

[0309] Here, we describe the 5'-UTR and 3'-UTR sequences that enhance the expression of coding sequences, including in the context of an mRNA vaccine vector.

[0310] The following examples further illustrate this disclosure, but should not be construed as limiting the scope of this disclosure in any way.

[0311] Example 1: Modification of 5'-UTR can affect eGFP mRNA Expression

[0312] Method :

[0313] UTR Cloning :

[0314] The 5'-UTR candidate sequence (shown in Table 2 below) was cloned together with eGFP and a reference 3'-UTR sequence (albumin, as used in the CureVac vector described, for example, EP2831240). Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'-UTR contains the T7 promoter sequence.

[0315] Use PCR to generate an in vitro transcription (IVT) template :

[0316] Templates for IVT were generated by PCR using Phusion PCR Mixture (NEB). The upstream primer contained the T7 promoter sequence, and the downstream primer contained the inverse complementary sequence at the 3'-UTR end of the corresponding clone, as well as the T80 sequence. The resulting PCR product contained sequences encoding the relevant sequences in the following order: T7 promoter-5'-UTR-eGFP-3'-UTR-A80. The PCR reaction was then treated with DPNI to digest the template DNA, and the DNA was purified using a PCR purification kit.

[0317] Use in vitro transcription (IVT) to perform mRNA synthesis :

[0318] The NEB IVT kit uses a template generated by PCR for IVT to prepare mRNA; this kit uses T7 RNA polymerase. The protocol will be modified to include... (AG). T7 polymerase incorporates at the start of each mRNA. (AG) is used to create a cap 1 structure at the 5' end. mRNA is generated using unmodified nucleotides or modified uridine (5'-methoxyuridine) (at a ratio of 25% to unmodified uridine). After the reaction, the DNA template is digested using a ribonuclease-free deoxyribonuclease. The mRNA transcript contains the coding sequence 5'-CAP-1-5'-UTR-eGFP-3'-UTR-A80. The mRNA is then purified using a silica column and resuspended in water.

[0319] Cell Transfection

[0320] Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and replated. The day before, 100,000 cells per well (96W plate) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well.

[0321] HeLa cells were grown in T175 flasks in HeLa complete medium (minimum basal medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested at 37°C for 5 minutes using cell digestion solution, then counted, washed, and re-seeded in collagen-treated plates. One day before transfection, 100,000 cells per well (96W plate, collagen-treated) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well.

[0322] Quantification of eGFP Expression :

[0323] eGFP fluorescence was detected using an incucyte machine, which captures images of live cells. Fluorescence was measured from the images in relative fluorescence units (RFU) using Incucyte software. The data reported in the figure show eGFP fluorescence 24 hours post-transfection.

[0324] Result :

[0325] Table 2 shows the expression levels of the candidate 5'-UTR. The sequences of the candidate 5'-UTR are shown in SEQ ID NO:1-8.

[0326] Table 2 .

[0327]

[0328] When compared with both control 5'-UTR (HSD17B4) and control 3'-UTR (albumin) eGFP expression, the inclusion of candidate 5'-UTR (Table 2) demonstrated an increase in eGFP-encoded mRNA expression in A549 cells. Figure 1 When compared with the expression of the control 5'-UTR (HSD17B4), the inclusion of GOT1, PRKACB, CHIT1, or GUSB 5'-UTR corresponded to 4-fold, 3-fold, 3-fold, and 3-fold increases in eGFP expression, respectively.

[0329] This result was confirmed in HeLa cells, where replacing the control 5'-UTR with the candidate 5'-UTR (Table 2) demonstrated an increase in the expression of eGFP-encoded mRNA. Figure 2 When compared with the expression of the control 5'-UTR (HSD17B4), the inclusion of GOT1, PRKACB, CHIT1, or GUSB 5'-UTR corresponded to 13-fold, 13-fold, 7-fold, and 14-fold increases in eGFP expression, respectively.

[0330] Example 2: Modification of 3'-UTR can affect eGFP mRNA Expression

[0331] Method :

[0332] UTR Cloning :

[0333] The 3'-UTR candidate sequence (shown in Table 3 below) was cloned together with eGFP and a reference 5'-UTR sequence (HSD17B4, as used in the CureVac vector described, for example, EP2831240). Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'-UTR contains the T7 promoter sequence.

[0334] Use PCR to generate an in vitro transcription (IVT) template :

[0335] Templates for IVT were generated by PCR using Phusion PCR Mixture (NEB). The upstream primer contained the T7 promoter sequence, and the downstream primer contained the inverse complementary sequence at the 3'-UTR end of the corresponding clone, as well as the T80 sequence. The resulting PCR product contained sequences encoding the relevant sequences in the following order: T7 promoter-5'-UTR-eGFP-3'-UTR-A80. The PCR reaction was then treated with DPNI to digest the template DNA, and the DNA was purified using a PCR purification kit.

[0336] Use in vitro transcription (IVT) to perform mRNA synthesis :

[0337] The NEB IVT kit uses a template generated by PCR for IVT to prepare mRNA; this kit uses T7 RNA polymerase. The protocol will be modified to include... (AG). T7 polymerase incorporates at the start of each mRNA. (AG) is used to create a cap 1 structure at the 5' end. mRNA is generated using unmodified nucleotides or modified uridine (5'-methoxyuridine) (at a ratio of 25% to unmodified uridine). After the reaction, the DNA template is digested using a ribonuclease-free deoxyribonuclease. The mRNA transcript contains 5'-CAP-1-5'-UTR-eGFP-3'-UTR-A80. The mRNA is then purified using a silica gel column and resuspended in water.

[0338] Cell Transfection

[0339] Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and replated. The day before, 100,000 cells per well (96W plate) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well.

[0340] HeLa cells were grown in T175 flasks in HeLa complete medium (minimum basal medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested at 37°C for 5 minutes using cell digestion solution, then counted, washed, and re-seeded in collagen-treated plates. One day before transfection, 100,000 cells per well (96W plate, collagen-treated) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well.

[0341] Quantification of eGFP Expression :

[0342] eGFP fluorescence was detected using an incucyte machine, which captures images of live cells. Fluorescence was measured from the images in relative fluorescence units (RFU) using Incucyte software. The data reported in the figure show eGFP fluorescence 24 hours post-transfection.

[0343] Result :

[0344] Table 3 shows the expression levels of candidate 3'-UTRs. The sequences of candidate 3'-UTRs are shown in SEQ ID NO:9 to 14.

[0345] Table 3 .

[0346]

[0347] When compared with both control 3'-UTR (albumin) and control 3'-UTR (HSD17B4) eGFP expression, the addition of candidate 3'-UTR (Table 3) demonstrated an increase in eGFP-encoded mRNA expression in A549 cells. Figure 3 When compared with the control 3'-UTR (albumin) expression, the addition of CHIT1, CS, or PKLR 3'-UTR resulted in a 3-fold, 3-fold, and 2-fold increase in eGFP expression, respectively.

[0348] This result was replicated in HeLa cells, where replacing the control 3'-UTR (albumin) with the candidate 3'-UTR (Table 3) demonstrated an increase in the expression of eGFP-encoded mRNA. Figure 4 Therefore, when compared with the control 3'-UTR (albumin) expression, the addition of CHIT1, CS, or PKLR 3'-UTR corresponds to a 7-fold, 4-fold, and 3-fold increase in eGFP expression, respectively.

[0349] Example 3: Combining modified 5'-UTR with modified 3'-UTR can increase eGFP mRNA Expression

[0350] The combinations of selected 5'-UTRs (Table 2) and selected 3'-UTRs (Table 3) were evaluated to determine whether the combined approaches could further increase eGFP mRNA expression. The following combinations were tested: GOT1 / CHIT1, GOT1 / CS, PRKACB / CHIT1, PRKACB / CS, CHIT1 / CHIT1, and CHIT1 / CS.

[0351] Method :

[0352] UTR Cloning :

[0353] The 5'-UTR and 3'-UTR were cloned together with eGFP as an ORF. Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'-UTR contains the T7 promoter sequence.

[0354] Use PCR to generate an in vitro transcription (IVT) template :

[0355] Templates for IVT were generated by PCR using Phusion PCR Mixture (NEB). The upstream primer contained the T7 promoter sequence, and the downstream primer contained the inverse complementary sequence at the 3'-UTR end of the corresponding clone, as well as the T80 sequence. The resulting PCR product contained sequences encoding the relevant sequences in the following order: T7 promoter-5'-UTR-ORF-3'-UTR-A80. The PCR reaction was then treated with DPNI to digest the template DNA, and the DNA was purified using a PCR purification kit.

[0356] Use in vitro transcription (IVT) to perform mRNA synthesis :

[0357] The NEB IVT kit uses a template generated by PCR for IVT to prepare mRNA; this kit uses T7 RNA polymerase. The protocol will be modified to include... (AG). T7 polymerase incorporates at the start of each mRNA. (AG) is used to create a cap 1 structure at the 5' end. mRNA is generated using unmodified nucleotides or modified uridine (5'-methoxyuridine) (at a ratio of 25% to unmodified uridine). After the reaction, the DNA template is digested using a ribonuclease-free deoxyribonuclease. The mRNA transcript contains 5'-CAP-1-5'-UTR-ORF-3'-UTR-A80. The mRNA is then purified using a silica gel column and resuspended in water.

[0358] Cell Transfection :

[0359] Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and replated. The day before, 100,000 cells per well (96W plate) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well.

[0360] Quantification of eGFP Expression :

[0361] eGFP fluorescence was detected using an incucyte machine, which captures images of live cells. Fluorescence was measured from the images in relative fluorescence units (RFU) using Incucyte software. The data reported in the figure show eGFP fluorescence 24 hours post-transfection.

[0362] Result :

[0363] Previous studies have shown that combining the selected 5'-UTR and 3'-UTR with the control 3'-UTR and 5'-UTR, respectively, can increase eGFP expression (see Examples 1 and 2).

[0364] The eGFP mRNA expression of these combinations of 5'-UTR and 3'-UTR was compared with the expression of the control 5'-UTR (HSD17B4) and 3'-UTR (albumin). In all cases, the eGFP mRNA expression of the candidate 5'-UTR and candidate 3'-UTR was increased compared with the control in A549 cells. Figure 5 and Figure 6 Interestingly, regardless of whether the mRNA contains modified bases ( Figure 6 It still does not contain modified bases. Figure 5 The overall trend for each combination remained consistent. In both cases, GOT / CS, PRKACB / CS, and CHIT / CS resulted in the largest increase in GFP fluorescence compared to the control mRNA (HSDB / Alb). Surprisingly, GOT / CS1 appeared to perform best in the context of modified mRNA. Figure 6 ), while when wild-type bases are used, PRKACB / CS results in the highest GFP fluorescence level.

[0365] Example 4: Combining modified 5'-UTR with modified 3'-UTR can increase the expression of scFv-Fc-encoding mRNA

[0366] To investigate whether combinations of UTRs also contribute to increased expression levels independently of the gene of interest, scFv expression of these combinations was tested. This was again performed on wild-type mRNA and modified mRNA.

[0367] Method :

[0368] UTR Cloning :

[0369] The 5'-UTR and 3'-UTR were cloned together with the scFv-Fc-encoding mRNA. Cloning was performed using restriction sites (REs) or seamlessly. The 5' end of the 5'-UTR contains the T7 promoter sequence.

[0370] Use PCR to generate an in vitro transcription (IVT) template :

[0371] Templates for IVT were generated by PCR using Phusion PCR Mixture (NEB). The upstream primer contained the T7 promoter sequence, and the downstream primer contained the inverse complementary sequence at the 3'-UTR end of the corresponding clone, as well as the T80 sequence. The resulting PCR product contained sequences encoding the relevant sequences in the following order: T7 promoter-5'-UTR-ORF-3'-UTR-A80. The PCR reaction was then treated with DPNI to digest the template DNA, and the DNA was purified using a PCR purification kit.

[0372] Use in vitro transcription (IVT) to perform mRNA synthesis :

[0373] The NEB IVT kit uses a template generated by PCR for IVT to prepare mRNA; this kit uses T7 RNA polymerase. The protocol will be modified to include... (AG). T7 polymerase incorporates at the start of each mRNA. (AG) is used to create a cap 1 structure at the 5' end. mRNA is generated using unmodified nucleotides or modified uridine (5'-methoxyuridine) (at a ratio of 25% to unmodified uridine). After the reaction, the DNA template is digested using a ribonuclease-free deoxyribonuclease. The mRNA transcript contains 5'-CAP-1-5'-UTR-ORF-3'-UTR-A80. The mRNA is then purified using a silica gel column and resuspended in water.

[0374] Cell Transfection

[0375] Lung A549 cells were grown in T175 flasks in A549 complete medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using accutase at 37°C for 5 minutes, then counted, washed, and replated. The day before, 100,000 cells per well (96W plate) were seeded in 100 μl of medium. On the day of transfection, the old medium was aspirated, and 140 μl of medium was added to each well. 1 μl of 100 ng / μl mRNA was diluted in 4 μl of OptiMEM, and 0.3 μl of Lipofectamine 2000 was diluted in 4.7 μl of OptiMEM. The mRNA and Lipofectamine (cationic liposome) complex were then mixed by vortexing and centrifuged, followed by incubation at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well. Collect 100 μl of supernatant at the specified time point.

[0376] scFv-Fc Quantification :

[0377] Cell supernatant (sup) was harvested from cells transfected with scFv-Fc mRNA 24 hours later. The supernatant was frozen at -80°C until quantification. For quantification, a cis-bio kit for Fc quantification was used. The principle of quantification is based on a competitive immunoassay using HTRF technology. An HfC-labeled protein (or antibody) can displace the binding between d2-labeled IgG and Cryptocate-labeled PAb anti-human Fc. The specific signal (i.e., energy transfer) is inversely proportional to the concentration of human Fc in the sample or standard. A standard curve was generated based on known concentrations of scFv-Fc, and the signal from the supernatant of scFv-Fc-transfected cells was interpolated using this standard curve to quantify the amount of scFv-Fc present in the supernatant. The concentration of scFv-Fc was measured in ng / mL. The data shown in the figure report the scFv-Fc levels 24 hours post-transfection.

[0378] Result :

[0379] Next, the expression of scFv mRNA containing these 5'-UTR and 3'-UTR combinations was compared with the expression of control 5'-UTR (HSD17B4) and 3'-UTR (albumin). Interestingly, similar to the eGFP fluorescence levels in Example 3, PRKACB / CHIT resulted in the highest expression levels when wild-type mRNA was used. Figure 7 However, apart from CHIT / CS, most other UTR combinations did not affect detectable scFv expression levels compared to the control (HSBD / Alb). In contrast, CHIT / CS and PRKACB / CHIT performed best compared to the control when modified mRNA was used as the expression substrate. Figure 8 ).

[0380] Therefore, among all the novel UTRs tested, whether alone or in combination, the CHIT / CS and PRKACB / CHIT combination produced the largest increase in expression levels, regardless of the gene of interest or whether the mRNA contained modified U or wild-type U.

[0381] Example 5: Enhancing in vitro protein expression of mRNA vaccine vectors

[0382] Next, using the UTR sequences reported by Andrew Fire and colleagues at Stanford University via GitHub on April 14, 2021, one of the best UTR combinations (CHIT / CS) was tested head-to-head against mRNAs containing putative UTR pairs from Moderna and Pfizer / BioNTech (mRNA Comp A and mRNA Comp B). This time, the modified mRNAs contained 100% 5'-methoxyuridine (instead of 25%, as in Examples 1-4).

[0383] EGFP Construct Cloning :

[0384] An mRNA construct (mRNA_AZ) encoding the EGFP reporter and paired with the CHIT1 5'-UTR (SEQ ID NO:19) was designed. Simultaneously, mRNA comparator A and mRNA comparator B constructs were designed to encode EGFP with putative UTR sequences flanked from each of the aforementioned COVID vaccines. Cloning was performed using a Gibson-based assembly method. Each plasmid has a T7 promoter sequence upstream of each 5'UTR and an 80-base-pair-long poly-A trajectory downstream of the 3'UTR, with a single BspQI site for subsequent linearization. All EGFP coding sequences are identical. The complete 5'-UTR and 3'-UTR sequences (SEQ ID NO:19 and 21, respectively) containing sequences derived from CHIT1 and CS UTRs are shown below.

[0385] Generate an in vitro transcription (IVT) template :

[0386] After purifying the plasmid from the bacterial cell in a manner similar to that outlined in Examples 1-4, a template for IVT was generated.

[0387] Use in vitro transcription (IVT) to perform mRNA synthesis :

[0388] The template generated for IVT was used in the NEB IVT kit to prepare mRNA; this kit uses T7 RNA polymerase. The protocol was modified to include... (AG). T7 polymerase incorporates at the start of each mRNA. (AG) is used to create a cap 1 structure at the 5' end. mRNA is generated using unmodified nucleotides or 100% modified uridine (N1-methylpseudouridine). Both mRNA comp A and mRNA comp B (containing mRNA with the putative UTR derived from the GitHub database) contain 100% modified U. After the reaction, the DNA template is digested using a ribonuclease-free deoxyribonuclease. The mRNA is then purified using a silica column and resuspended in water.

[0389] Quantification of EGFP Expression :

[0390] Following the manufacturer's instructions, purified mRNA was transfected into BHK-21 or HEK293 cells using Lipofectamine MessengerMAX transfection reagent (ThermoFisher). EGFP fluorescence was detected using an IncuCyte instrument, which captures images of live RNA-transfected cells over a 96-hour period. Fluorescence was measured from the images using IncuCyte software, as relative fluorescence units (arbitrary units).

[0391] Result :

[0392] Compared to two competing mRNA molecules (mRNA Comp A and mRNA Comp B), mRNAs using the 5'-UTR CHIT1 (SEQ ID NO:19) and 3'-UTR CS (SEQ ID NO:21) UTR groups showed better performance in BHK-21 cells. Figure 9A ) and HEK293 cells ( Figure 9B The two produce the highest levels of EGFP expression. Maximum mRNA expression depends on the incorporation of modified nucleotides (such as N1-methylpseudouridine (pseudoU)) to evade the host cell antiviral response in HEK293 cells. Figure 9B ).

[0393] Example 6—Efficacy of mRNA Vaccine Vectors Containing Antigen-Linker-Ferritin Sequences

[0394] The overall objective of this study was to determine the immunogenicity of a candidate SARS-CoV-2 mRNA vaccine comprising the mCHIT / CS UTR combination in mice and untreated nonhuman primates. The mRNA vaccine encodes a stable spike (S) protein-ferritin subunit fusion protein that assembles into nanoparticles upon expression for high-concentration antigen display.

[0395] Method

[0396] Mouse Study

[0397] LNP-formulated mRNA vaccine was administered twice (21 days apart) to untreated BALB / c mice (n=6 per group) via intramuscular injection of 50 μl into the thigh muscle. Fourteen days after the second vaccination, mice were exsanguinated and serum was collected for neutralization assays based on SARS-CoV-2 pseudoviruses. A set of pseudoviruses carrying the following SARS-CoV-2 spike proteins of interest were used to assess the amplitude and extent of neutralizing antibody (nAb) responses between different groups: Delta, Wuhan (D614G), BA.1, BA.2, and BA.4 / 5. S-shaped curves were generated, and the average of three replicates at each serum dilution was calculated, with the 50% (ID) value calculated. 50 Neutralization activity was assessed by considering uninfected cells as representing 100% neutralization and cells transduced solely with the virus as representing 0% neutralization.

[0398] Non-Human Primate Study

[0399] LNP-formulated mRNA vaccine was administered twice (28 days apart) to untreated cynomolgus nonhuman primates (NHP; n=6 per group), 1 ml intramuscularly each time. Each animal received a 10 μg dose per vaccination. Fourteen days after the second vaccination, NHPs were exsanguinated and serum was obtained for neutralization assays based on SARS-CoV-2 pseudoviruses. The amplitude and extent of neutralizing antibody (nAb) responses between different groups were assessed using a set of pseudoviruses carrying the following SARS-CoV-2 spike proteins of interest: Delta, Wuhan (D614G), BA.1, BA.2, and BA.4 / 5. S-shaped curves were generated, and the average of three replicates at each serum dilution was taken, with the 50% (ID) calculated. 50 Neutralization activity was assessed by considering uninfected cells as representing 100% neutralization and cells transduced solely with the virus as representing 0% neutralization.

[0400] In both mouse and NHP studies, groups were administered either the Delta S protein ferritin construct (Delta FLVLP) or Delta S protein-encoded mRNA only (Delta FL Spike). This allowed for comparison of the immunogenicity and reactivity characteristics of mRNA-based nanoparticle vaccine approaches compared to the spike-only approaches used in tozinameran and elasomeran. Both the Delta FL VLP and Delta FL Spike constructs contain the CHIT1 / CS UTR combination disclosed herein.

[0401] Result

[0402] Figure 10 (mice) and Figure 11 The results shown in (NHP) indicate that the magnitude of the neutralizing antibody response against the homolog (Delta) was significantly higher in animals vaccinated with Delta FL VLP compared to animals vaccinated with Delta FL Spike. Interestingly, the breadth of the neutralizing antibody response was also greater for all tested variants. Figure 10 and Figure 11 ).

[0403] Table 4 below proves Figure 11 The fold change of mRNA VLP / FL spike protein ratio in the data.

[0404] Table 4

[0405] Virus mRNA VLP / FL Spike Fold Change 1 <![CDATA[mRNA VLP / FL Spike p-value 2 > Delta 9 0.002 D614G 15 <0.001 BA.1 29 <0.001 BA.2 18 <0.001 BA.4 / 5 >8 <0.001

[0406] 1 Fold change = mRNA VLP geometric mean titer (GMT) / mRNA FL spike GMT

[0407] 2 p-value calculated using a two-tailed t-test

[0408] This demonstrates that when using mRNA to initiate nanoparticle antigens, a broad neutralizing response can be obtained from a single vaccine construct. This could lead to better efficacy against multiple existing and potentially future variants of interest without requiring the independent generation of a booster each time a new dominant variant emerges.

[0409] Sequence

[0410] 5'-UTR modified (m)CHIT1

[0411] DNA sequence (SEQ ID 1):

[0412] ATTGTGCTGCATC

[0413] RNA sequence (SEQ ID 2):

[0414] AUUGUGCUGCAUC

[0415] 5'-UTR PRKACB

[0416] DNA sequence (SEQ ID 3):

[0417] ATTCTGCTGTTTGCTCCTTGCCAGGTTCAAC

[0418] RNA sequence (SEQ ID 4):

[0419] AUUCUGCUGUUUGCUCCUUGCCAGGUUCAAC

[0420] 5'-UTR of GOT1

[0421] DNA sequence (SEQ ID 5):

[0422] AAAATCCTTGATTCCTAGTCTCTCGAT

[0423] RNA(SEQ ID 6):

[0424] AAAAUCUCUUGAUUCCUAGUCUCUCGAU

[0425] 5'-UTR of GUSB

[0426] DNA sequence (SEQ ID 7):

[0427] ATCCTCAACCAAGCGCCGCGAGACGGTGGCCGAGCGGGGGACCGGGAAGGC

[0428] RNA(SEQ ID 8):

[0429] AUCCUCAACCAAGCGCCCGCAGACGGUGGCCGAGCGGGGGACCGGGAAGGC

[0430] 3'-UTR CS

[0431] DNA sequence (SEQ ID 9):

[0432] AACTGGAGACTGGGTGAAAGTGACTACCAGAAAGTGAGGAAGCCTAAATAAA

[0433] RNA(SEQ ID 10):

[0434] AACUGGAGACUGGGUGAAAGUGACUACCAGAAAGUGAGGAAGCCUAAAUAAA

[0435] 3'-UTR of CHIT1

[0436] DNA sequence (SEQ ID 11):

[0437] GTCGCTAAAGCCCCTCCAGTCCCAGCTTTGAGGCTGGGCCCAGGATCACTCTACAGCCTGCCTCCTGGGTTTTCCCTGGGGGCCGCAATCTGGCTCCTGCAGGCCTTTCTGTGGTCTTCCTTTATCCAGGCTTTCTGCTCTCAGCCTTGCCTTCCTTTTTTCTGGGTCTCCTGGGCTGCCCCTTTCACTTGCAAAATAAA

[0438] RNA sequence (SEQ ID 12):

[0439] GUCGCUAAAGCCCCUCCAGUCCCAGCUUUGAGGCUGGGCCCAGGAUCACUCUACAGCCUGCCUCCUGGGUUUUCCCUGGGGGCCGCAAUCUGGCUCCUGCAGGCCUUUCUGUGGUCUUCCUUUAUCCAGGCUUUCUGCUCUCAGCCUUGCCUUCCUUUUUUCUGCGACUCCUGGGCUGCCCCUUUCACUUGCAAAAUAAA

[0440] 3'-UTR PKLR

[0441] DNA sequence (SEQ ID 13):

[0442] GACGCCCCTCCCTCCTCTGGAGTCTACGTTCTCCAGCCCACACCCCTCCAAAGCCCCACCTTTAAGTCCTCTCTTCTCTATTCCTGACCCTCCCTACCTGAGGCCTATCTGAGACTATAACTGTCATCTAGCCCCTTCGAGGTTGCCCCTTCCCCATCTCCATTTCACACAGGTCCTGAAAGTCTGTGTCCAATTATGCACTGGCCACCCAACAGCACCAATTGTACATTCCCTGCATCCAATCTGCTCAGCAGGCCCTAAGATGCCTTGAGTCTTTAATCCCA

[0443] RNA sequence (SEQ ID 14):

[0444] GACGCCCCUCCCUCCUGGAGUCUACGUUCUCCAGCCCACACCCCUCCAAAGCCCCACCUUUAAGUCCUCUCUUCUCUAUUCCCUGACCCUCCCUACCUGAGGCCUAUCUGAGACUAUAACUGUCAUCUAGCCCCUUCGAGG UUGCCCCUUCCCCAUCUCCAUUUCACACAGGUCCUGAAAGUCUGUGUCCAAUUAUGCACUGGCCACCCAACAGCACCAAUUGUACAUUCCCUGCAUCCAAUCUGCUCAGCAGGCCCUAAGAUGCCUUGAGUCUUUAAUCCCA

[0445] T7 promoter sequence (SEQ ID 15):

[0446] TAATACGACTCACTATAAGG

[0447] 3'UTR albumin sequence (SEQ ID 16):

[0448] GCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT

[0449] 5'UTR HSDB sequence (SEQ ID 17):

[0450] GTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATT

[0451] 5'-UTR CHIT1 with additional sequence elements (mCHIT1 5'-UTR sequence with underscore)

[0452] DNA sequence (SEQ ID 18):

[0453] AGG ATTGTGCTGCATC AAGCTTGCCGCCACC

[0454] RNA sequence (SEQ ID 19):

[0455] AGG AUUGUGCUGCAUC AAGCUUGCCGCCACC

[0456] 3'-UTR CS and additional sequence elements (CS 3'-UTR sequence with underscores)

[0457] DNA sequence (SEQ ID 20):

[0458] TGATGATAATAGGACTAGTGGATCC AACTGGAGACTGGGTGAAAGTGACTACCAGAAAGTGAGGAAGCC TAAATAAA CCTAGCGTACGTAAAAAATGGAAAGAACCTAGCGTACG

[0459] RNA sequence (SEQ ID 21):

[0460] UGAUGAUAAUAGGACUAGUGGAUCC ACUGGGGGGGGGGGGGGGCC WHAWOOOOOOOOOOOOOOO. CCUAGCGUACGUAAAAAAUGGAAAGAACCUAGCGUACG

[0461]

[0462] SARS-CoV-2 Wuhan D614G spike protein-linker-ferritin RNA sequence (SEQ ID NO:23)

[0463]

[0464] SARS-CoV-2 Delta-linker-ferritin DNA sequence (SEQ ID NO:24)

[0465]

[0466]

[0467] SARS-CoV-2 Omicron BA.4 / 5-linker-ferritin DNA sequence (SEQ ID NO:26)

[0468]

[0469] SARS-CoV-2 Omicron BA.4 / 5-linker-ferritin RNA sequence (SEQ ID NO:27)

[0470]

[0471] Linker amino acid sequence: GSGSGS (SEQ ID NO:28).

[0472] Linker DNA sequence: GGTTCAGGTGGATCAGGT (SEQ ID NO:29)

[0473] Linker RNA sequence: GGUUCAGGUGGAUCAGGU (SEQ ID NO:30)

[0474] Ferritin subunit, DNA sequence (SEQ ID NO:31):

[0475] GATATAGAAAAACTCCTCAATGAACAAGTAAATAAGGAGATGCAAAGTTCTAACCTGTACATGAGCATGTCTTCTTGGTGTTACACCCATAGCCTCGATGGAGCGGGATTGTTCCTTTTGACCACGCTGCGGAGGAGTATGAGCATGCTAAAAAGCTGATAATATTTCTCCAACGAGAATAATGTTCCAGTGCAATTGACAAGTATATCCGCCCTGAGCATAAGTTTGAAGGGCTCACACAAA TTTTCCAAAAGGCATACGAACACGAACAGCACATTAGCGAGTCTATTAACAACATTGTTGATCATGCAATCAAGTCCAAAGATCACGCCACGTTTAATTTCCTCCAGTGGTATGTAGCTGAGCAACATGAGGAAGAAGTGTTGTTTAAGGATATTCTTGATAAAATTGAACTTATTGGAAATGAGAACCATGGCCTCTATCTTGCGGACCAATACGTCAAGGGAATTGCCAAGTCCCGCAAGAGT

[0476] Ferritin subunit, RNA sequence (SEQ ID NO:32):

[0477] GAUAUAGAAAAACUCCUCAAUGAACAAGUAAAUAAGGAGAUGCAAAGUUCUAACCUGUACAUGAGCAUGUCUUCUUGGUGUUACACCCAUAGCCUCGAUGGAGCGGGAUUGUUCCUUUUUGA CCACGCUGCGGAGGAGUAUGAGCAUGCUAAAAAGCUGAUAAUAUUUCUCAACGAGAAUAAUGUUCCAGUGCAAUUGACAAGUAUAUCCGCCCCUGAGCAUAAGUUUGAAGGGCUCACACAAA UUUUCCAAAAGGCAUACGAACACGAACAGCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUGCAAUCAAGUCCAAAGAUCACGCCACGUUUAAUUUCCUCCAGUGGUAUGUAGCUGAG CAACAUGAGGAAGAAGUGUUGUUUAAGGAUAUUCUUGAUAAAAUUGAACUUAUUGGAAAUGAGAACCAUGGCCUCUAUCUUGCGGACCAAUACGUCAAGGGAAUUGCCAAGUCCCGCAAGAGU

[0478] Ferritin subunit, protein sequence (SEQ ID NO:33)

[0479] DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0480] Leader sequence: MPLLLLLPLLWAGALA (SEQ ID NO:34).

[0481] The upstream ORF of CHIT1, a relatively long sequence: ATGGGCTGCAGCCTGCCGCTGA (SEQ ID NO:35)

[0482] The mRNA construct sequence encoding the SARS-CoV-2 Wuhan D614G spike-ferritin fusion protein (SEQ ID NO:36)

[0483]

[0484] mRNA construct sequence encoding the SARS-CoV-2 Delta spike-ferritin fusion protein (SEQ ID NO: 37)

[0485]

[0486] The mRNA construct sequence encoding the SARS-CoV-2 Omicron BA.4 / 5 spike-ferritin fusion protein (SEQ ID NO:38)

[0487]

[0488] SARS-CoV2 Wuhan D614G spike-ferritin fusion protein polypeptide sequence (SEQ ID NO:39)

[0489]

[0490] SARS-CoV-2 Delta spike-ferritin fusion protein polypeptide sequence (SEQ ID NO:40)

[0491]

[0492] SARS-CoV-2 Omicron BA.4 / 5 spike-ferritin fusion protein polypeptide sequence (SEQ ID NO:41) (lead sequence underlined)

[0493]

[0494] SARS-CoV-2XBB.1.5 spike protein-linker-ferritin RNA sequence (SEQ ID NO:42)

[0495]

[0496] mRNA construct sequence encoding the SARS-CoV-2XBB.1.5 spike-ferritin fusion protein (SEQ ID NO:43)

[0497]

[0498] SARS-CoV2 Omicron XBB.1.5 spike-ferritin fusion protein polypeptide sequence (lead sequence underlined) (SEQ ID NO:44)

[0499] MPLLLLLPDISEASE

Claims

1. A nucleic acid molecule comprising a 5' untranslated region (5'-UTR), a coding sequence, and a 3' untranslated region (3'-UTR), wherein the coding sequence is operatively linked to the 5'-UTR and the 3'-UTR, and wherein... (i) The 5'-UTR contains a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human citrate synthase (CS); (ii) The 5'-UTR contains a sequence of the 5'-UTR derived from the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains a sequence of the 3'-UTR derived from human chitinase-1 (CHIT1); (iii) The 5'-UTR contains a sequence of the 5'-UTR derived from the catalytic subunit β (PRKACB) of human protein kinase cAMP activation, and the 3'-UTR contains a sequence of the 3'-UTR derived from human citrate synthase (CS); (iv) The 5'-UTR contains a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1), wherein the coding sequence is not derived from human chitinase-1 (CHIT1). (v) The 5'-UTR contains a sequence derived from the 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human citrate synthase (CS); or (vi) The 5'-UTR contains a sequence derived from the 5'-UTR of human aspartate aminotransferase 1 (GOT1), and the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1).

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule, and (i) the 5'-UTR contains the sequence of SEQ ID NO:1 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (ii) the 5'-UTR contains the sequence of SEQ ID NO:3 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains (iii) the 5'-UTR contains the sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains (iv) The 5'-UTR contains the sequence of SEQ ID NO: 9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO: 11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; (v) The 5'-UTR contains the sequence of SEQ ID NO: 5 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO: 9 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; or (vi) The 5'-UTR contains the sequence of SEQ ID NO:5 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:11 or a sequence having at least 80%, 85%, 90%, or 95% identity with it.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a ribonucleic acid molecule, and (i) the 5'-UTR contains the sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains SEQ ID NO:

2. (ii) the 5'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it. (iii) the 5'-UTR contains the sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO:4 or a sequence having at least 80%, 85%, 90%, or 95% identity with it. (iv) The 5'-UTR contains the sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO:2 or a sequence having at least 80%, 85%, 90%, or 95% identity with it. (v) The 5'-UTR contains the sequence of SEQ ID NO:12 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; and the 3'-UTR contains the sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, or 95% identity with it. The sequence of SEQ ID NO:10 or a sequence having at least 80%, 85%, 90%, or 95% identity with it; or (vi) the 5'-UTR contains the sequence of SEQ ID NO:6 or a sequence having at least 80%, 85%, 90%, or 95% identity with it, and the 3'-UTR contains the sequence of SEQ ID NO:

10. The sequence with ID NO:12 or a sequence that has at least 80%, 85%, 90%, or 95% identity with it.

4. The nucleic acid molecule according to claim 1 or claim 3, wherein the 5'-UTR comprises or is composed of the sequence of SEQ ID NO:2, and the 3'-UTR comprises or is composed of the nucleic acid sequence of SEQ ID NO:

10.

5. The molecule according to any of the preceding claims, wherein the coding sequence comprises a sequence encoding a therapeutic protein or peptide.

6. The molecule of claim 5, wherein the therapeutic protein or peptide is a wild-type sequence of a human protein.

7. The molecule according to claim 5 or claim 6, wherein the therapeutic protein or peptide is an antibody or an antigen-binding fragment thereof.

8. The molecule of claim 5, wherein the therapeutic protein or peptide comprises a disease-associated antigen (DAA).

9. The molecule of claim 8, wherein the disease-associated antigen is a tumor-associated antigen, a viral antigen, or a bacterial antigen.

10. The molecule of claim 9, wherein the tumor-associated antigen is not expressed in normal tissues or is mutated in tumor cells.

11. The molecule according to any one of claims 1 to 10, wherein the nucleic acid molecule is mRNA.

12. The molecule according to any one of claims 8 to 11, wherein the coding sequence further encodes a multipolymerization unit (MU).

13. The molecule according to claim 12, wherein the polymerizing unit (MU) is ferritin.

14. The molecule according to claim 12 or claim 13, wherein the coding sequence further encodes a linker, optionally a glycine-serine linker.

15. The molecule of claim 14, wherein the linker is encoded between the DAA and the MU to encode a DAA-MU fusion protein.

16. The molecule according to any one of claims 9 to 15, wherein the viral antigen is a coronavirus antigen.

17. The molecule of claim 16, wherein the coronavirus is selected from SARS-CoV-1 and / or SARS-CoV-2.

18. The molecule according to claim 16 or claim 17, wherein the coronavirus antigen is a coronavirus spike (S) protein or an antigenic fragment thereof.

19. The molecule of claim 18, wherein the S protein is stable in its pre-fusion conformation.

20. The molecule of claim 18 or claim 19, wherein the S protein comprises the K986P mutation and the V987P mutation.

21. The molecule of claim 18, wherein the antigen fragment thereof is a receptor-binding domain (RBD).

22. A composition comprising a first nucleic acid carrier comprising a molecule according to any one of claims 11 to 21, wherein the disease-associated antigen is a Wuhan variant spike (S) protein or a Delta variant spike (S) protein.

23. The composition of claim 22, wherein the first nucleic acid vector comprises the coding sequence shown in SEQ ID NO:23 or SEQ ID NO:

25.

24. The composition according to claim 22 or 23, further comprising a second nucleic acid carrier comprising a molecule according to any one of claims 11 to 21, wherein the disease-associated antigen is an Omicron variant spike (S) protein, optionally variant BA.2, BA.4 / 5, or XBB.1.

5.

25. The composition of claim 24, wherein the second nucleic acid vector comprises the coding sequence shown in SEQ ID NO:27 or SEQ ID NO:

42.

26. The composition according to any one of claims 22 to 25, wherein the first nucleic acid carrier and / or the second nucleic acid carrier comprises a polyadenylated sequence comprising 60 to 100 adenine nucleotides.

27. The composition according to any one of claims 22 to 26, wherein the first nucleic acid carrier and / or the second nucleic acid carrier comprises N1-methylpseudouridine at 80% to 100% of the uridine sites.

28. The composition according to any one of claims 22 to 27, wherein the first nucleic acid vector and / or the nucleic acid vector comprises a 5'-cap structure, optionally a cap 1 structure.

29. The composition according to any one of claims 22 to 28, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence shown in SEQ ID NO:19 and the 3'-UTR sequence shown in SEQ ID NO:

21.

30. A composition comprising a nucleic acid vector comprising a molecule according to any one of claims 11 to 21, wherein the disease-associated antigen is an Omicron variant spike (S) protein, optionally variant BA.2, BA.4 / 5, or XBB.1.

5.

31. The composition of claim 30, wherein the nucleic acid vector comprises the coding sequence shown in SEQ ID NO:27 or SEQ ID NO:

42.

32. The composition according to any one of claims 30 to 31, wherein the nucleic acid vector comprises a polyadenylated sequence comprising 60 to 100 adenine nucleotides.

33. The composition according to any one of claims 30 to 32, wherein the first nucleic acid carrier and / or the second nucleic acid carrier comprises N1-methylpseudouridine at 80% to 100% of the uridine sites.

34. The composition according to any one of claims 30 to 33, wherein the first nucleic acid vector and / or the nucleic acid vector comprises a 5'-cap structure, optionally a cap 1 structure.

35. The composition according to any one of claims 30 to 34, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence shown in SEQ ID NO:19 and the 3'-UTR sequence shown in SEQ ID NO:

21.

36. The molecule or composition according to any one of claims 11 to 35, wherein the nucleic acid carrier is formulated in lipid nanoparticles (LNPs).

37. The composition or nucleic acid molecule according to any of the preceding claims, wherein the composition or nucleic acid molecule is used in a pharmaceutical product.

38. A vaccine comprising a nucleic acid molecule or composition according to any of the preceding claims.

39. The composition or vaccine according to any one of claims 22 to 38, wherein the composition or vaccine is used in a method for preventing and / or treating an infectious disease, optionally a disease caused by a coronavirus.

40. A method for preventing and / or treating an infectious disease in a subject, optionally a disease caused by a coronavirus, the method comprising administering to the subject an effective amount of a composition or vaccine according to any one of claims 22 to 38.

41. A method of vaccinating a subject against an infectious disease, optionally a disease caused by a coronavirus, the method comprising administering an effective amount of the composition or vaccine according to any one of claims 22 to 38.

42. A nucleic acid vector comprising a 5' cap 1 structure, a 5'-UTR sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), a coding sequence encoding a Wuhan variant spike (S) protein fused with a ferritin sequence or a Delta variant spike (S) protein fused with a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylated sequence comprising 70 to 90 adenine nucleotides.

43. A nucleic acid vector comprising a 5' cap 1 structure, a 5'-UTR sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), a coding sequence encoding the spike (S) protein of an Omicron variant BA.2, BA.4 / 5, or XBB.1.5 fused to a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylated sequence comprising 70 to 90 adenine nucleotides.

44. A composition comprising the nucleic acid vector according to claim 42 and the nucleic acid vector according to claim 43.

45. A nucleic acid vector comprising the sequence shown in SEQ ID NO:

36.

46. ​​A nucleic acid vector comprising the sequence shown in SEQ ID NO:

37.

47. A nucleic acid vector comprising the sequence shown in SEQ ID NO:

38.

48. A nucleic acid vector comprising the sequence shown in SEQ ID NO:

43.

49. A composition comprising the carrier according to claim 45 and the carrier according to claim 46.

50. A composition comprising the carrier according to claim 45 or claim 46 and the carrier according to claim 47 or claim 48.

51. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:

39.

52. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:

40.

53. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:

41.

54. A nucleic acid vector comprising a coding sequence that encodes a polypeptide having the sequence shown in SEQ ID NO:

44.

55. The nucleic acid vector according to any one of claims 51 to 54, wherein the nucleic acid vector further comprises 5'-UTR and 3'-UTR as claimed in any one of claims 1-4 or 29.

56. The nucleic acid vector according to any one of claims 51 to 55, wherein the nucleic acid vector is mRNA.

57. The nucleic acid vector of claim 56, wherein the mRNA comprises any of the features described in claims 26 to 28.

58. A composition comprising the carrier according to claim 51 and the carrier according to claim 52.

59. A composition comprising the carrier according to claim 51 or claim 52 and the carrier according to claim 53 or claim 54.

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