Nucleic acid molecules

Abstract

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

Description

[Technical Field]

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

[0002] Nucleic acid vectors have multiple applications in both the treatment and testing of human diseases. In molecular biology, nucleic acid vectors can be used to introduce disease-related genes or proteins of interest into cell or animal models as assay tools. Furthermore, there are currently countless therapeutic situations in which expression of nucleic acid sequences is desirable, including the expression of functional proteins to treat diseases caused by the lack of functional proteins or as nucleic acid-based vaccines. Nucleic acid vectors generally contain a simple structure consisting of a coding sequence encoding a peptide or protein of interest, 5' and 3' untranslated regions (UTRs) that aid in the translation and stability of the vector, and a polyadenylation signal that protects the vector from enzymatic degradation and plays an important role in translation. Components of nucleic acid vectors can be modified to treat or model specific diseases.

[0003] The recent success of nucleic acid vaccines during the COVID-19 pandemic has sparked renewed interest in the field of nucleic acid-based therapeutics, particularly those aimed at expressing heterologous proteins of interest. For example, there is currently interest in universal mRNA vaccines that simultaneously administer antigens from multiple disease-associated pathogens to confer general immunity against multiple seasonal pathogens (e.g., clinical trial NCT05596S734 evaluating mRNA vaccine candidates against COVID-19 and influenza).

[0004] Universal vaccines would be greatly aided by improvements in nucleic acid vectors. For example, by maximizing or increasing translation efficiency from vectors, high neutralizing responses to antigens could be achieved at lower mRNA dose levels. This would allow for the simultaneous administration of multiple vectors encoding multiple antigens without exceeding the recommended mRNA dose per vaccination. Furthermore, increasing translation efficiency could reduce the costs associated with producing mRNA therapeutics by enabling effective responses at lower dose levels. Such advantages are applicable to any nucleic acid-based expressed biologic therapy (e.g., in vivo expressed antibodies, wild-type proteins, cancer neoantigens, and traditional anti-pathogen vaccines). Summary of the Invention

[0005] The present disclosure relates to nucleic acid molecules comprising at least one coding sequence adjacent to and operably linked to a 5'-UTR and / or a 3'-UTR. The UTR(s) are engineered to increase translation of a protein of interest encoded by the coding sequence. The coding sequence(s) 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 mutants. The molecule may also include coding sequences for multimerization units such that upon assembly, a multimeric complex is formed.

[0006] Specific examples of the present disclosure are summarized below: This list is illustrative only and is not intended to be an exhaustive list of all examples provided by the present 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 operably linked to the 5'-UTR and the 3'-UTR; and (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 derived from the 5'-UTR of human protein kinase cAMP-activated catalytic subunit beta (PRKACB), and the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1); (iii) the 5'-UTR contains a sequence derived from the 5'-UTR of human protein kinase cAMP-activated catalytic subunit beta (PRKACB) and the 3'-UTR contains a sequence derived from the 3'-UTR of human citrate synthase (CS); (iv) the 5'-UTR contains a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), the 3'-UTR contains a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1), and the coding sequence is not derived from human chitinase-1 (CHIT1); (v) the 5'-UTR comprises a sequence derived from the 5'-UTR of human glutamic oxaloacetic transaminase 1 (GOT1), and the 3'-UTR comprises a sequence derived from the 3'-UTR of human citrate synthase (CS); or (vi) A nucleic acid molecule, the 5'-UTR of which comprises a sequence derived from the 5'-UTR of human glutamic oxaloacetic transaminase 1 (GOT1) and the 3'-UTR of which comprises a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1).

[0008] 2. The nucleic acid molecule of paragraph 1, wherein the nucleic acid molecule is a deoxyribonucleic (DNA) molecule and (i) the 5'-UTR comprises the sequence of SEQ ID NO: 1, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (ii) the 5'-UTR comprises the sequence of SEQ ID NO: 3, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO: 11, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; or (iii) the 5'-UTR comprises the sequence of SEQ ID NO: 3, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, or 95% identical thereto. 90% or 95% identical thereto; (iv) the 5'-UTR comprises the sequence of SEQ ID NO: 1 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3'-UTR comprises the sequence of SEQ ID NO: 11 or a sequence at least 80%, 85%, 90% or 95% identical thereto; (v) the 5'-UTR comprises the sequence of SEQ ID NO: 5 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3'-UTR comprises the sequence of SEQ ID NO: 9 or a sequence at least 80%, 85%, 90% or 95% identical thereto; or (vi) the 5'-UTR comprises the sequence of SEQ ID NO: 5 or a sequence at least 80%, 85%, 90% or 95% identical thereto and the 3'-UTR comprises the sequence of SEQ ID NO: 11 or a sequence at least 80%, 85%, 90% or 95% identical thereto.

[0009] 3. The nucleic acid molecule of paragraph 1, wherein the nucleic acid molecule is a ribonucleic acid molecule (RNA) and (i) the 5'-UTR comprises the sequence of SEQ ID NO:2, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:10, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (ii) the 5'-UTR comprises the sequence of SEQ ID NO:4, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:12, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; or (iii) the 5'-UTR comprises the sequence of SEQ ID NO:4, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:10, or a sequence at least 80%, 85%, 90%, or 95% identical thereto. or 95% identical thereto; (iv) the 5'-UTR comprises the sequence of SEQ ID NO:2, or a sequence at least 80%, 85%, 90% or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:12, or a sequence at least 80%, 85%, 90% or 95% identical thereto; (v) the 5'-UTR comprises the sequence of SEQ ID NO:6, or a sequence at least 80%, 85%, 90% or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:10, or a sequence at least 80%, 85%, 90% or 95% identical thereto; or (vi) the 5'-UTR comprises the sequence of SEQ ID NO:6, or a sequence at least 80%, 85%, 90% or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO:12, or a sequence at least 80%, 85%, 90% or 95% identical thereto.

[0010] 4. The nucleic acid molecule of paragraph 1 or paragraph 3, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 2 and the 3'-UTR comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.

[0011] 5. The molecule of any one of paragraphs 1 to 4, wherein the coding sequence comprises a sequence encoding a therapeutic protein or peptide.

[0012] 6. The molecule of paragraph 5, wherein the therapeutic protein or peptide is the wild-type sequence of a human protein.

[0013] 7. The molecule of paragraph 5 or 6, wherein the therapeutic protein or peptide is an antibody or antigen-binding fragment thereof.

[0014] 8. The molecule described in paragraph 5, wherein the therapeutic protein or peptide comprises a disease-associated antigen (DAA).

[0015] 9. The molecule of paragraph 8, wherein the disease-associated antigen is a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0016] 10. The molecule described in paragraph 9, wherein the tumor-associated antigen is not expressed in normal tissue or is mutated in tumor cells.

[0017] 11. The molecule of any one of paragraphs 1 and 3 to 10, wherein the nucleic acid molecule is mRNA.

[0018] 12. The molecule of any one of paragraphs 8 to 11, wherein the coding sequence further encodes a multimerization unit (MU).

[0019] 13. The molecule described in paragraph 12, wherein the multimerization unit (MU) is ferritin.

[0020] 14. The molecule of paragraph 12 or 13, wherein the coding sequence further encodes a linker, optionally a glycine-serine linker.

[0021] 15. The molecule of paragraph 14, wherein a linker is encoded between the DAA and the MU, thereby encoding a DAA-MU fusion protein.

[0022] 16. The molecule of any one of paragraphs 9 to 15, wherein the viral antigen is a coronavirus antigen.

[0023] 17. The molecule according to paragraph 16, wherein the coronavirus is selected from SARS-CoV-1 and / or SARS-CoV-2.

[0024] 18. The molecule of paragraph 16 or 17, wherein the coronavirus antigen is a coronavirus spike (S) protein or an antigenic fragment thereof.

[0025] 19. The molecule of paragraph 18, wherein the S protein is stabilized in a pre-fusion conformation.

[0026] 20. The molecule of paragraph 18 or 19, wherein the S protein comprises a K986P mutation and a V987P mutation.

[0027] 21. The molecule described in paragraph 18, wherein the antigenic fragment is a receptor binding domain (RBD).

[0028] 22. A composition comprising a first nucleic acid vector comprising a molecule described in any one of paragraphs 11 to 21, wherein the disease-associated antigen is the Wuhan variant spike (S) protein or the Delta variant spike (S) protein.

[0029] 23. The composition described in paragraph 22, wherein the first nucleic acid vector comprises the antigen-linker-ferritin sequence set forth in SEQ ID NO: 23 or SEQ ID NO: 25.

[0030] 24. The composition of any one of paragraphs 22-23, further comprising a second nucleic acid vector comprising the molecule of any one of paragraphs 111-21, wherein the disease-associated antigen is omicron mutant spike (S) protein, optionally mutant BA.2, BA.4 / 5, or XBB.1.5.

[0031] 25. The composition of any one of paragraphs 22 to 24, wherein the first nucleic acid vector and / or the second nucleic acid vector(s) comprises a polyadenylation sequence comprising 60 to 100 adenine nucleotides.

[0032] 26. The composition of any one of paragraphs 22 to 25, wherein the first nucleic acid vector and / or the second nucleic acid vector(s) contain N1-methylpseudouridine at 80% to 100% of the uridine positions.

[0033] 27. The composition of any one of paragraphs 22 to 26, wherein the first vector and / or nucleic acid vector(s) comprises a 5'-cap structure, optionally a Cap 1 structure.

[0034] 28. The composition described in any one of paragraphs 22 to 27, wherein the first nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence set forth in SEQ ID NO: 19 and the 3'-UTR sequence set forth in SEQ ID NO: 21.

[0035] 29. The composition of any one of paragraphs 24 to 28, wherein the second nucleic acid vector comprises the antigen-linker-ferritin sequence set forth in SEQ ID NO: 27.

[0036] 30. The composition of any one of paragraphs 24 to 28, wherein the second nucleic acid vector comprises the antigen-linker-ferritin sequence set forth in SEQ ID NO: 42.

[0037] 31. A composition comprising a nucleic acid vector comprising the molecule of any one of paragraphs 11 to 21, wherein the disease-associated antigen is omicron mutant spike (S) protein, optionally mutant BA.2, BA.4 / 5, or XBB.1.5.

[0038] 32. The composition described in paragraph 31, wherein the first nucleic acid vector comprises the antigen-linker-ferritin sequence set forth in SEQ ID NO: 27 or SEQ ID NO: 42.

[0039] 33. The composition of any one of paragraphs 31 to 32, wherein the nucleic acid vector comprises a polyadenylation sequence comprising 60 to 100 adenine nucleotides.

[0040] 34. The composition of any one of paragraphs 31 to 33, wherein the nucleic acid vector contains N1-methylpseudouridine at 80% to 100% of the uridine positions.

[0041] 35. The composition of any one of paragraphs 31 to 34, wherein the nucleic acid vector comprises a 5'-cap structure, optionally a Cap 1 structure.

[0042] 36. The composition described in any one of paragraphs 31 to 35, wherein the nucleic acid vector and / or the second nucleic acid vector comprises or consists of the 5'-UTR sequence set forth in SEQ ID NO: 19 and the 3'-UTR sequence set forth in SEQ ID NO: 21.

[0043] 37. The molecule or composition of any one of paragraphs 11 to 36, wherein the nucleic acid vector is formulated in a lipid nanoparticle (LNP).

[0044] 38. A composition or nucleic acid molecule according to any one of paragraphs 1 to 37 for use in medicine.

[0045] 39. A vaccine comprising the nucleic acid molecule or composition described in any one of paragraphs 1 to 38.

[0046] 40. A composition or vaccine according to any one of paragraphs 22 to 39 for use in a method for the prevention and / or treatment of an infectious disease, optionally a disease caused by a coronavirus.

[0047] 41. A method for preventing and / or treating an infectious disease in a subject, comprising administering to the subject an effective amount of a composition or vaccine described in any one of paragraphs 22 to 39, optionally for a disease caused by a coronavirus.

[0048] 42. A method for vaccinating a subject against an infectious disease, optionally a disease caused by a coronavirus, comprising administering an effective amount of a composition or vaccine described in any one of paragraphs 22 to 39.

[0049] 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 mutant spike (S) protein fused with a ferritin sequence or a delta mutant spike (S) protein fused with a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylation sequence comprising 70 to 90 adenine nucleotides.

[0050] 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 an omicron variant BA.2, BA.4 / 5, or XBB.1.5 spike (S) protein fused to a ferritin sequence, a 3'-UTR sequence derived from the 3'-UTR of human citrate synthase (CS), and a polyadenylation sequence comprising 70 to 90 adenine nucleotides.

[0051] 45. A composition comprising the nucleic acid vector of paragraph 43 and the nucleic acid vector of paragraph 44.

[0052] 46. ​​A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 36.

[0053] 47. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 37.

[0054] 48. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 38.

[0055] 49. A nucleic acid vector comprising the sequence set forth in SEQ ID NO: 43.

[0056] 50. A composition comprising a vector described in paragraph 46 or 47 and a vector described in paragraph 48 or 49.

[0057] 51. A nucleic acid vector comprising a coding sequence encoding a polypeptide having the sequence set forth in SEQ ID NO:39.

[0058] 52. A nucleic acid vector comprising a coding sequence encoding a polypeptide having the sequence set forth in SEQ ID NO:40.

[0059] 53. A nucleic acid vector comprising a coding sequence encoding a polypeptide having the sequence set forth in SEQ ID NO:41.

[0060] 54. A nucleic acid vector comprising a coding sequence encoding a polypeptide having the sequence set forth in SEQ ID NO:44.

[0061] 55. The nucleic acid vector of any one of paragraphs 51 to 54, further comprising a 5'-UTR and a 3'-UTR of any one of paragraphs 1 to 4 or 28.

[0062] 56. The nucleic acid vector of any one of paragraphs 51 to 55, which is an mRNA.

[0063] 57. The nucleic acid vector of paragraph 56, wherein the mRNA comprises any of the features described in paragraphs 26 to 28.

[0064] 58. A composition comprising the vector described in paragraph 51 and the vector described in paragraph 52.

[0065] 59. A composition comprising a vector described in paragraph 51 or 52 and a vector described in paragraph 53 or 54. [Brief explanation of the drawings]

[0066] Those skilled in the art will understand that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way.

[0067] [Figure 1]This is a bar graph comparing the expression (in A549 cells) of mRNA encoding eGFP using a constant 3'-UTR region (albumin) and candidate 5'-UTRs (UTR-11, UTR-37, UTR-52, and UTR-53, as identified in the Examples). The black bars represent vectors with a control 5'-UTR region (HSD17B4). The selected 5'-UTRs increase the fluorescence intensity of eGFP in A549 cells. The candidate 5'-UTRs demonstrate the ability to increase eGFP expression in A549 cells compared to the control 5'-UTR (HSD17B4). Data are representative of three independent experiments, with three to four technical replicates performed for each experiment. [Figure 2] This is a bar graph comparing the expression (in HeLa cells) of mRNA encoding eGFP using the constant 3'-UTR region (albumin) and candidate 5'-UTRs (UTR-11, UTR-37, UTR-52, UTR-53). Black bars represent vectors with a control 3'-UTR (albumin) and a control 5'-UTR (HSD17B4). The candidate 5'-UTRs demonstrate the ability to increase eGFP expression in HeLa cells compared to the control 5'-UTR (HSD17B4). Data are representative of three independent experiments, with three to four technical replicates performed for each experiment. [Figure 3] 1 is a bar graph comparing the expression of eGFP mRNA (in A549 cells) using candidate 3'-UTR regions (UTR-3, UTR-4, UTR-36 as identified in the Examples) and a control 5'-UTR (HSD17B4). Black bars represent the control 3'-UTR (albumin) and 5'-UTR (HSD17B4). The candidate 3'-UTRs can increase eGFP mRNA expression in A549 cells compared to the control 3'-UTR. Data are representative of two independent experiments, with three technical replicates performed for each experiment. [Figure 4]This is a bar graph comparing the expression of eGFP mRNA (in HeLa cells) using candidate 3'-UTRs (UTR-3, UTR-4, UTR-36) and a control 5'-UTR (HSD17B4). The candidate 3'-UTRs can increase the expression of eGFP mRNA in HeLa cells compared to the control 3'-UTR. Black bars represent the control 3'-UTR (albumin) and control 5'-UTR (HSD17B4). Data are representative of two independent experiments, with three technical replicates performed for each experiment. [Figure 5] This is a bar graph comparing the expression of eGFP mRNA (in A549 cells) using combinations of candidate 3'-UTRs (CHIT-1, CS) and candidate 5'-UTRs (GOT1, PRKACB, CHIT1) with a control consisting of a control 3'-UTR (albumin) and control 5'-UTR (HSD17B4). Black bars represent eGFP mRNA expression in the control. Combining candidate 3'-UTRs and candidate 5'-UTRs can increase eGFP mRNA expression in A549 cells. Data are representative of three independent experiments, with three technical replicates performed for each experiment. [Figure 6] This is a bar graph showing the expression (in A549 cells) of modified mRNA encoding eGFP using combinations of candidate 5' (GOT1, PRKACB, CHIT) and candidate 3'-UTRs (CHIT, CS). Black bars represent the expression of eGFP encoding modified mRNA using the control 5' (HSD17B4) and 3'-UTR (albumin) regions. Combinations of candidate 5'-UTRs and candidate 3'-UTRs increase the expression of eGFP encoding modified mRNA compared to expression using the control 5'-UTR and control 3'-UTR. Data are representative of three independent experiments, with three technical replicates performed for each experiment. [Figure 7]Figure 1 shows antibody expression in A549 cells using scFv-Fc-encoding mRNA with candidate or control 5'-UTR and 3'-UTR combinations. Black bars represent expression of scFv-Fc-encoding mRNA with the 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) resulted in higher expression of scFv-Fc-encoding mRNA than expression with the control 5'-UTR and 3'-UTR regions. Data are representative of three independent experiments, with three technical replicates performed for each experiment. [Figure 8] Figure 1 shows antibody expression in A549 cells using modified scFv-Fc-encoding mRNAs with candidate or control 5'-UTR and 3'-UTR combinations (in A549 cells). The modified mRNAs contain a modified uridine (5-methoxyuridine). Black bars represent expression of scFv-Fc-encoding mRNAs using the control 5'-UTR (HSD17B4) and control 3'-UTR (albumin). The combinations 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) resulted in higher expression of scFv-Fc-encoding mRNAs than those using the control 5'-UTR and 3'-UTR regions. Data are representative of three independent experiments, with three technical replicates performed for each experiment. [Figure 9A] This figure shows that mRNA with the 5'-UTR CHIT1 and 3'-UTR CS UTRs produced the highest levels of EGFP expression in both BHK-21 cells (Panel A) and HEK293 cells (Panel B) compared with two competitor mRNA molecules. mRNA_AZ, mRNA comp A, and mRNA comp B all contain 100% pseudo-U. For comparison, mRNA_AZ, which contains 0% pseudo-U, was included. [Figure 9B]This figure shows that mRNA with the 5'-UTR CHIT1 and 3'-UTR CS UTRs produced the highest levels of EGFP expression in both BHK-21 cells (Panel A) and HEK293 cells (Panel B) compared with two competitor mRNA molecules. mRNA_AZ, mRNA comp A, and mRNA comp B all contain 100% pseudo-U. For comparison, mRNA_AZ, which contains 0% pseudo-U, was included. [Figure 10] An mRNA molecule according to the present disclosure encoding the delta antigen fused to ferritin via a linker shows improved pan-mutant immunogenicity in mice compared to that encoding the native spike. [Figure 11] We show that mRNA molecules according to the present disclosure encoding the delta antigen fused to ferritin via a linker have improved pan-variant immunogenicity in non-human primates compared to those encoding the native spike. DETAILED DESCRIPTION OF THE INVENTION

[0068] All references cited are incorporated herein by reference in their entirety.

[0069] Many modifications and other examples of the disclosures set forth herein will come to mind to one skilled in the art to which these disclosures pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is to be understood, therefore, that the disclosure is not to be limited to the particular examples disclosed, and that modifications and other examples are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0070] Units, prefixes, and symbols may be shown in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their commonly accepted one-letter codes. The terms defined below are more fully defined by reference to the specification in its entirety.

[0071] definition The term "nucleic acid sequence" is intended to encompass polymers of DNA or RNA, i.e., polynucleotides, which may be single- 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, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule and thus include double- and single-stranded DNA, as well as double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, including, but not limited to, methylated and / or capped polynucleotides. Nucleic acids are typically linked via phosphate linkages to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, etc.).

[0072] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. These terms encompass amino acid polymers that are naturally occurring or modified by any other manipulation or modification, such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Also included within the definition are polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the present disclosure are based on antibodies, it is understood that in some aspects the polypeptides can occur as single chains or associated chains.

[0073] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning the two sequences and introducing gaps to maximize identity between the sequences. Percent identity should generally be calculated between nucleic acids of the same type, i.e., DNA or RNA sequences. Thus, when a DNA sequence "corresponds" to an RNA sequence, or an RNA sequence "corresponds" to a DNA sequence, it is understood that the first step is to convert the RNA sequence to the corresponding DNA sequence (particularly by substituting uracil (U) with thymidine (T) throughout the sequence), or vice versa (particularly by substituting T with U throughout the sequence). Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed using the blastn program set to default parameters (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. Only perfect matches are counted in identity scoring. The length excludes gaps at the ends of the sequences, but includes internal gaps.

[0074] "5'-untranslated region (5'-UTR)" has the usual meaning recognized by those skilled in the art. It is a region of a nucleic acid molecule located 5' of a coding sequence that is not translated into protein. The 5'-UTR usually begins at the transcription start site and ends before the start codon of the coding sequence.

[0075] "3'-untranslated region (3'-UTR)" has the usual meaning recognized by those skilled in the art. It is a region of a nucleic acid molecule located 3' of a coding sequence and not translated into protein. The 3'-UTR is usually 3' of the coding sequence. If the molecule contains a polyadenylation signal, the 3'-UTR is usually between the coding sequence and the polyadenylation signal.

[0076] A "coding sequence" is a contiguous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). A coding sequence typically encodes a polypeptide. The coding sequences disclosed herein are operably linked to the 5' UTR and 3' UTR described herein.

[0077] "Messenger RNA (mRNA)" is any RNA that encodes (at least one) protein (a polymer of naturally occurring, non-naturally occurring, or modified amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Those of skill in the art will understand that, unless otherwise specified, the nucleic acid sequences described in this application may recite a "T" in a representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" is replaced with a "U." Thus, any DNA disclosed herein and identified by a specific sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to that DNA, in which each "T" in the DNA sequence is replaced with a "U."

[0078] "Nucleoside" refers to a compound containing a sugar molecule (e.g., pentose or ribose) or its derivative in combination with an organic base (e.g., a purine or pyrimidine) or its derivative (also referred to herein as a "nucleobase"). Nucleic acids can contain one or more regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acid comprises a region of nucleotides ("nucleotide" refers to a nucleoside containing a phosphate group).

[0079] "Expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing), and (3) translation of the RNA into a polypeptide or protein.

[0080] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. The composition may be sterile.

[0081] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some embodiments, 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, etc.). In some embodiments, a subject is a human.

[0082] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0083] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the words "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprises," "comprising," "containing," and "having," etc., may mean "includes," "including," etc. "Consisting essentially of" or "consists essentially of" is open-ended and allows for the presence of more than what is recited, but excludes prior art embodiments, so long as the presence of more than what is recited does not alter basic or novel characteristics of what is recited.

[0084] Unless specifically stated or clear from the context, as used herein, the term "or" is understood to be inclusive. When used herein in a phrase such as "A and / or B," it is intended to include "A and B," "A or B," "A," and "B." Similarly, when used in a phrase such as "A, B, and / or C," it is intended to encompass each of the following aspects: 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).

[0085] Any molecule, vector, composition, use, or method provided herein can be combined with any one or more of the other molecules, vectors, compositions, uses, or methods provided herein.

[0086] Untranslated Regions (UTRs) The present 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 operably linked to the 5'-UTR and the 3'-UTR; and (i) the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), and the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of human citrate synthase (CS); (ii) the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human protein kinase cAMP-activated catalytic subunit beta (PRKACB), and the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1); (iii) the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human protein kinase cAMP-activated catalytic subunit beta (PRKACB), and the 3'-UTR consists of a sequence derived from the 3'-UTR of human citrate synthase (CS); (iv) the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human chitinase-1 (CHIT1), the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1), and the coding sequence is not derived from human chitinase-1 (CHIT1); (v) the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human glutamic oxaloacetic transaminase 1 (GOT1), and the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of human citrate synthase (CS); or (vi) A nucleic acid molecule is provided, wherein the 5'-UTR comprises or consists of a sequence derived from the 5'-UTR of human glutamic oxaloacetic transaminase 1 (GOT1), and the 3'-UTR comprises or consists of a sequence derived from the 3'-UTR of human chitinase-1 (CHIT1).

[0087] The nucleic acid molecules of the present disclosure are nucleic acid vectors.

[0088] The 5'-UTR and 3'-UTR sequences of the present disclosure are particularly useful for increasing the translation level of coding sequences. The examples show that the combination of the 5'-UTR and 3'-UTR disclosed herein enhances the translation level of multiple proteins of interest, including GFP and scFv-Fc (e.g., therapeutic proteins). The translation levels were higher than those achieved by clinically validated UTR combinations derived from regulatory-approved mRNA vaccine products (see, e.g., Figure 10). Thus, the present disclosure provides nucleic acid molecules capable of enhancing the translation of proteins of interest, potentially paving the way for reducing the dosing regimens of mRNA vaccines currently available in the art. This may enable the development of combination mRNA vaccines against multiple pathogens that can be administered as a single dose. Furthermore, the UTR combinations described herein may be preferred for use in conjunction with therapeutic proteins expressed in vivo, such as antibodies (or variants thereof) targeting intracellular targets. The UTRs disclosed herein may enhance the dynamic range of in vivo expression yields, thus allowing a larger dose range to be explored to identify the most effective dose.

[0089] The level of translation obtained with the UTRs of the present disclosure can be assessed by any suitable assay available to one of skill in the art, for example, by measuring the concentration of the protein of interest encoded by the coding sequence, for example, by detecting a marker protein such as GFP, or a therapeutic protein such as scFv-Fc, or a vaccine antigen such as the SARS-CoV-2 spike protein.

[0090] The 5'-UTRs and 3'-UTRs of the present disclosure can increase translation of a coding sequence, optionally in HeLa cells or A549 cells, compared to a 5'-UTR sequence from albumin, which optionally comprises or consists of the sequence set forth in SEQ ID NO: 16 or the corresponding RNA sequence, and a 3'-UTR sequence from HSD17B4, which optionally comprises or consists of the sequence set forth in SEQ ID NO: 17 or the corresponding RNA sequence.

[0091] In one example, the 5'-UTR derived from the 5'-UTR of CHIT1 does not include a sequence containing ATG, and optionally, the 5'-UTR derived from the 5'-UTR of CHIT1 does not include a sequence consisting of ATGGGCTGCAGCCTGCCGCTGA (SEQ ID NO: 35) or a corresponding RNA sequence.

[0092] The present disclosure further provides a deoxyribonucleic (DNA) molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR; and (i) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 1, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (ii) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 3, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 11, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (iii) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 3, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (iv) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 1, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 11, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (v) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 5, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 9, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; or (vi) A deoxyribonucleic (DNA) molecule is provided, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 5, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises the sequence of SEQ ID NO: 11, or a sequence at least 80%, 85%, 90%, or 95% identical thereto.

[0093] The present disclosure further provides a ribonucleic (RNA) molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR; and (i) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 2, or a sequence at least 80%, 85%, 90% or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 10, or a sequence at least 80%, 85%, 90% or 95% identical thereto; (ii) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 4, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 12, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (iii) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 4, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 10, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (iv) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 2, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 12, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; (v) the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 6, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 10, or a sequence at least 80%, 85%, 90%, or 95% identical thereto; or (vi) A ribonucleic (RNA) molecule is provided, wherein the 5'-UTR comprises or consists of the sequence of SEQ ID NO: 6, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and the 3'-UTR comprises or consists of the sequence of SEQ ID NO: 12, or a sequence at least 80%, 85%, 90%, or 95% identical thereto.

[0094] In one example, a nucleic acid molecule of the present disclosure comprises a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21, or a sequence at least 80%, 85%, 90%, or 95% identical thereto. The present disclosure further provides a nucleic acid molecule comprising a 5'-UTR, a coding sequence, and a 3'-UTR, the 5'-UTR comprises a sequence derived from the 5'-UTR of any one of human chitinase-1 (CHIT1), glucuronidase beta (GUSB1), human protein kinase cAMP-activated catalytic subunit beta (PRKACB), and glutamic oxaloacetic transaminase 1 (GOT1); and / or the 3'-UTR comprises a sequence derived from the 3'-UTR of any one of human chitinase-1 (CHIT1), pyruvate kinase L / R (PKLR), and human citrate synthase (CS); The coding sequence is operably linked to a 5'-UTR and a 3'-UTR, and the coding sequence is not CHIT1, GUSB1, PRKACB, GOT1, PKLR, or CS.

[0095] The examples show that these 5'-UTRs and / or these 3'UTRs are capable of increasing translation of a coding sequence as assessed according to the present disclosure.

[0096] The present disclosure further provides a deoxyribonucleic 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:1, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, 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 at least 80%, 85%, 90%, or 95% identical thereto.

[0097] The present disclosure further 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 at least 80%, 85%, 90%, or 95% identical thereto, 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 at least 80%, 85%, 90%, or 95% identical thereto.

[0098] In one example, a 5'-UTR of the present disclosure comprises a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from any one of SEQ ID NOs: 1-8, which sequence can increase translation of a coding sequence, optionally in HeLa cells or A549 cells, compared to a reference 5'-UTR, optionally HSD17B4 5'-UTR (SEQ ID NO: 17), when both molecules contain a constant 3'-UTR sequence. A constant 3'-UTR sequence means that the same 3'-UTR sequence is paired with the 5'-UTR of the present disclosure and the reference 5'-UTR.

[0099] In one example, a 3'-UTR of the present disclosure comprises a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from any one of SEQ ID NOs: 9-14, which sequence can increase translation of a coding sequence, optionally in HeLa cells or A549 cells, compared to a reference 3'-UTR, optionally an albumin 3'-UTR (SEQ ID NO: 16), when both molecules contain a constant 5'-UTR sequence. A constant 5'-UTR sequence means that the same 5'-UTR sequence is paired with the 3'-UTR of the present disclosure and the reference 3'-UTR.

[0100] In one example, a nucleic acid molecule of the disclosure comprises a 5'-UTR comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NO: 1 or the corresponding RNA sequence, and a 3'-UTR comprising a sequence at least 80%, 85%, 90%, or 95% identical to a sequence selected from SEQ ID NO: 9 or the corresponding RNA sequence, wherein the 5'-UTR and 3'-UTR sequences are capable of increasing translation of a coding sequence, optionally in HeLa cells or A549 cells, compared to translation by a nucleic acid molecule comprising a reference 5'-UTR, optionally HSD17B4 5'-UTR (SEQ ID NO: 17 or the corresponding RNA sequence), and a reference 3'-UTR, optionally albumin 3'-UTR (SEQ ID NO: 16 or the corresponding RNA sequence), operably linked to the coding sequence.

[0101] In one example, the 5'-UTR consists of a sequence selected from any one of SEQ ID NOs: 1-8, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, which is capable of increasing translation of the coding sequence as assessed in accordance with the present disclosure, and / or the 3'-UTR consists of a sequence selected from any one of SEQ ID NOs: 9-14, or a sequence at least 80%, 85%, 90%, 95%, or 98% identical thereto, which is capable of increasing translation of the coding sequence as assessed in accordance with the present disclosure.

[0102] In one example, a nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, the coding sequence being operably linked to the 5'-UTR and the 3'-UTR, and the 5'-UTR comprises a sequence derived from human chitinase-1 (CHIT1) 5'-UTR, optionally a sequence according to SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence at least 80%, 85%, 90%, or 95% identical thereto. The examples show that this 5'-UTR can increase translation of the coding sequence as assessed according to the present disclosure.

[0103] In one example, a nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, the coding sequence being operably linked to the 5'-UTR and the 3'-UTR, and the 3'-UTR comprising a sequence derived from human citrate synthase (CS) 3'-UTR, optionally a sequence according to SEQ ID NO: 9 or SEQ ID NO: 10, or a sequence at least 80%, 85%, 90%, or 95% identical thereto. The examples show that this 3'-UTR can increase translation of the coding sequence as assessed according to the present disclosure.

[0104] In one example, a nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, the coding sequence being operably linked to the 5'-UTR and the 3'-UTR, wherein the 5'-UTR comprises a sequence derived from the human chitinase 1 (CHIT1) 5'UTR and the 3'-UTR comprises a sequence derived from the human citrate synthase (CS) 3'-UTR. The Examples show that nucleic acid molecules comprising these 5'-UTRs and 3'-UTRs result in the highest translation levels of multiple proteins of interest in multiple cell lines compared to clinically validated 5'-UTR and 3'-UTR combinations.

[0105] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from human protein kinase cAMP-activated catalytic subunit beta (PRKACB) 5'UTR, optionally a sequence according to SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0106] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR, and wherein the 3'-UTR comprises a sequence derived from human chitinase-1 (CHIT1) 3'-UTR, optionally a sequence according to SEQ ID NO: 11 or SEQ ID NO: 12, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0107] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR, wherein the 5'-UTR comprises a sequence derived from human protein kinase cAMP-activated catalytic subunit beta (PRKACB) 5'-UTR and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure, and the 3'-UTR comprises a sequence derived from chitinase-1 (CHIT1) 3'-UTR, optionally a sequence according to SEQ ID NO: 5, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0108] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from glutamic oxaloacetic transaminase 1 (GOT1) 5'-UTR, optionally a sequence according to SEQ ID NO: 5 or SEQ ID NO: 6, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0109] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably linked to the 5'-UTR and the 3'-UTR, and wherein the 5'-UTR comprises a sequence derived from glucuronidase beta (GUSB1) 5'UTR, optionally a sequence according to SEQ ID NO: 7 or SEQ ID NO: 8, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0110] In one example, the nucleic acid molecule comprises a 5'-UTR, a coding sequence, and a 3'-UTR, wherein the coding sequence is operably 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 a sequence according to SEQ ID NO: 13 or SEQ ID NO: 14, or a sequence at least 80%, 85%, 90%, or 95% identical thereto, and is capable of increasing expression of the coding sequence as assessed in accordance with the present disclosure.

[0111] Table 1 shows the UniProt (release 2022_04) codes of the genes referred to in accordance with the present disclosure.

[0112] [Table 1]

[0113] In one example, the 5'-UTR sequence and / or 3'-UTR sequence of the present disclosure comprises a sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence disclosed herein, which sequence is capable of increasing expression of a coding sequence as assessed according to the present disclosure.

[0114] The nucleic acid molecule of the present disclosure can be, for example, a plasmid, an episome, a cosmid, or a phage. Suitable vectors and methods for vector 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)).

[0115] In one example, the nucleic acid molecules of the present disclosure are closed circular or linear molecules.

[0116] In various examples, a nucleic acid molecule according to the present disclosure includes, in the 5' to 3' direction of transcription, a promoter, 5'-UTRs and 3'-UTRs flanking the coding sequence, and a polyadenylation signal.

[0117] In one example, the nucleic acid molecule of the present disclosure further comprises a 5'-cap structure, optionally a Cap 1 structure. Further suitable cap structures and approaches for generating suitable cap structures are described in WO 2017 / 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 is completed simultaneously during the in vitro transcription reaction using the following chemical RNA cap analog to create a 5'-guanosine cap structure: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap] according to the manufacturer's protocol. G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of the modified RNA may be completed post-transcriptionally using vaccinia virus capping enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both vaccinia virus capping enzyme and 2'-O-methyltransferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure, followed by 2'-O-methylation of the third nucleotide from the 5' end using 2'-O-methyltransferase. The Cap 3 structure may be generated from the Cap 2 structure, followed by 2'-O-methylation of the fourth nucleotide from the 5' end using a 2'-O-methyl-transferase. The enzyme may be derived from a recombinant source. Further suitable means for generating suitable cap structures are disclosed in WO 2016 / 193226, which is incorporated herein by reference.

[0118] In one example, a nucleic acid molecule of the present disclosure includes a promoter that is any promoter for a DNA-dependent RNA polymerase, such as T7 (optionally comprising or consisting of the sequence TAATACGACTCACTATAAGG (SEQ ID NO: 15)), T3, SP6, or Syn5 RNA polymerase.

[0119] In some examples, the nucleic acid molecules disclosed herein contain a polyadenylation signal (polyA tail). The polyA tail is a long sequence of adenine residues at the 3' end of the molecule. The polyA tail serves two purposes: it is essential for translation, and poly(A)-binding protein (PABP) recruits translation factors to enhance translation levels. Furthermore, the polyA tail improves the stability of the nucleic acid molecule by binding poly(A) in mRNA, protecting it from exonuclease digestion. In mRNA, the polyA tail is also known to play an important role in transporting mRNA from the nucleus to the ribosome (Shlake, T. et al., RNA Biol., (2012), 9(11), 1319-1330). In one example, a nucleic acid molecule disclosed herein contains a polyA tail of about 50 to about 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 examples, the poly-A tail contains 50-250 adenosines. In some examples, the poly-A tail contains 60-100 adenosines. In some examples, the poly-A tail contains 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 adenosines. In some examples, the poly-A tail contains 77 adenosines.

[0120] In one example of the present disclosure, the nucleic acid molecule comprises a split poly(A) tail, which can include at least two adenosine-containing elements, optionally containing 30-60 adenosines each, optionally separated by a spacer of 1-25 nucleotides.

[0121] In one example, a nucleic acid molecule of the present disclosure is a ribonucleic acid (RNA) molecule.

[0122] In one example, the ribonucleic acid (RNA) molecule is mRNA.

[0123] Leader sequence 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 the expressed therapeutic protein. In such instances, the leader sequence and the gene of interest are within the same open reading frame (ORF).

[0124] Signal peptides comprise the N-terminal 15–60 amino acids of a protein and are typically required for translocation across membranes along the secretory pathway, controlling the entry of most proteins into the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) directs ribosomes to the rough endoplasmic reticulum (ER) and initiates transport of the growing peptide chain across it for processing. ER processing produces the mature protein, and the signal peptide, at least for secreted proteins, is typically cleaved by a resident signal peptidase.

[0125] The signal peptide may have a length of 15 to 60 amino acids. For example, the signal peptide may have a length of 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 some examples, the signal peptide may have a length of 20 to 60, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 4 ... 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, 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 length. In some examples, the signal peptide has the following sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 34).

[0126] Nucleosides and nucleotides In some examples, the nucleic acid molecules provided herein are not chemically modified and contain the standard nucleotides adenine (A), thymine (T), or uracil (U), and the nucleic acid molecule is RNA, guanine (G), or cytosine (C).

[0127] In some examples, the nucleic acid molecule comprises a modified nucleotide. Many modified nucleotides are known in the art, as disclosed in International Publication No. WO 2007 / 024708, which is incorporated herein by reference. Modifications can include either naturally occurring or non-naturally occurring modifications. Modifications can include those in the sugar, backbone, or nucleobase of the nucleotide and / or nucleoside, as is known in the art.

[0128] In some examples, the nucleic acid molecules herein can include natural (i.e., standard) nucleotides or nucleosides, non-natural or naturally occurring modified nucleotides or nucleosides, or any combination thereof.

[0129] In one example where the nucleic acid molecule is RNA, the RNA can include standard A, G, and C nucleotides, as well as modified U nucleotides.

[0130] In some instances, nucleic acid molecules comprising modified nucleosides or nucleotides (e.g., "modified RNA nucleic acid molecules") exhibit reduced immunogenicity in a cell or organism compared to an unmodified RNA nucleic acid molecule comprising the same sequence.

[0131] In some examples, modified nucleosides in nucleic acid molecules provided herein (e.g., RNA nucleic acid molecules such as mRNA) include N1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1Ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (Ψ). In some examples, modified nucleotides in nucleic acid molecules (e.g., RNA nucleic acid molecules such as mRNA) include 5-methoxymethyluridine, 5-methylthiouridine, 1-methoxymethylpseudouridine, 5-methylcytidine, and / or 5-methoxycytidine. In some examples, RNA nucleic acid molecules include a combination of at least two (e.g., two, three, four, or more) of any of the foregoing modified nucleobases.

[0132] In some examples, the nucleic acid molecules provided herein contain N1-methyl-pseudouridine (m1Ψ) at one or more or all uridine positions of the nucleic acid molecule.

[0133] In some examples, the nucleic acid molecule comprises 5-methoxy-uridine (mo5U) at one or more or all uridine positions of the nucleic acid molecule.

[0134] In some examples, a nucleic acid molecule contains from about 1% to about 100% modified nucleotides (either with respect to overall nucleotide content or with respect to one or more types of nucleotides (i.e., any one or more of A, G, U, T, or C)). In some examples, a nucleic acid molecule contains any intervening percentage of modified nucleotide content, e.g., 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%, 20% ~80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%. The remaining percentages are made up of unmodified A, G, U, T, or C.

[0135] A nucleic acid molecule can contain as little as 1% and as much as 100% modified nucleotides, or any intervening percentage, e.g., at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, a nucleic acid can contain modified pyrimidines such as modified uracil or cytosine. In some examples, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in a nucleic acid are substituted with modified uracils (e.g., 5-substituted uracils). The modified uracils can be substituted with a compound having a single unique structure, or with multiple compounds having different structures (e.g., two, three, four, or more unique structures). In some examples, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the nucleic acid are substituted with modified cytosines (e.g., 5-substituted cytosines). The modified cytosines can be substituted with a compound having a single unique structure or with multiple compounds having different structures (e.g., two, three, four, or more unique structures).

[0136] In some examples, the nucleic acid molecule is an mRNA in which uridines are replaced by compounds having a single unique structure. In some examples, the single unique structure is N1-methyl-pseudouridine. In some examples, 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.

[0137] In one example, the mRNA comprises modified nucleobases. In some examples, the modified nucleobases are modified adenine (A), cytosine (C), uracil (U), and guanine (G).

[0138] In one example, the modified nucleobase is a modified U. In some examples, the modified U is 1-methylpseudouridine (m1Ψ) and pseudouridine (Ψ), as disclosed in WO 2007 / 024708, which is incorporated herein by reference.

[0139] In one example, a nucleic acid molecule of the present disclosure comprises a UTR sequence that includes 5-methoxy-uridine (mo5U) at one or more or all uridine positions of the nucleic acid molecule. The molecule can include a ratio of at least 25% modified to unmodified uridines, including 25%-50%, or at least 50%.

[0140] The examples show that modification of uridines in the 5'-UTR sequence derived from the human CHIT1 5'-UTR and the 3'-UTR sequence derived from the human citrate synthase (CS) 3'-UTR results in a particularly substantial increase in translation.

[0141] In one example, a nucleic acid molecule of the present disclosure comprises a sequence that includes N1-methyl-pseudouridine (m1Ψ) at one or more or all uridine positions of the nucleic acid molecule. The molecule can contain at least a 75% ratio of modified uridines to unmodified uridines, including 100%.

[0142] Code Sequence In one example, a nucleic acid molecule of the present disclosure comprises a coding sequence that encodes a therapeutic protein or peptide, optionally the wild-type sequence of a human protein, or an antibody or antigen-binding fragment thereof.

[0143] Thus, the nucleic acid molecules of the present disclosure may be useful in gene therapy.

[0144] In some instances, the coding sequence is not CHIT1, GUSB1, PRKACB, GOT1, PKLR, or CS.

[0145] In one example, a nucleic acid molecule of the present disclosure comprises a coding sequence that encodes a disease-associated antigen (DAA). This molecule of the present disclosure is a vaccine vector.

[0146] Vaccine Vector Nucleic acid vaccine vectors offer considerable advantages over traditional vaccination approaches. From a safety perspective, RNA-based vaccines are noninfectious, unlike live or live-attenuated vaccination approaches. Furthermore, RNA vaccines do not integrate into the genome and are therefore free of the risk of mutagenesis. Furthermore, DNA- and RNA-based vaccines have been shown to be highly effective against numerous infectious agents, including Zika, influenza, rabies, and SARS-CoV-2. Nucleic acid-based vaccines offer an inexpensive, rapid, and easily scalable alternative to traditional vaccination approaches (Pardi, N., et al., Nature Reviews, (2018), 17, 261-279).

[0147] In one example of the present disclosure, the disease-associated antigen may be a viral antigen, a bacterial antigen, or a tumor-associated antigen.

[0148] Once inside the cell, the coding sequence of the DNA molecule may be transcribed and translated, or the coding sequence of the RNA molecule may be translated, to produce an antigenic protein of the antigen or a fragment thereof.

[0149] During the production of an antigen, exposure of the host immune system to the protein or protein fragment can stimulate an immune response. This immune response can include stimulating antibody production by B cells and producing memory B cells that can produce antibodies against the antigen or fragment thereof of a specific infectious agent. Upon infection with the same infectious agent, the host immune system is primed against this antigen or antigenic protein fragment, shortening the duration of the immune response to the infectious agent. Thus, the onset of symptoms in response to the infection is reduced or prevented.

[0150] In one example of the present disclosure, the nucleic acid molecule comprises a sequence that encodes a nanoantigen particle. The antigen may be an antigen described anywhere herein.

[0151] In one example, the nucleic acid molecule includes a coding sequence encoding a multimerization unit. In one example, the multimerization unit is a ferritin protein. The multimerization unit can be a scaffold for a nanoantigen particle. In some examples, the ferritin is Helicobacter pylori ferritin. In some examples, the nucleic acid molecule includes sequences encoding an antigen protein and a ferritin protein, and the antigen protein and ferritin assemble to form the nanoantigen particle.

[0152] In one example of the present disclosure, the coding sequence further encodes a linker, which may be encoded between the DAA and the multimerization unit, optionally a ferritin protein, such that the DAA is fused to the multimerization unit in the encoded molecule.

[0153] In one example, the nucleic acid molecule comprises an RNA 5'-UTR sequence and a 3'-UTR sequence, as well as an RNA coding sequence, optionally an mRNA sequence.

[0154] coronavirus vaccine The recent Covid-19 pandemic has created an urgent need for improved vaccines targeting coronaviruses of concern. To date, several variants of SARS-CoV-2 have been identified, with some of the most infectious being the delta and omicron variants. All currently approved vaccines for the treatment of SARS-CoV-2 involve stimulating immunity to the SARS-CoV-2 spike protein. However, several mutations in the receptor-binding domain of the spike protein have been identified in new variants of SARS-CoV-2, which are thought to have led to increased vaccine resistance in these newly emerged variants (Zhao, J. et al., Environmental research, (2022), 206 (112240)). Research estimates that current vaccines are approximately 3-5 times less effective against the delta variant of SARS-CoV-2 than the alpha variant (Planas, D. et al., Nature, (2021), 596, 276-280).

[0155] Thus, there is a continuing need for improved vaccines in general, including those useful for the prevention and treatment of coronaviruses.

[0156] Thus, one example of the present disclosure provides a nucleic acid molecule described herein that encodes a coronavirus (CoV) antigen. In one example, the nucleic acid molecule includes RNA 5'-UTR and 3'-UTR sequences and an RNA coding sequence. In one example, the coronavirus antigen can 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 of the following variants: 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.

[0157] Coronavirus virions contain multiple glycosylated spike (S) proteins that protrude from the surface of the virion. These S proteins form trimeric structures and mediate viral entry into host cells, making them prime targets for vaccine design.

[0158] The coronavirus spike protein is 1,273 amino acids long and contains a signal peptide and two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD) that recognizes and binds to a specific host cell receptor, angiotensin-converting enzyme 2 (ACE2). The S2 subunit mediates virus-cell membrane fusion.

[0159] Thus, in one example, the nucleic acid molecule comprises a sequence encoding the S protein or an antigenic fragment thereof. Upon delivery to a host cell, the S protein is translated and processed in the host cell, resulting in the display of trimerized S protein on the host cell surface.

[0160] In some examples, the nucleic acid molecule comprises sequences encoding a CoV S protein and a ferritin protein, wherein the CoV S protein and ferritin assemble to form the nanoantigen particle.

[0161] The S protein may be stabilized in the pre-fusion conformation. Additionally, the S protein may contain the K986P and / or V987P mutations.

[0162] In one example, the nucleic acid molecule of the present disclosure encodes an antigenic fragment thereof that is a receptor binding domain (RBD). In one example, the antigenic fragment is the RBD of the SARS-CoV-2 S protein. In one example, the antigenic fragment is the RBD of the SARS-CoV-1 S protein.

[0163] In some instances, it may be advantageous to fuse a fragment of an antigenic protein to the ferritin in the nanoantigen particle. In one example, the fragment of the antigenic protein may be an RBD. The RBDs in the nanoantigen particle may be derived from antigens of the same infectious agent, i.e., monovalent, or the RBDs of the nanoantigen particle may be derived from antigens of two or more infectious agents, i.e., multivalent.

[0164] Polymerization Unit The nucleic acid molecules provided herein, in some examples, encode fusion proteins that include a vaccine antigen linked to a multimerization unit. In some examples, such multimerization units confer desired properties to the antigen encoded by the nucleic acid molecule. For example, the Examples show that multimerization units improve the immunogenicity of an antigen (e.g., COVID spike protein) compared to the immunogenicity of the same antigen expressed without the multimerization unit. Furthermore, the multimerization units provided herein improve pan-variant responses to the antigen. For example, nucleic acid molecules provided herein that include a coding sequence encoding a COVID spike protein-multimerization unit fusion protein elicit a broader immune response to SARs-CoV-2 variants compared to the spike protein alone.

[0165] In some examples, the multimerization unit is a protein that can self-assemble into highly symmetric, stable, and structurally organized protein nanoparticles with diameters of 10 to 150 nm, a size range well suited for optimal interaction with various cells of the immune system. In some examples, 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 examples, the multimerization unit is hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of approximately 22 nm, lacks nucleic acid, and is therefore non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58-68). In some examples, the multimerization unit is hepatitis B core antigen (HBcAg), which self-assembles into particles with diameters of 24 to 31 nm, similar to viral cores obtained from HEY-infected human livers. The produced HBcAg self-assembles into two classes of nanoparticles of different sizes, with diameters of 300 A and 360 A, corresponding to 180 or 240 protomers. In some instances, an antigen is fused to HBsAG or HBcAG to promote self-assembly of antigen-presenting nanoparticles.

[0166] In some examples, the multimerization unit is selected from the following self-assembling proteins: ferritin, lumazine synthase, and encapsulin.

[0167] Ferritin is a protein whose primary function is intracellular iron storage. It consists of 24 subunits, each consisting of four alpha-helical bundles that 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. Helicobacter pylori ferritin is composed of 24 identical protomers, while animal ferritin light and heavy chains exist that can assemble independently or bind to particles of 24 subunits in different ratios (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 to carrying and exposing antigens.

[0168] Lumazine synthase (LS) is also highly suitable as a nanoparticle platform for antigen presentation. LS, responsible for the penultimate catalytic step in riboflavin biosynthesis, is an enzyme present in a wide variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber, S.E.Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014). LS monomers are 150 amino acids long and consist of a beta sheet flanked by tandem alpha helices. Numerous different quaternary structures have been reported for LS, showing morphological diversity ranging from a homopentamer to a symmetric assembly of 12 pentamers forming a capsid with a diameter of 150 Å. LS cages of over 100 subunits have even been described (Zhang, X. et al., J. Mol. Biol. 2006;362:753-770).

[0169] Encapsulin, a novel protein cage nanoparticle isolated from the thermophilic bacterium Thermotoga maritima, can also be used as a platform for presenting antigens on the surface of self-assembling nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers with a thin, icosahedral, T=1 symmetric cage structure, with inner and outer diameters of 20 nm and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15:939-947). The exact function of encapsulin in T. maritima is not yet clearly understood, but its crystal structure was recently elucidated and its function was hypothesized as a cellular compartment that encapsulates proteins such as DyP (dye decolorizing peroxidase) and Flp (ferritin-like protein) involved in the oxidative stress response (Rahmanpour R. et al. FEES J. 2013, 280:2097-2104).

[0170] In some examples, the nucleic acid molecules provided herein comprise a coding sequence encoding a coronavirus antigen (e.g., a SARS-CoV-2 spike (S) protein) fused to a ferritin subunit.

[0171] Linker In some examples, the nucleic acid molecules disclosed herein encode a fusion protein. In such cases, each of the domains of the fusion protein (e.g., the antigen and the multimerization unit) can be separated by a coding sequence encoding a linker sequence. The linker sequence can be self-cleaving. In other words, the linker can be a self-cleaving linker. In other examples, the linker can be a protease-sensitive linker. In some examples, the linker can be a glycine-serine linker.

[0172] In some examples, the self-cleaving linker is selected from an F2A linker, a P2A linker, a T2A linker, an E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, called 2A peptides, has been described in the art (see, e.g., Kim, J. et al. (2011) PLoS ONE 6:e18556).

[0173] In some examples, the glycine-serine linker has the following amino acid sequence: GSGGSG (SEQ ID NO: 28). In some examples, the glycine-serine linker is encoded by SEQ ID NO: 29 or SEQ ID NO: 30.

[0174] Those of skill in the art will understand that other art-recognized linkers may be suitable for use in the constructs of the present disclosure (e.g., encoded by the nucleic acid molecules provided herein). Those of skill in the art will similarly understand that other polycistronic constructs (nucleic acid molecules encoding two or more antigens / polypeptides separately within the same molecule) may be suitable for use as provided herein.

[0175] manufacturing The nucleic acid vaccine vector of the present disclosure can be produced by in vitro transcription. In vitro transcription of RNA is known in the art and is described in International Publication No. 2014 / 152027, the entire contents of which are incorporated herein by reference. In some examples, the RNA of the present disclosure is prepared according to any one or more of the methods described in International Publication Nos. 2018 / 053209 and 2019 / 036682, each of which is incorporated herein by reference. In summary, a DNA template is typically generated as a linearized plasmid, and then in vitro transcription to synthesize RNA is performed in parallel with or after capping.

[0176] The 5' cap can be added by a multi-step enzymatic reaction or via co-transcription. In co-transcriptional capping, a cap analog such as CleanCap® 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.

[0177] After purification, the mRNA product can be encapsulated in lipid nanoparticles (LNPs).

[0178] composition The present disclosure also provides a pharmaceutical composition comprising a nucleic acid molecule or LNP as defined anywhere herein and a pharmaceutical carrier.

[0179] In one example, the pharmaceutical composition is a monovalent composition comprising a nucleic acid molecule according to the present disclosure encoding a first antigen, or an immunogenic fragment or immunogenic variant thereof.

[0180] In one example, the pharmaceutical composition is a bivalent composition comprising an additional nucleic acid molecule according to the present disclosure encoding a second antigen, or an immunogenic fragment or immunogenic variant thereof, wherein the second antigen is different from the first antigen.

[0181] The present disclosure further provides a composition comprising a first nucleic acid molecule according to the present 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.

[0182] In one example, a composition according to the present disclosure may further comprise a second nucleic acid molecule encoding an Omicron mutant S protein, optionally mutant 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.

[0183] In one example, the second nucleic acid molecule encodes the omicron mutant S protein BA.4 / 5.

[0184] In one example, the second nucleic acid molecule encodes the omicron mutant S protein XBB.1.5.

[0185] In one example of the present disclosure, the composition comprises: 1) a first nucleic acid molecule comprising a 5'-UTR operably linked to a coding sequence, the 5'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 19, and a 3'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 21, wherein the coding sequence comprises a sequence encoding a delta mutant S protein, a linker, and a ferritin protein, and the encoded protein is a delta mutant S protein-ferritin fusion protein; 2) a second nucleic acid molecule comprising a 5'-UTR operably linked to a coding sequence, the 5'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 19, and a 3'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 21, wherein the coding sequence comprises a sequence encoding an omicron mutant S protein, a linker, and a ferritin protein, and the encoded protein is an omicron mutant S protein-ferritin fusion protein.

[0186] In one example of the present disclosure, the composition comprises: 1) a first nucleic acid molecule comprising a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19 and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21 operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding a delta mutant S protein, a linker, and a ferritin protein, and the encoded protein is a delta mutant S protein-ferritin fusion protein; 2) a second nucleic acid molecule comprising a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19 and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21 operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding an omicron mutant S protein, a linker, and a ferritin protein, and the encoded protein is an omicron mutant S protein-ferritin fusion protein.

[0187] In one example of the present disclosure, the first nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:25 and / or the second nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:27.

[0188] In one example of the present disclosure, the first nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:25 and / or the second nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:42.

[0189] In one example of the present disclosure, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 37, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 38. In one example, the first nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 37, and the second nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 38.

[0190] In one example of the present disclosure, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 37, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 43. In one example, the first nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 37, and the second nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 43.

[0191] In one example, the first nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:40, and the second nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0192] In one example, the first nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0193] In one example, the first nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:40, and the second nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0194] In one example, the first nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:40, and the second nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0195] In one example of the present disclosure, the composition comprises: 1) a first nucleic acid molecule comprising a 5'-UTR operably linked to a coding sequence, the 5'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 19, and a 3'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 21, wherein the coding sequence comprises a sequence encoding a Wuhan variant S protein, a linker, and a ferritin protein, and the encoded protein is a Wuhan variant S protein-ferritin fusion protein; 2) a second nucleic acid molecule comprising a 5'-UTR operably linked to a coding sequence, the 5'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 19, and a 3'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 21, wherein the coding sequence comprises a sequence encoding an omicron mutant S protein, a linker, and a ferritin protein, and the encoded protein is an omicron mutant S protein-ferritin fusion protein.

[0196] In one example of the present disclosure, the composition comprises: 1) a first nucleic acid molecule comprising a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19 and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21, operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding a Wuhan variant S protein, a linker, and a ferritin protein, and the encoded protein is a Wuhan variant S protein-ferritin fusion protein; 2) a second nucleic acid molecule comprising a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19 and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21 operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding an omicron mutant S protein, a linker, and a ferritin protein, and the encoded protein is an omicron mutant S protein-ferritin fusion protein.

[0197] In one example of the present disclosure, the first nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:23, and the second nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:27.

[0198] In one example of the present disclosure, the first nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:23, and the second nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:42.

[0199] In one example of the present disclosure, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 36, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 38. In one example, the first nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 36, and the second nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 38.

[0200] In one example of the present disclosure, the first nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 36, and the second nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 43. In one example, the first nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 36, and the second nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 43.

[0201] In one example, the first nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:39, and the second nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0202] In one example, the first nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0203] In one example, the first nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:39, and the second nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0204] In one example, the first nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:39, and the second nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0205] In one example, the first nucleic acid molecule and / or the second nucleic acid molecule of any composition of the present disclosure comprises 80% to 100% N1-methyl-pseudouridine (m1Ψ) at the uridine positions of the nucleic acid molecule.

[0206] In one example, the first vector and / or nucleic acid vector(s) comprises a 5'-cap structure, optionally a Cap 1 structure.

[0207] In one example, the first nucleic acid molecule and / or the second nucleic acid molecule of the composition comprises a T7 promoter sequence, optionally an RNA sequence corresponding to the sequence set forth in SEQ ID NO:15.

[0208] In one example, the first nucleic acid molecule and / or the second nucleic acid molecule of the composition encodes a glycine-serine linker, optionally having the sequence of SEQ ID NO:28.

[0209] In one example, the first nucleic acid molecule and / or the second nucleic acid molecule of the composition comprises a poly-A tail of 70-90 adenosine nucleotides.

[0210] In one example, the first nucleic acid molecule and / or the second nucleic acid molecule of the composition comprises a leader sequence. The leader sequence is cleaved from the mature expressed antigen. In one example, the leader sequence encodes the following amino acid sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 34).

[0211] The present disclosure further provides a monovalent composition comprising a nucleic acid molecule according to the present 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.

[0212] In one example, the nucleic acid molecule encodes the omicron mutant S protein BA.4 / 5.

[0213] In one example, the nucleic acid molecule encodes the omicron mutant S protein XBB.1.5.

[0214] In one example of the present disclosure, a composition includes a nucleic acid molecule comprising a 5'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 19, and a 3'-UTR comprising a sequence at least 95% identical to the sequence of SEQ ID NO: 21, operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding an omicron mutant S protein, a linker, and a ferritin protein, and the encoded protein is a delta mutant S protein-ferritin fusion protein.

[0215] In one example of the present disclosure, a composition includes a nucleic acid molecule comprising a 5'-UTR comprising or consisting of the sequence of SEQ ID NO: 19 and a 3'-UTR comprising or consisting of the sequence of SEQ ID NO: 21 operably linked to a coding sequence, wherein the coding sequence comprises a sequence encoding an Omicron S protein, a linker, and a ferritin protein, and the encoded protein is a delta mutant S protein-ferritin fusion protein.

[0216] In one example of the present disclosure, the nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:27.

[0217] In one example of the present disclosure, the nucleic acid molecule comprises the coding sequence set forth in SEQ ID NO:42.

[0218] In one example of the present disclosure, the nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 38. In one example, the nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 38.

[0219] In one example of the present disclosure, the nucleic acid molecule comprises the sequence set forth in SEQ ID NO: 43. In one example, the nucleic acid molecule consists of the sequence set forth in SEQ ID NO: 43.

[0220] In one example, the nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0221] In one example, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:41.

[0222] In one example, the nucleic acid molecule comprises a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0223] In one example, the nucleic acid molecule contains a coding sequence that encodes the polypeptide sequence set forth in SEQ ID NO:44.

[0224] In one example, the nucleic acid molecules of the composition contain 80% to 100% N1-methyl-pseudouridine (m1Ψ) at the uridine positions of the nucleic acid molecule.

[0225] In one example, the nucleic acid vector comprises a 5'-cap structure, optionally a Cap 1 structure.

[0226] In one example, the nucleic acid molecule of the composition comprises a T7 promoter sequence, optionally an RNA sequence corresponding to the sequence set forth in SEQ ID NO:15.

[0227] In one example, the nucleic acid molecule of the composition encodes a glycine-serine linker, optionally having the sequence of SEQ ID NO:28.

[0228] In one example, the nucleic acid molecule of the composition comprises a poly-A tail of 70-90 adenosine nucleotides.

[0229] In one example, the nucleic acid molecule of the composition comprises a leader sequence. The leader sequence is cleaved from the mature expressed antigen. In one example, the leader sequence encodes the following amino acid sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 34).

[0230] In all examples of this disclosure, the antigen-encoding sequence can be further optimized via mutation to improve protein stability (such as the structure of the CoV spike protein or RBD), maximize protein translation, and reduce unwanted side effects.

[0231] The composition can include an effective amount of a nucleic acid molecule as defined herein. The effective amount of a nucleic acid molecule used therapeutically will depend, for example, on the therapeutic objectives, the route of administration, and the condition of the patient. In one example, an effective amount of a nucleic acid molecule as defined anywhere herein in a pharmaceutical composition is effective to treat or prevent a disease associated with a coronavirus infection.

[0232] The composition is a pharmaceutically acceptable (e.g., physiologically acceptable) composition and includes a carrier, preferably a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. A pharmaceutically acceptable carrier may include one or more excipients. Pharmaceutically acceptable excipients are known and include carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Any suitable carrier can be used within the context of the present disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The physiologically acceptable excipient may be a pH-buffered aqueous solution. Examples of physiologically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, 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 dextrins; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols, such as mannitol and sorbitol; salt-forming counterions, such as sodium; and / or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0233] The composition can be optionally sterilized. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier before use. The composition can be produced according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0234] The compositions may be administered intravenously. The compositions may also be administered parenterally or subcutaneously.

[0235] Methods of administering the pharmaceutical compositions defined herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In particular examples, the pharmaceutical compositions are administered intranasally, intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, intranasal mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local. Each dose may or may not be administered by the same route of administration.

[0236] Delivery System 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, construction of nucleic acids as part of retroviruses or other vectors, etc. In addition, pulmonary administration can also be used, e.g., by use of an inhaler or nebulizer and formulation with an aerosolizing agent.

[0237] The present disclosure relates to nucleic acid molecules that may be suitable for use as vaccine vectors.

[0238] Lipid nanoparticles (LNPs) may be used as a platform for vaccine vector delivery. LNPs may contain ionizable cationic lipids, cholesterol, phospholipids (such as distearoylphosphatidylcholine), and polyethylene glycol (PEG)-lipids. The ionizable cationic lipids are involved in nanoparticle packaging by interacting with negatively charged RNA molecules. Upon administration, LNPs are rapidly cleared from injected tissues and are therefore unlikely to induce inflammation and tissue damage.

[0239] Therefore, in one example of the present disclosure, the nucleic acid molecule described anywhere herein is packaged in a delivery system. In one example, the delivery system is an LNP. Thus, the present disclosure also relates to an LNP comprising the nucleic acid molecule described anywhere herein.

[0240] In one example, the LNP comprises a nucleic acid molecule described anywhere herein, wherein the nucleic acid molecule encodes an antigenic protein. In one example, the LNP comprises one or more nucleic acid molecules described anywhere herein, wherein the molecule encodes a CoV S protein.

[0241] use The present disclosure provides the nucleic acid molecules described herein for use in medicine.

[0242] In one example, a nucleic acid molecule of the present disclosure that encodes a therapeutic protein or peptide is useful for treating a disease or condition characterized by a lack of that therapeutic protein or peptide.

[0243] In one example, the nucleic acid molecules of the present disclosure that encode disease-associated antigens are useful as nucleic acid vaccine vectors.

[0244] After administration to a patient, the coding sequence is transcribed and translated, if it is a DNA sequence, or translated, if it is an RNA sequence, into the antigenic protein or fragment of the antigenic protein it encodes. The production of these antigenic proteins or fragments of antigenic proteins stimulates an immune response, resulting in the production of neutralizing antibodies. Upon infection with the corresponding infectious agent, the presence of neutralizing antibodies and memory B cells increases the rate of the immune response and minimizes the severity and duration of the onset of symptoms.

[0245] The vaccine vectors of the present disclosure may be used as prophylactic treatments against disease-causing target antigens. In one example of the present disclosure, the vaccine vectors may be used to prevent CoV, particularly SARS-CoV-2.

[0246] The vaccine vectors of the present disclosure may also be used as treatments against target antigens that have infected a subject. In one example of the present disclosure, the vaccine vectors may be used to treat CoV, particularly SARS-CoV-2.

[0247] The present disclosure further provides a method of preventing or treating a disease or condition, comprising administering to a patient in need thereof a nucleic acid molecule described anywhere herein. The present disclosure also relates to a nucleic acid molecule described anywhere herein for use in a method of manufacturing a medicament useful for preventing or treating a disease. In one example, the disease is caused by a CoV. In one example, the disease is COVID-19.

[0248] Furthermore, the present disclosure relates to a method of inducing an immune response in a subject, comprising administering to the subject a nucleic acid molecule, combination, composition, pharmaceutical composition, or formulation described anywhere herein.

[0249] In one example, the subject is a human. [Example]

[0250] Here we describe 5'-UTR and 3'-UTR sequences that enhance expression of coding sequences, including in the context of mRNA vaccine vectors.

[0251] The following examples further illustrate the present disclosure, but should not be construed as in any way limiting its scope.

[0252] Example 1: Modifications of the 5'-UTR can affect eGFP mRNA expression in A549 and HeLa cells method: UTR cloning: Candidate 5'-UTR sequences (shown in Table 2 below) were cloned together with eGFP and a reference 3'-UTR sequence (e.g., albumin, used in the CureVac vector described in EP 2831240). Cloning was performed using restriction sites (RE) or seamlessly. The 5' end of the 5'-UTR contained a T7 promoter sequence.

[0253] Generation of in vitro transcription (IVT) templates using PCR: Templates for IVT were generated by PCR using Phusion PCR mix (NEB). The upstream primer contained a T7 promoter sequence, and the downstream primer contained the reverse complement of the 3'-UTR end of each clone along with a T80 sequence. The resulting PCR products contained the relevant coding 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 purified using a PCR purification kit.

[0254] mRNA synthesis using in vitro transcription (IVT): Using templates generated for IVT using PCR, mRNA was prepared using the NEB IVT kit with T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, creating a Cap 1 structure at the 5' end. mRNA was generated using unmodified nucleotides or modified uridine (5' methoxyuridine) at a 25% ratio to unmodified uridine. After the reaction was completed, the DNA template was digested using RNase-free DNase. The mRNA transcript contained 5'-CAP-1-5'-UTR-eGFP coding sequence-3'-UTR-A80. The mRNA was then purified using a silica column and resuspended in water.

[0255] Cell transfection Lung A549 cells were grown in T175 flasks in complete A549 medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated. The day before, 100,000 cells were seeded into each well (96W plate) 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mixture of mRNA and Lipofectamine was added to each well.

[0256] HeLa cells were grown in T175 flasks in complete HeLa medium (Minimum Essential Medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated onto collagen-treated plates. The day before transfection, 100,000 cells in 100 μl of medium were seeded into each well (96W plates, collagen-treated). 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mixture of mRNA and Lipofectamine was added to each well.

[0257] Quantification of eGFP expression: eGFP fluorescence was detected using an Incucyte machine that captures images from live cells. Fluorescence was measured from images in relative fluorescence units (RFU) using Incucyte software. Data reported in the figures show eGFP fluorescence 24 hours after transfection.

[0258] result: The expression levels of the candidate 5'-UTRs are shown in Table 2. The sequences of the candidate 5'-UTRs are shown in SEQ ID NOs: 1 to 8.

[0259] [Table 2]

[0260] Inclusion of candidate 5'-UTRs (Table 2) was shown to increase expression of mRNA encoding eGFP in A549 cells compared with eGFP expression using both the control 5'-UTR (HSD17B4) and the control 3'-UTR (albumin) (Figure 1). Inclusion of the GOT1, PRKACB, CHIT1, or GUSB 5'-UTR corresponded to a 4-fold, 3-fold, 3-fold, and 3-fold increase in eGFP expression, respectively, compared with expression using the control 5'-UTR (HSD17B4).

[0261] This result was confirmed in HeLa cells, where replacement of the control 5'-UTR with the candidate 5'-UTR (Table 2) was shown to increase expression of the mRNA encoding eGFP (Figure 2). Inclusion of the GOT1, PRKACB, CHIT1, or GUSB 5'-UTR corresponded to a 13-fold, 13-fold, 7-fold, and 14-fold increase in eGFP expression, respectively, compared to expression using the control 5'-UTR (HSD17B4).

[0262] Example 2: Modifications of the 3'-UTR can affect eGFP mRNA expression in A549 and HeLa cells method: UTR cloning: Candidate 3'-UTR sequences (shown in Table 3 below) were cloned together with eGFP and a reference 5'-UTR sequence (e.g., HSD17B4, used in the CureVac vector described in EP 2831240). Cloning was performed using restriction sites (RE) or seamlessly. The 5' end of the 5'-UTR contained a T7 promoter sequence.

[0263] Generation of in vitro transcription (IVT) templates using PCR: Templates for IVT were generated by PCR using Phusion PCR mix (NEB). The upstream primer contained a T7 promoter sequence, and the downstream primer contained the reverse complement of the 3'-UTR end of each clone along with a T80 sequence. The resulting PCR products contained the relevant coding 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 purified using a PCR purification kit.

[0264] mRNA synthesis using in vitro transcription (IVT): Using templates generated for IVT using PCR, mRNA was prepared using the NEB IVT kit with T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, creating a Cap 1 structure at the 5' end. mRNA was generated using unmodified nucleotides or modified uridine (5' methoxyuridine) at a 25% ratio to unmodified uridine. After the reaction was completed, the DNA template was digested using RNase-free DNase. The mRNA transcript contained 5'-CAP-1-5'-UTR-eGFP-3'-UTR-A80. The mRNA was then purified using a silica column and resuspended in water.

[0265] Cell transfection Lung A549 cells were grown in T175 flasks in complete A549 medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated. The day before, 100,000 cells were seeded into each well (96W plate) 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mixture of mRNA and Lipofectamine was added to each well.

[0266] HeLa cells were grown in T175 flasks in complete HeLa medium (Minimum Essential Medium MEM supplemented with 10% FBS and 1% non-essential amino acids) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated onto collagen-treated plates. The day before transfection, 100,000 cells in 100 μl of medium were seeded into each well (96W plates, collagen-treated). 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mixture of mRNA and Lipofectamine was added to each well.

[0267] Quantification of eGFP expression: eGFP fluorescence was detected using an Incucyte machine that captures images from live cells. Fluorescence was measured from images in relative fluorescence units (RFU) using Incucyte software. Data reported in the figures show eGFP fluorescence 24 hours after transfection.

[0268] result: The expression levels of the candidate 3'-UTRs are shown in Table 3. The sequences of the candidate 3'-UTRs are shown in SEQ ID NOs: 9 to 14.

[0269] [Table 3]

[0270] Addition of candidate 3'-UTRs (Table 3) was shown to increase the expression of mRNA encoding eGFP in A549 cells compared with both the control 3'-UTR (albumin) and the control 3'-UTR (HSD17B4) (Figure 3). Addition of CHIT1, CS, or PKLR 3'-UTR resulted in a 3-fold, 3-fold, and 2-fold increase in eGFP expression, respectively, compared with the control 3'-UTR (albumin).

[0271] This result was replicated in HeLa cells, where replacement of the control 3'-UTR (albumin) with the candidate 3'-UTRs (Table 3) was shown to increase expression of the mRNA encoding eGFP (Figure 4). Thus, addition of the CHIT1, CS, or PKLR 3'-UTR was consistent with a 7-fold, 4-fold, and 3-fold increase in eGFP expression, respectively, compared with expression using the control 3'-UTR (albumin).

[0272] Example 3: Combination of modified 5'-UTR and modified 3'-UTR can increase eGFP mRNA expression Combinations of selected 5'-UTRs (Table 2) and selected 3'-UTRs (Table 3) were evaluated to determine whether a combinatorial approach could further increase eGFP mRNA expression. The following combinations were assayed: GOT1 / CHIT1, GOT1 / CS, PRKACB / CHIT1, PRKACB / CS, CHIT1 / CHIT1, and CHIT1 / CS.

[0273] method: UTR cloning: The 5'-UTR and 3'-UTR were cloned together with the ORF eGFP. Cloning was performed using restriction sites (RE) or seamlessly. The 5' end of the 5'-UTR contained a T7 promoter sequence.

[0274] Generation of in vitro transcription (IVT) templates using PCR: Templates for IVT were generated by PCR using Phusion PCR mix (NEB). The upstream primer contained a T7 promoter sequence, and the downstream primer contained the reverse complement of the 3'-UTR end of each clone along with a T80 sequence. The resulting PCR products contained the coding sequences for 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 purified using a PCR purification kit.

[0275] mRNA synthesis using in vitro transcription (IVT): Using templates generated for IVT using PCR, mRNA was prepared using the NEB IVT kit with T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, creating a Cap 1 structure at the 5' end. mRNA was generated using unmodified nucleotides or modified uridine (5' methoxyuridine) at a 25% ratio to unmodified uridine. After the reaction was completed, the DNA template was digested using RNase-free DNase. The mRNA transcript contained 5'-CAP-1-5'-UTR-ORF-3'-UTR-A80. The mRNA was then purified using a silica column and resuspended in water.

[0276] Cell transfection: Lung A549 cells were grown in T175 flasks in complete A549 medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated. The day before, 100,000 cells were seeded into each well (96W plate) 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mixture of mRNA and Lipofectamine was added to each well.

[0277] Quantification of eGFP expression: eGFP fluorescence was detected using an Incucyte machine that captures images from live cells. Fluorescence was measured from images in relative fluorescence units (RFU) using Incucyte software. Data reported in the figures show eGFP fluorescence 24 hours after transfection.

[0278] result: It has previously been shown that it is possible to increase eGFP expression by incorporating selected 5'-UTRs and 3'-UTRs together with control 3'-UTRs and 5'-UTRs, respectively (see Examples 1 and 2).

[0279] We compared the expression of eGFP mRNA using these 5'-UTR and 3'-UTR combinations with that using the control 5'-UTR (HSD17B4) and 3'-UTR (albumin). In both cases, eGFP mRNA expression using the candidate 5'-UTR and 3'-UTR combinations was increased compared to the control in A549 cells (Figures 5 and 6). Interestingly, the general trend for each combination was preserved regardless of whether the mRNA contained modified bases (Figure 6) or not (Figure 5). In both cases, GOT / CS, PRKACB / CS, and CHIT / CS produced the greatest increases in GFP fluorescence compared to the control mRNA (HSDB / Alb). Surprisingly, GOT / CS1 appeared to perform best in conjunction with the modified mRNA (Figure 6), while PRKACB / CS produced the highest GFP fluorescence levels when using wild-type bases.

[0280] Example 4: Combination of modified 5'-UTR and modified 3'-UTR can increase expression of mRNA encoding scFv-Fc To test whether UTR combinations act similarly to increase expression levels independent of the gene of interest, these combinations were tested on scFv expression, again with both wild-type and modified mRNA.

[0281] method: UTR cloning: The 5'-UTR and 3'-UTR were cloned together with the mRNA encoding the scFv-Fc. Cloning was performed using restriction sites (RE) or seamlessly. The 5' end of the 5'-UTR contained a T7 promoter sequence.

[0282] Generation of in vitro transcription (IVT) templates using PCR: Templates for IVT were generated by PCR using Phusion PCR mix (NEB). The upstream primer contained a T7 promoter sequence, and the downstream primer contained the reverse complement of the 3'-UTR end of each clone along with a T80 sequence. The resulting PCR products contained the coding sequences for 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 purified using a PCR purification kit.

[0283] mRNA synthesis using in vitro transcription (IVT): Using templates generated for IVT using PCR, mRNA was prepared using the NEB IVT kit with T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, creating a Cap 1 structure at the 5' end. mRNA was generated using unmodified nucleotides or modified uridine (5' methoxyuridine) at a 25% ratio to unmodified uridine. After the reaction was completed, the DNA template was digested using RNase-free DNase. The mRNA transcript contained 5'-CAP-1-5'-UTR-ORF-3'-UTR-A80. The mRNA was then purified using a silica column and resuspended in water.

[0284] Cell transfection Lung A549 cells were grown in T175 flasks in complete A549 medium (Ham's F-12K supplemented with 10% FBS) at 37°C and 5% CO2. Cells were harvested using Accutase for 5 minutes at 37°C, then counted, washed, and replated. The day before, 100,000 cells were seeded into each well (96W plate) 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 complexes were then mixed by vortexing, spun down, and incubated at room temperature for 5–10 minutes. 10 μl of the mRNA and Lipofectamine mixture was added to each well, and 100 μl of supernatant was collected at specific time points.

[0285] Quantitation of scFv-Fc: After 24 hours, cell supernatants (sup) were collected from cells transfected with scFv-Fc mRNA. The sup was frozen at -80°C until quantification. The cis-bio kit for Fc quantification was used. The principle of quantification is based on a competitive immunoassay using HTRF technology. hFc-tagged proteins (or antibodies) can displace the binding between d2-labeled IgG and cryptate-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 from known concentrations of scFv-Fc, and the signal from the sup of scFv-Fc-transfected cells was interpolated using this standard curve to quantify the amount of scFv-Fc present in the sup. The concentration of scFv-Fc was measured in ng / mL. The data shown in the figure report the scFv-Fc levels 24 hours after transfection.

[0286] result: Again, the expression of scFv mRNA using these 5'-UTR and 3'-UTR combinations was compared with that using the 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 level when wild-type mRNA was used (Figure 7). However, with the exception of CHIT / CS, most of the other UTR combinations did not affect the detectable scFv expression level 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).

[0287] Thus, of all the novel UTRs tested, whether alone or in combination, the CHIT / CS and PRKACB / CHIT combinations produced the greatest increase in expression levels, regardless of the gene of interest or whether the mRNA contained a modified or wild-type U.

[0288] Example 5 - Enhanced protein expression from mRNA vaccine vectors in vitro Next, using the UTR sequences reported by Andrew Fire et al. of Stanford University via GitHub on April 14, 2021, one of the optimal UTR combinations (CHIT / CS) was directly tested against mRNAs containing the putative UTR pairs from Moderna and Pfizer / BioNTech (mRNA Comp A and mRNA Comp B). This time, the modified mRNA contained 100% 5' methoxyuridine (rather than 25% as in Examples 1-4).

[0289] EGFP construct cloning: An mRNA construct was designed (mRNA_AZ) that encodes an EGFP reporter and uses the CHIT1 5'-UTR (SEQ ID NO: 19) paired with the CS 3'-UTR (SEQ ID NO: 21). Simultaneously, mRNA comparator A and mRNA comparator B constructs were designed to encode EGFP adjacent to the predicted UTR sequences from each of the COVID vaccines listed above. Cloning was performed using a Gibson-based assembly method. Each plasmid contained a T7 promoter sequence upstream of each 5'-UTR and an 80-base pair polyA track downstream of the 3'-UTR, along with a single BspQI site for subsequent linearization. All EGFP coding sequences were identical. The complete 5'-UTR and 3'-UTR sequences, including sequences derived from the CHIT1 and CS UTRs, are shown below (SEQ ID NOs: 19 and 21, respectively).

[0290] Generation of in vitro transcription (IVT) templates: Templates for IVT were generated after plasmid purification from bacterial cells in a similar manner as outlined in Examples 1-4.

[0291] mRNA synthesis using in vitro transcription (IVT): Using the generated IVT template, mRNA was prepared using the NEB IVT kit with T7 RNA polymerase. The protocol was modified to include CleanCap® (AG). T7 polymerase incorporates CleanCap® (AG) at the start of each mRNA, creating a Cap 1 structure at the 5' end. mRNA was generated using either unmodified nucleotides or 100% modified uridine (N1-methylpseudouridine). Both mRNA comp A and mRNA comp B (mRNAs containing predicted UTRs derived from the GitHub database) contained 100% modified U. After the reaction was completed, the DNA template was digested using RNase-free DNase. The mRNA was then purified using a silica column and resuspended in water.

[0292] Quantification of EGFP expression: Purified mRNA was transfected into either BHK-21 or HEK293 cells using Lipofectamine MessengerMAX transfection reagent (ThermoFisher) according to the manufacturer's protocol. EGFP fluorescence was detected over a 96-hour period using an IncuCyte instrument that captures images from live RNA-transfected cells. Fluorescence was measured from the images as relative fluorescence units (arbitrary units) using IncuCyte software.

[0293] result: mRNA with a set of 5'-UTR CHIT1 (SEQ ID NO: 19) and 3'-UTR CS (SEQ ID NO: 21) UTRs resulted in the highest levels of EGFP expression compared to two competitor mRNA molecules (mRNA Comp A and mRNA Comp B) in both BHK-21 cells (Figure 9A) and HEK293 cells (Figure 9B). Maximum expression from the mRNA depended on the incorporation of modified nucleotides, such as N1-methylpseudouridine (pseudo-U), to circumvent the host cell antiviral response in HEK293 cells (Figure 9B).

[0294] Example 6 - Efficacy of mRNA vaccine vectors containing antigen-linker-ferritin sequences The overall objective of this study was to determine the immunogenicity of a candidate SARS-CoV-2 mRNA vaccine containing the mCHIT / CS UTR combination in mice and naive non-human primates. The mRNA vaccine encoded a stabilized spike (S) protein-ferritin subunit fusion protein that, upon expression, assembled into nanoparticles for high-density antigen presentation.

[0295] method Mouse studies Groups of naive BALB / c mice (n=6 per group) received two 50 μl injections of the LNP-formulated mRNA vaccine intramuscularly (21 days apart) into the thigh muscle. Fourteen days after the second vaccination, mice were bled, serum was collected, and a SARS-CoV-2 pseudovirus-based neutralization assay was performed. Both the magnitude and breadth of neutralizing antibody (nAb) responses among different groups were assessed using a panel of pseudoviruses bearing the following SARS-CoV-2 spike proteins of interest: Delta, Wuhan (D614G), BA.1, BA.2, and BA.4 / 5. Sigmoidal curves were constructed, averaging triplicates at each serum dilution, to determine the 50% (ID ). 50 ) Neutralizing activity was calculated, with uninfected cells considered to represent 100% neutralization and cells transduced with virus alone considered to represent 0% neutralization.

[0296] Non-human primate studies Groups of naive cynomolgus non-human primates (NHPs; n=6 per group) received two intramuscular doses (28 days apart) of the LNP-formulated mRNA vaccine as 1 mL injections. Each animal received a dose of 10 μg per vaccination. 14 days after the second vaccination, blood was drawn from the NHPs, and serum was collected for SARS-CoV-2 pseudovirus-based neutralization assays. Both the magnitude and breadth of neutralizing antibody (nAb) responses among different groups were assessed using a panel of pseudoviruses bearing the following SARS-CoV-2 spike proteins of interest: Delta, Wuhan (D614G), BA.1, BA.2, and BA.4 / 5. Sigmoidal curves were generated by averaging triplicates at each serum dilution, and a 50% ( ID50 ) Neutralizing activity was calculated, with uninfected cells considered to represent 100% neutralization and cells transduced with virus alone considered to represent 0% neutralization.

[0297] In both the mouse and NHP studies, groups were administered either a DeltaS protein ferritin construct (DeltaFL VLP) or mRNA encoding only the DeltaS protein (DeltaFL spike). This allowed for a comparison of the immunogenicity and reactogenicity profiles of the mRNA-based nanoparticle vaccine approach compared to the spike-only approach used in Tojinameran and Elasomeran. Both the DeltaFL VLP construct and the DeltaFL spike construct contained the CHIT1 / CS UTR combination disclosed herein.

[0298] result The results shown in Figure 10 (mice) and Figure 11 (NHPs) demonstrate that the amplitude of the neutralizing antibody response to the cognate variant (Delta) was significantly greater in animals inoculated with DeltaFL VLP compared to animals administered DeltaFL spike. Interestingly, the breadth of the neutralizing antibody response was also greater for all variants tested (Figures 10 and 11).

[0299] Table 4 below shows the fold change of mRNA VLP / FL spikes for the data in FIG.

[0300] [Table 4] 1 Fold change = mRNA VLP geometric mean titer (GMT) / mRNA FL spike GMT 2 p-values ​​calculated using two-tailed t-tests

[0301] This suggests that when mRNA is used to deliver nanoparticle antigens, a broad neutralizing response can be obtained from a single vaccine construct, which may result in better efficacy against multiple existing and possibly future variants of concern, without the need to independently generate boosters each time a new dominant variant emerges.

[0302] array 5'-UTR modification (m)CHIT1 DNA sequence (SEQ ID NO: 1): ATTGTGCTGCATC RNA sequence (SEQ ID NO:2): AUUGUGCUGCAUC 5'-UTR PRKACB DNA sequence (SEQ ID NO: 3): ATTCTGCTGTTTGCTCCTTGCCAGGTTCAAC RNA sequence (SEQ ID NO: 4): AUUCUGCUGUUUGCUCCUUGCCAGGUUCAAC 5'-UTR GOT1 DNA sequence (SEQ ID NO: 5): AAAATCTCTTGATTCCTAGTCTCTCGAT RNA sequence (SEQ ID NO: 6): AAAAUCUCUUGAUUCCUAGUCUCUCGAU 5'-UTR GUSB DNA sequence (SEQ ID NO: 7): ATCCTCAACCAAGCGCCGCAGACGGTGGCCGAGCGGGGGACCGGGAAGC RNA sequence (SEQ ID NO: 8): AUCCUCAACCAAGCGCCGCAGACGGUGGCCGAGCGGGGGACCGGGAAGC 3'-UTR CS DNA sequence (SEQ ID NO: 9): AACTGGAGACTGGGTGAAAGTGACTACCAGAAAGTGAGGAAGCCTAAATAAA RNA sequence (SEQ ID NO: 10): AACUGGAGACUGGGGUGAAAGUGACUACCAGAAAGUGAGGAAGCCUAAAUAAA 3'-UTR CHIT1 DNA sequence (SEQ ID NO: 11): GTCGCTAAAGCCCCTCCAGTCCCAGCTTTGAGGCTGGGCCCAGGATCACTCTACAGCCTGCCTCCTGGGTTTTCCCTGGGGGCCGCAATCTGGCTCCTGCAGGCCTTTCTGTGGTCTTCCTTTATCCAGGCTTTCTGCTCTCAGCCTTGCCTTCCTTTTTTCTGGGTCTCCTGGGCTGCCCCTTTCACTTGCAAAATAAA RNA sequence (SEQ ID NO: 12): GUCGCUAAAGCCCCUCCAGUCCCAGCUUUGAGGCUGGGCCCAGGAUCACUCUACAGCCUGCCUCCUGGGUUUUCCCUGGGGGCCGCAAUCUGGCUCCUGCAGGCCUUUCUGUGGUCUUCCUUUAUCCAGGCUUUCUGCUCUCAGCCUUGCCUUCCUUUUUUCUGCGACUCCUGGGCUGCCCCUUUCACUUGCAAAAUAAA 3’-UTR PKLR DNA sequence (SEQ ID NO: 13): GACGCCCCTCCCTCCTCTGGAGTCTACGTTCTCCAGCCCACACCCCTCCAAAGCCCCACCTTTAAGTCCTCTCTTCTCTATTCCTGACCCTCCCTACCTGAGGCCTATCTGAGACTATAACTGTCATCTAGCCCCTTCGAGGTTGCCCCTTCCCCATCTCCATTTCACACAGGTCCTGAAAGTCTGTGTCCAATTATGCACTGGCCACCCAACAGCACCAATTGTACATTCCCTGCATCCAATCTGCTCAGCAGGCCCTAAGATGCCTTGAGTCTTTAATCCCA RNA sequence (SEQ ID NO: 14): GACGCCCCUCCCUCCUCUGGAGUCUACGUUCUCCAGCCCACACCCCUCCAAAGCCCCACCUUUAAGUCCUCUUCUCUAUUCCUGACCCUCCCUACCUGAGGCCUAUCUGAGACUAUAACUGUCAUCUAGCCCCUUCGAGG UUGCCCCUUCCCCAUCUCCAUUUCACACAGGUCCUGAAAGUCUGUGUCCAAUUAUGCACUGGCCACCCAACAGCACCAAUUGACAUUCCCUGCAUCCAAUCUGCUCAGCAGGCCCUAAGAUGCCUUGAGUCUUUAAUCCCA T7 promoter sequence (SEQ ID NO: 15): TAATACGACTCACTATAAGG 3'UTR albumin sequence (SEQ ID NO: 16): GCATCACATTTAAAAGCATCTCAGCCTACCATGAGAATAAGAGAAAGAAAATGAAGATCAATAGCTTATTCATCTCTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAACCT 5'UTR HSDB sequence (SEQ ID NO: 17): GTCCCGCAGTCGGCGTCCAGCGGCTCTGCTTGTTCGTGTGTGTGTCGTTGCAGGCCTTATT 5'-UTR CHIT1 and additional sequence elements (mCHIT1 5'-UTR sequence underlined) DNA sequence (SEQ ID NO: 18):

[0303] [Table 5] RNA sequence (SEQ ID NO: 19):

[0304] [Table 6] 3'-UTR CS and additional sequence elements (CS 3'-UTR sequence underlined) DNA sequence (SEQ ID NO: 20):

[0305] [Table 7] RNA sequence (SEQ ID NO:21):

[0306] [Table 8] SARS-CoV-2 Wuhan D614G spike protein-linker-ferritin DNA sequence (SEQ ID NO: 22) SARS-CoV-2 Wuhan D614G spike protein-linker-ferritin RNA sequence (SEQ ID NO: 23) SARS-CoV-2 delta-linker-ferritin DNA sequence (SEQ ID NO: 24) SARS-CoV-2 delta-linker-ferritin RNA sequence (SEQ ID NO: 25) SARS-CoV-2 Omicron BA.4 / 5-Linker-Ferritin DNA Sequence (SEQ ID NO: 26) SARS-CoV-2 Omicron BA.4 / 5-linker-ferritin RNA sequence (SEQ ID NO: 27) Linker amino acid sequence: GSGGSG (SEQ ID NO: 28). Linker DNA sequence: GGTTCAGGTGGATCAGGT (SEQ ID NO: 29) Linker RNA sequence: GGUUCAGGUGGAUCAGGU (SEQ ID NO: 30) Ferritin subunit, DNA sequence (SEQ ID NO: 31): GATATAGAAAAACTCCTCAATGAACAAGTAAATAAGGAGATGCAAAGTTCTAACCTGTACATGAGCATGTCTTCTTGGTGTTACACCCATAGCCTCGATGGAGCGGGATTGTTCCTTTTTGACCACGCTGCGGAGGAGTATGAGCATGCTAAAAAGCTGATAATATTTCTCAACGAGAATAATGTTCCAGTGCAATTGACAAGTATATCCGCCCCTGAGCATAAGTTTGAAGGGCTCACACAAA TTTTCCAAAAGGCATACGAACACGAACAGCACATTAGCGAGTCTATTAAACAACATTGTTGATCATGCAATCAAGTCCAAAGATCACGCCACGTTTAATTTCCTCCAGTGGTATGTAGCTGAGCAACATGAGGAAGAAGTGTTGTTTAAGGATATTCTTGATAAAATTGAACTTATTGGAAATGAGAGAACCATGGCCTCTATCTTGCGGACCAATACGTCAAGGGAATTGCCAAGTCCCGCAAGAGT Ferritin subunit, RNA sequence (SEQ ID NO: 32): GAUAUAGAAAAACUCCUCAAUGAACAAGUAAAUAAGGAGAUGCAAAGUUCUAACCUGUACAUGAGCAUGUCUUCUUGGUGUUACACCCAUAGCCUCGAUGGAGCGGGAUUGUUCCUUUUUGA CCACGCUGCGGAGGAGUAUGAGCAUGCUAAAAAGCUGAUAAUUUUCUCAACGAGAAUAAUGUUCCAGUGCAAUUGACAAGUAUAUCCGCCCCUGAGCAUAAGUUUGAAGGGCUCACACAAA UUUUCCAAAAGGCAUACGAACACGAACAGCACAUUAGCGAGUCUAUUAACAACAUUGUUGAUCAUGCAAUCAAGUCCAAAGAUCACGCCACGUUUAAUUUCCUCCAGUGGUAUGUAGCUGAG CAACAUGAGGAAGAAGUGUGUUUAAGGAUAUUCUUGAUAAAAUUGAACUUAUUGGAAAUGAGAACCAUGGCCUCUAUCUUGCGGACCAAUACGUCAAGGGAAUUGCCAAGUCCCGCAAGAGU Ferritin subunit, protein sequence (SEQ ID NO: 33) DIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS Leader sequence: MPLLLLLPLLWAGALA (SEQ ID NO: 34). CHIT1 upstream ORF, longer sequence: ATGGGCTGCAGCCTGCCGCTGA (SEQ ID NO: 35) mRNA construct sequence encoding SARS-CoV-2 Wuhan D614G spike-ferritin fusion protein (SEQ ID NO: 36) mRNA construct sequence encoding SARS-CoV-2 deltaspike-ferritin fusion protein (SEQ ID NO: 37) mRNA construct sequence encoding SARS-CoV-2 omicron BA.4 / 5 spike-ferritin fusion protein (SEQ ID NO: 38) SARS-CoV2 Wuhan D614G spike-ferritin fusion protein polypeptide sequence (SEQ ID NO: 39)

[0307] [Table 9] SARS-CoV-2 deltaspike-ferritin fusion protein polypeptide sequence (SEQ ID NO: 40)

[0308] [Table 10] SARS-CoV-2 Omicron BA.4 / 5 spike-ferritin fusion protein polypeptide sequence (SEQ ID NO: 41) (leader sequence underlined)

[0309] [Table 11] SARS-CoV-2 XBB.1.5 spike protein-linker-ferritin RNA sequence (SEQ ID NO: 42) mRNA construct sequence encoding SARS-CoV-2 XBB.1.5 spike-ferritin fusion protein (SEQ ID NO: 43) SARS-CoV2 Omicron XBB.1.5 Spike-Ferritin Fusion Protein Polypeptide Sequence (Leader Sequence Underlined) (SEQ ID NO: 44)

[0310] [Table 12]

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