CN116042657A

CN116042657A - Self-replicating mRNA vaccine

Info

Publication number: CN116042657A
Application number: CN202310061944.9A
Authority: CN
Inventors: 宫悦; 刘晓虎; 余志斌; 徐江; 丁隽; 雍丹妮; 刘根盛; 贾为国
Original assignee: Shanghai Funuojian Biotechnology Co ltd
Current assignee: Shanghai Funuojian Biotechnology Co ltd
Priority date: 2023-01-16
Filing date: 2023-01-16
Publication date: 2023-05-02
Also published as: WO2024152996A1

Abstract

The present invention provides a messenger ribonucleic acid (mRNA) molecule comprising: expression cassette 1, which contains the coding sequence for the RNA replicase, and expression cassette 2, which contains the coding sequence for the antigen. The invention also provides an mRNA vaccine obtained by encapsulating the mRNA molecules of the invention into Lipid Nanoparticles (LNPs). The invention also provides DNA molecules which are transcribed into mRNA molecules of the invention. The invention also provides the use of an mRNA molecule of the invention in the manufacture of an mRNA vaccine that elicits an anti-HER 2 immune response. The present invention also provides a method for producing an mRNA molecule, comprising: (1) A viral-based genomic DNA sequence in which the portion following the promoter of the structural polyprotein in the coding sequence of the structural polyprotein is replaced with the coding sequence of the antigen; (2) A promoter site for RNA polymerase is added to the 5 'side of the 5' UTR; (3) adding poly A to the 3 'side of the 3' UTR; and (4) transcribing the DNA molecule constructed in steps (1) to (3) into an mRNA molecule.

Description

Self-replicating mRNA vaccine

[ field of technology ]

The present invention relates to self-replicating messenger ribonucleic acid (mRNA) molecules, self-replicating mRNA vaccines, DNA molecules for transcription into self-replicating mRNA molecules, the use of self-replicating mRNA molecules in the manufacture of self-replicating mRNA vaccines that elicit an anti-HER 2 immune response, and methods of manufacturing self-replicating mRNA molecules.

[ background Art ]

Human epidermal growth factor receptor 2 (HER 2) is a functional receptor expressed on the surface of normal cells, but is a tumor-associated antigen that is abnormally high in 30% of breast cancer patients' tumor tissues. In patients with high HER2 expression, tumors will show a stronger tumor aggressiveness and a higher recurrence frequency. The clinical data at present show that the indexes such as survival time or recurrence probability of a patient population generating cytotoxic immune response against HER2 in the organism are obviously improved, and the feasibility of enabling the organism to obtain long-term immunotherapy or prevention effect is suggested by expressing HER2 to attack and kill tumor cells which highly express the antigen by inducing the organism to generate specific immune response against HER2 and simultaneously forming immune memory.

Among the various forms of tumor vaccines based on immune cells, polypeptides, viral vectors and nucleic acid molecules, mRNA vaccines present unique advantages. The mRNA vaccine enters cytoplasm to complete the translation expression of antigen, and is not integrated into genome, thus avoiding the risk of antigen gene insertion. mRNA vaccines demonstrate the ability to induce both humoral and cellular immunity, inducing a strong immune response in vivo. The mRNA vaccine uses the cell-free preparation method of in-vitro transcription and microfluidic mixed encapsulation nano lipid particles, gets rid of the dependence production of living cells, is convenient for mass production, shortens the production period and simplifies the production and purification process.

At present, linear mRNA vaccines are divided into non-replication and self-replication, the self-replication mRNA is obtained by modifying the genome structure of RNA virus, and the number of mRNA molecules expressing antigens is efficiently amplified by using viral replicase, so that the aim of expressing a large number of antigen proteins by a small number of mRNA molecules is fulfilled, and the vaccination quantity of the mRNA can be greatly reduced. In addition, since self-replicating mRNA can potentially activate a natural immune response, the immune response of the body can be further enhanced, forming an adjuvant effect.

mRNA tumor vaccines based on tumor-associated antigens that have entered clinical trials are classified as either recombinant viral RNA replicon particle vaccines produced by cells or non-replicating mRNA vaccines based on lipid nanoparticles synthesized by cells. The former can produce recombinant viral RNA replicon particles with single infection capacity in cells, and can effectively deliver self-replicating mRNA molecules through the interaction of viral proteins and cell receptors, and can realize high-level expression of tumor-related antigens to stimulate the body immunity due to the amplification effect of the self-replicating mRNA molecules, but can possibly lead to unexpected immune response against the viral vector due to the immunogenicity of the viral vector, so that the future application of continuous use of the same viral vector for booster inoculation can be prevented. In addition, the recombinant viral vector has higher technical requirements and complex production process, and can limit the rapid application of viral vector tumor vaccines to a certain extent.

Non-replicating mRNA vaccines based on lipid nanoparticle delivery are expressed by the delivery of non-replicating mRNA encoding tumor-associated antigens to cells with potential lipid receptor interactions by the nanolipid particles. Because the synthesis of mRNA molecules and the loading of mRNA molecules to the nano lipid particles are carried out through the in vitro transcription and encapsulation of acellular matrix, the preparation process is simple, and the rapid synthesis and use of vaccines are facilitated. However, the amount of non-replicating mRNA expressed antigen is proportional to the amount of mRNA successfully delivered during immunization and the half-life is short, so that in order to achieve the antigen expression levels necessary to elicit adequate immunoprotection in vivo, either more doses of vaccine or repeated vaccination are required.

[ invention ]

The invention relates to the following embodiments:

1. a messenger ribonucleic acid (mRNA) molecule comprising:

expression cassette 1 comprising coding sequence for RNA replicase, and

expression cassette 2 comprising coding sequences for antigens.

2. The mRNA molecule of embodiment 1, wherein the RNA replicase is a viral RNA replicase.

3. The mRNA molecule of embodiment 1, wherein the expression cassette 1 is derived from a coding sequence of a viral non-structural polyprotein.

4. The mRNA molecule according to embodiment 2 or 3, wherein the virus is a virus of the Togaviridae family (Togaviridae).

5. The mRNA molecule according to embodiment 4, wherein the virus of the Togaviridae family (Togaviridae) is a virus of the Alphavirus genus (Alphavirus).

6. The mRNA molecule according to embodiment 4, wherein the virus of the Alphavirus genus is venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV).

7. The mRNA molecule of embodiment 1, wherein the antigen is a HER2 antigen or variant thereof.

8. The mRNA molecule of embodiment 7, wherein the HER2 antigen variant has an engineered intracellular domain (ICD) in which a Tyrosine Kinase (TK) active site is inactivated mutated.

9. The mRNA molecule of embodiments 7 or 8, wherein the HER2 antigen variant has a major histocompatibility complex 1 (MHC 1) transport domain (MITD) that replaces the transmembrane domain (TMD) of the HER2 antigen.

10. The mRNA molecule of embodiment 9, wherein the HER2 antigen variant has a Signal Peptide (SP) of MITD that replaces the SP of HER2 antigen.

11. The mRNA molecule of

embodiments

8 or 10, wherein the HER2 antigen variant has the sequence of SEQ ID NO:2 or 4.

12. The mRNA molecule of embodiment 11, wherein the coding sequence of the HER2 antigen variant has the sequence of SEQ ID NO:1 or 3.

13. The mRNA molecule of embodiment 7, wherein the 5' side of the coding sequence of the HER2 antigen or variant thereof has a Kozak sequence.

14. The mRNA molecule of embodiment 13, wherein the Kozak sequence is GCCACCAUGG.

15. The mRNA molecule of embodiment 1, wherein the expression cassette 2 has a promoter, the coding sequence for the antigen being located downstream of the promoter.

16. The mRNA molecule according to embodiment 15, wherein the promoter is derived from the species from which the expression cassette 1 is derived.

17. The mRNA molecule of embodiment 15 or 16, wherein the promoter is a 26S promoter.

18. The mRNA molecule of embodiment 1, wherein

The upstream of the expression cassette 1 is also provided with a promoter site and a 5' UTR of RNA polymerase; and is also provided with

The expression cassette 2 also has a 3' UTR and a poly A downstream.

19. The mRNA molecule of embodiment 18, wherein the RNA polymerase is a T7 RNA polymerase.

20. The mRNA molecule of embodiment 18, wherein the 5'utr and 3' utr are derived from a species from which the expression cassette 1 is derived.

21. The mRNA molecule of

embodiment

19 or 20, which corresponds to a DNA sequence having the sequence of SEQ ID NO:5 or 6.

22. The mRNA molecule of embodiment 1, wherein the uridine triphosphate is natural Uridine Triphosphate (UTP) or N1-methyl pseudouridine triphosphate (N1-Me-pUTP).

23. A messenger ribonucleic acid (mRNA) vaccine obtained by encapsulating the mRNA molecule according to any one of embodiments 1 to 22 into Lipid Nanoparticles (LNP).

A DNA molecule transcribed into an mRNA molecule according to any of embodiments 1 to 22.

25. Use of an mRNA molecule according to any one of embodiments 1 to 22 in the manufacture of a messenger ribonucleic acid (mRNA) vaccine that elicits an anti-HER 2 immune response.

26. A method of making a messenger ribonucleic acid (mRNA) molecule comprising:

(1) A viral-based genomic DNA sequence in which the portion following the promoter of the structural polyprotein in the coding sequence of the structural polyprotein is replaced with the coding sequence of the antigen;

(2) A promoter site for RNA polymerase is added to the 5 'side of the 5' UTR;

(3) Adding poly-A to the 3 'side of the 3' UTR; and

(4) Transcribing the DNA molecule constructed in steps (1) to (3) into an mRNA molecule.

27. The method of embodiment 26, wherein the virus is a virus of the Togaviridae family (Togaviridae).

28. The method of embodiment 27, wherein the virus of the Togaviridae family (Togaviridae) is a virus of the Alphavirus genus (Alphavirus).

29. The method of embodiment 28, wherein the virus of the genus Alphavirus (Alphavirus) is venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV).

30. The method of embodiment 26, wherein the antigen is a HER2 antigen or variant thereof.

31. The method of embodiment 30, wherein the HER2 antigen variant has an engineered intracellular domain (ICD) in which a Tyrosine Kinase (TK) active site is inactivated mutated.

32. The method of embodiment 30 or 31, wherein the HER2 antigen variant has a major histocompatibility complex 1 (MHC 1) transport domain (MITD) that replaces the transmembrane domain (TMD) of the HER2 antigen.

33. The method of embodiment 32, wherein the HER2 antigen variant has a Signal Peptide (SP) of MITD that replaces the SP of HER2 antigen.

34. The method of embodiment 31 or 33, wherein the HER2 antigen variant has the amino acid sequence of SEQ ID NO:2 or 4.

35. The method of embodiment 34, wherein the coding sequence of the HER2 antigen variant has the sequence of SEQ ID NO:1 or 3.

36. The method of embodiment 30, wherein the 5' side of the coding sequence of the HER2 antigen or variant thereof has a Kozak sequence.

37. The method of embodiment 36, wherein the Kozak sequence is GCCACCATGG.

38. The method of embodiment 26, wherein the DNA molecule constructed by steps (1) - (3) has the sequence of SEQ ID NO:5 or 6.

39. The method of embodiment 26, wherein the transcription is in vitro transcription.

40. The method of embodiment 39, wherein the in vitro transcription uses:

nucleic acid vectors for in vitro transcription, in which the DNA molecules constructed in steps (1) to (3) are inserted,

adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate, and

RNA polymerase.

41. The method of embodiment 40, wherein the uridine triphosphate is natural Uridine Triphosphate (UTP) or N1-methyl pseudouridine triphosphate (N1-Me-pUTP).

42. The method of embodiment 40, wherein the RNA polymerase is T7 RNA polymerase.

[ technical Effect ]

Through the implementation mode, the invention at least achieves the following technical effects:

(1) When the self-replicating mRNA enters cells for expression replication, the full-length self-replicating mRNA which jointly codes antigen molecules and non-structural polyprotein and independently codes antigen can be replicated at high level, but the self-replicating mRNA which independently codes antigen molecules can have higher replication level, so that the high-efficiency expression of the antigen can be realized under the condition of avoiding immune response and toxicity caused by the expression of virus components as much as possible.

(2) Self-replicating mRNA vaccines activate a stronger immune response against antigens and tumor-specific killing effect than non-replicating mRNA vaccines.

(3) Compared with non-replicating mRNA, only 1/10 dose of self-replicating mRNA tumor vaccine can realize the same level of immunostimulating effect, so that mRNA immunity dose can be reduced, adverse reaction caused by mRNA dose can be reduced, and safety can be improved.

(4) The invention utilizes an in vitro transcription method to prepare self-replicating mRNA molecules and utilizes a microfluidic technology to encapsulate the mRNA into nano lipid particles, thereby realizing the cell-free preparation of the self-replicating mRNA tumor vaccine.

[ brief description of the drawings ]

FIG. 1A is a schematic representation of the structure of the antigen variant VG-HR1 of HER 2.

FIG. 1B is a schematic representation of the structure of the antigen variant VG-HR13 of HER 2.

FIG. 2A is a schematic representation of the structure of a self-replicating mRNA expression cassette expressing VG-HR 1.

FIG. 2B is a schematic representation of the structure of a self-replicating mRNA expression cassette expressing VG-HR 13.

FIG. 3A is a schematic representation of the structure of a non-self-replicating mRNA expression cassette expressing VG-HR 1.

FIG. 3B is a schematic representation of the structure of a non-self-replicating mRNA expression cassette expressing VG-HR 13.

FIG. 4 shows a map of plasmid PSaRNA-VG-HR13 containing the expression cassette for self-replicating mRNA obtained by inserting the expression cassette for self-replicating mRNA for VG-HR13 into pUC57-Kan plasmid.

FIG. 5 shows the denaturing agarose gel electrophoresis pattern (FIG. 5A) and capillary electrophoresis integrity test results (FIG. 5B) using linearized PSaRNA-VG-HR1 as IVT template.

FIG. 6 shows Western Blot (Western Blot) assay results after transfection of HEK-293T cells with LNP encapsulating self-replicating mRNA expressing VG-HR1 and non-self-replicating mRNA.

FIG. 7 shows the relative changes in copy number (copy number 6-48 hours post-transfection/copy number 2 hours post-transfection) of the non-structural polyprotein coding sequence portion of the self-replicating mRNA expressing VG-HR1 (or the full length of the self-replicating mRNA expressing VG-HR 1) and the VG-HR1 coding sequence portion (FIGS. 7A and 7B) and the schematic representation of the self-replication principle (FIG. 7C).

FIG. 8 shows the change in body weight of mice administered with groups (groups 2-8) of LNPs encapsulating VG-HR13 expressing self-replicating mRNA and non-self-replicating mRNA.

FIG. 9 shows the concentration of specific anti-HER 2 antibodies in the sera of mice in

weeks

2 and 4 of groups (groups 2-8) administered LNP encapsulating VG-HR13 expressing self-replicating mRNA and non-self-replicating mRNA.

FIG. 10 granzyme B levels (CD 8) at week 4 in mice administered groups (groups 2-8) of LNPs encapsulating VG-HR13 expressing self-replicating mRNA and non-self-replicating mRNA ⁺ T cell immunity level).

[ detailed description ] of the invention

Human epidermal growth factor receptor-2 (HER 2) and its variant antigens

Wild-type HER2 is a 185kD transmembrane glycoprotein consisting of 1255 amino acids, an important member of the tumorigenic pathway. HER2 can be broadly divided into a cysteine-rich extracellular domain (extracellular domain, ECD), a lipophilic transmembrane domain (transmembrane domain, TMD) and an intracellular domain with tyrosine kinase catalytic activity (Intracellular domain, ICD).

In particular embodiments, HER2 variant antigens may be produced by engineering wild-type HER 2. In a specific embodiment, the HER2 variant antigen is produced by engineering ICD of wild-type HER 2. The JAK-STAT signaling pathway is the primary signaling mechanism for a variety of cytokines and growth factors. JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis. These cellular events are critical for hematopoiesis, immune development, mammary gland development and lactation, adipogenesis, ampholytic growth and other processes. Thus, in a further embodiment, the DNA sequence of the antigenic variant of HER2 is obtained by inactivating mutation of the Tyrosine Kinase (TK) active site in the coding sequence of the intracellular domain (ICD) of wild type HER2 to inhibit the unintended activation of the JAK-STAT signaling pathway.

In a further embodiment, the HER2 variant antigen is made by replacing the TMD of wild-type HER2 with another domain. The major histocompatibility complex1 (major histocompatibility complex, MHC 1) transport domain (MHC 1 trafficking domain, MITD) is a domain that directs Cytotoxic T Lymphocyte (CTL) epitopes of antigens onto MHC1 molecules located on the endoplasmic reticulum to increase antigen presentation efficiency. Thus, in one embodiment, HER2 variant antigen is produced by replacing TMD of wild-type HER2 with MITD.

In a further embodiment, the HER2 variant antigen is produced by replacing the Signal Peptide (SP) of wild-type HER2 with another SP. In a specific embodiment, the HER2 variant antigen is produced by replacing the SP of wild-type HER2 with the SP of MITD.

In a preferred embodiment, the amino acid sequence of the HER2 variant antigen is as set forth in SEQ ID NO:2 or 4.

In a preferred embodiment, the coding sequence of the HER2 variant antigen is human codon optimized. In a preferred embodiment, the HER2 variant antigen has a coding sequence as set forth in SEQ ID NO:1 or 3. In a preferred embodiment, the 5' end of the coding sequence of the HER2 variant antigen is linked to a Kozak sequence. In a specific embodiment, the Kozak sequence is as shown in GCCACCATGG (DNA sequence) or GCCACCAUGG (RNA sequence).

[ RNA replicase ]

RNA replicases are RNA-dependent RNA polymerases (RNA dependent RNA polymerase, rdRp) that synthesize RNA using RNA as a template, also known as RNA synthetases. RdRp is present in most RNA viruses and serves to replicate viral RNA as well as synthesize mRNA.

In one embodiment, the mRNA vaccines of the present invention contain coding sequences for RNA replicase enzymes to effect mRNA self-replication. In one embodiment, the mRNA vaccine of the invention comprises a coding sequence for a viral non-structural polyprotein containing a coding sequence for an RNA replicase to effect mRNA self-replication.

[ expression cassette derived from Virus ]

HER2 variant antigens may preferably be expressed in expression cassettes derived from viruses. In a preferred embodiment, the virus is a virus of the Togaviridae family (Togaviridae). In a further preferred embodiment, the virus is of the genus Alphavirus (Alphavirus), which may be selected, for example, from the group consisting of: the virus may be selected from the group consisting of the Olra virus (Aura virus), the Ba Ma Senlin virus (Barmah Forest virus), the Brucella virus (Bebaru virus), the Carbaw European virus (Cabassou virus), the chikungunya virus (Chikungunya virus), the Oriental equine encephalitis virus (Eastern equine encephalitis virus), the palustris virus (Everglas virus), the Morganburg virus (Fort Morgan virus), the Getah virus (Getah virus), the Gaosdi J virus (Highlands J virus), the Ma Yaluo virus (Mayaro virus), the Middelburg virus (Middelburg virus), the Mo Sida Rebaud virus (Mosso das Pedras virus), the Mu Kanbu virus (Mucambo virus), the En Du Mu virus (Ndeu virus), the Ounig-nyong virus (O' gung virus), the Nairudin Pi Shuna virus (Pixuna virus), the Neo virus (Ross), the Ross River virus (Ross River virus), the Fossian virus (3949), the West River virus (Focus virus), the West Hance virus (Focus virus) (Salmon pancreas disease virus), the Hance virus (Tornalia virus (Wedner virus) and the Wedner virus (Tornalia virus) (35), the Wedner Hance virus (Tornalia virus) (Teis), the Wedner Hans virus (Dura virus) (Weldmanna virus) (35), the Weldmanna virus (Tornalia virus) (Weldmanna virus) (35), the Weldmanna virus (Du virus) (Weldmanna virus) and the Weldmanna virus (Teh virus). In a further preferred embodiment, the virus is venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV). In a further preferred embodiment, the virus is the VEEV TC-83 strain.

The genome of VEEV is substantially composed of the following parts: a 5 'untranslated sequence (UTR), a coding sequence for an unstructured polyprotein (nonstructural polyprotein), a coding sequence for a structured polyprotein (structural polyprotein), and a 3' UTR. Among them, the nonstructural polyproteins include enzymes that maintain viral replication, and the like, and the structural polyproteins are mainly viral envelope proteins.

In a preferred embodiment, the expression cassette derived from the virus is preferably made by replacing the coding sequence of the structural polyprotein in the viral genome (the part following the promoter of the structural polyprotein) with the coding sequence of the HER2 variant antigen. In a preferred embodiment, the 5' end of the coding sequence of the HER2 variant antigen is linked to a Kozak sequence. In a specific embodiment, the Kozak sequence is as shown in GCCACCATGG (DNA sequence) or GCCACCAUGG (RNA sequence). In a further embodiment, an RNA polymerase promoter site is appended 5 'to the 5' UTR. In a further embodiment, a polyadenylation (poly A) and restriction endonuclease site is appended 3 'to the 3' UTR. In a preferred embodiment, the viral-derived expression cassette comprises in this order from the 5 'end to the 3' end: RNA polymerase promoter site, 5'utr, coding sequence for non-structural polyprotein, promoter sequence for structural polyprotein, coding sequence for HER2 variant antigen, 3' utr, poly a and restriction enzyme site. In a further preferred embodiment, the RNA polymerase is preferably T7 RNA polymerase. In a further preferred embodiment, the promoter of the structural polyprotein is the 26S promoter of a virus of the genus Alphavirus (Alphavirus). In a further preferred embodiment, the promoter of the structural polyprotein is the 26S promoter of VEEV. In a further preferred embodiment, the cleavage site of the restriction enzyme may be any cleavage site of a restriction enzyme, preferably a cleavage site of a BspQI restriction enzyme.

[ 5' hat ]

The 5 'end of eukaryotic mRNA typically has a bridged 7-methylguanosine (m 7G) Cap structure (Cap 0), in which the 2' hydroxyl group of the first nucleoside following m7G is methylated to form the Cap1 structure (m 7 GpppmN). Prior studies have found that the 5' end cap structure can regulate the shear maturation of mRNA and help the RNA transcription products pass through selective channels of the nuclear membrane into the cytoplasm. In addition, the 5' cap structure also protects the mRNA from exonuclease degradation, works in concert with the translation initiation factor protein, recruits ribosomes, and aids in ribosome binding to the mRNA, allowing translation to begin from AUG. In general, cap structures can recognize eukaryotic initiation factor 4E (eIF 4E) during the initiation phase of translation, enabling subsequent translation processes, while Cap1 structures can greatly reduce mRNA immunogenicity in vivo.

The 5' cap that can be used in the present invention is not particularly limited as long as it does not interfere with the achievement of the technical effect of the present invention. In a preferred embodiment, the 5 'cap for self-replication is m7G (5') ppp (5 ') (2' -OMeA) pU having the formula C ₃₁ H ₄₂ N ₁₂ O ₂₅ P ₄ The structural formula is as follows;

there are different "capping" methods for preparing mRNA by in vitro transcription, including enzymatic capping, co-transcriptional capping, and the like.

The enzymatic capping is a more traditional capping mode, the method needs to obtain uncapped mRNA by purification after the IVT reaction involving T7 polymerase is finished, cap0 is produced by vaccinia virus capping enzyme (with RNA triphosphatase activity, guanylate transferase activity and guanine methyltransferase activity), and the final mRNA is obtained by converting the mRNA into Cap1 by 2' -O-methyltransferase and S-adenosylmethionine and purifying the mRNA again.

The Cap analogue is directly added into an IVT reaction system participated by T7 polymerase, so that mRNA containing Cap1 structure is obtained by a one-step method, and only one purification is needed in the whole process. The method reduces the preparation steps, thereby effectively shortening the overall treatment time, simplifying the purification steps and reducing the quantity of required enzymes. Therefore, the chemical method co-transcription capping is relatively simple in process, less in impurity introduction and capable of rapidly improving the productivity of mRNA vaccines and medicines. Currently, one-step co-transcription capping is becoming the dominant technological route for mRNA preparation.

[ template for self-replicating mRNA ]

The template is not particularly limited as long as it can be used for self-replication of mRNA. In a preferred embodiment, the template for self-replication of mRNA may begin at 5' -) TAATACGACTCACTATAAT … -3', wherein the underlined region is the promoter region of RNA polymerase, more preferably the promoter region of T7 RNA polymerase.

Because self-replicating mRNA template sequences are typically longer, IVT yields and product integrity are lower than conventional templates.

[ Uridine Triphosphate (UTP) ]

The uridine triphosphate which can be used in the present invention is not particularly limited as long as it does not hinder the achievement of the technical effects of the present invention, and may be natural uridine triphosphate or any modified uridine triphosphate commonly used in the art. In a preferred embodiment, UTP is N1-methyl pseudouridine triphosphate (N1-Me-pUTP, commonly denoted as "ψ"), of formula C ₁₀ H ₁₄ N ₂ Na ₃ O ₁₅ P ₃ The structural formula is as follows:

the incorporation of N1-methyl pseudouridine triphosphate in mRNA vaccine and drug manufacturing processes increases translation efficiency of mRNA and reduces immunogenicity of mRNA in vivo.

[ 5'UTR and 3' UTR ]

The 5'UTR and the 3' UTR which can be used in the present invention are not particularly limited as long as the achievement of the technical effects of the present invention is not hindered. In a preferred embodiment, the 5'UTR and the 3' UTR are derived from a species from which the expression cassette comprising the coding sequence for the RNA replicase is derived.

[ encapsulation vector for mRNA ] and delivery method using the same

Since naked mRNA cannot enter cells of an organism effectively to carry out protein expression, and mRNA has poor stability and is easy to degrade, the mRNA vaccine of the invention is preferably encapsulated in a protective carrier. The encapsulating carrier of the mRNA which can be used in the present invention is not particularly limited as long as it is sufficient to keep the mRNA vaccine of the present invention from degrading for a long enough period of time and does not hinder achievement of the technical effect of the present invention. In a preferred embodiment, nanoparticle-type carriers are used in the present invention to encapsulate mRNA. In a further preferred embodiment, lipid-containing nanoparticles (also referred to as "lipid nanoparticles (lipid nanopartical, LNP)") are used in the present invention to encapsulate mRNA. In further preferred embodiments, the LNP may include, but is not limited to, liposomes and micelles. In particular embodiments, the lipid nanoparticle may include cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, pegylated lipids, and/or structural lipids.

In a specific embodiment, the LNP may comprise one or more (e.g., 1,2, 3, 4, 5, 6, 7, or 8) cations and/or ionizable lipids. "cationic lipid" generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH). The cationic lipids may include, but are not limited to, SM102, 3- (didodecylamino) -N1, N1, 4-thirtieth-1-piperazineethylamine (KL 10), N1- [2- (didodecylamino) ethyl ] -N1, N4, N4-thirtieth-1, 4-piperazineethylamine (KL 22), 14, 25-tricosyl-15,18,21,24-tetraazaocta-ne (KL 25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, octyl-CLindMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA, and DMRIE.

In certain embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, e.g., about 40-65%, about 40-60%, about 45-55%, or about 48-53%.

In one embodiment, the LNP can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids. The non-cationic lipid may comprise an anionic lipid. Anionic lipids suitable for use in the lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphospholipid ethanolamine, N-succinylphospholipid ethanolamine, N-glutaryl phosphatidyl phosphoethanoi, and other neutral lipids to which anionic groups are attached.

In more specific embodiments, the non-cationic lipid may include a neutral lipid, which may include, for example, a phospholipid, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), palmitoyl phosphatidylethanolamine (POPE), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (sop), or a mixture thereof. In addition, lipids having a mixture of saturated and unsaturated fatty acid chains may be used. For example, the neutral lipids described herein may be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine.

In certain embodiments, the molar ratio of the phospholipid in the lipid nanoparticle is about 5-20%.

In certain embodiments, the LNP can comprise lipid conjugates, e.g., polyethylene glycol (PEG) modified lipids and derivatized lipids. PEG modified lipids can include, but are not limited to, polyethylene glycol chains up to 5kDa in length covalently linked to lipids having alkyl chains of C6-C20 length. The addition of these components can prevent lipid aggregation, can also increase circulation duration, facilitate delivery of the lipid-nucleic acid composition to target cells, or rapid release of nucleic acid. For example, the polyethylene glycol (PEG) modified lipid molecule may be a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In certain embodiments, the molar ratio of the polyethylene glycol (PEG) modified lipid molecules in the lipid nanoparticle is about 0.5-2%, e.g., about 1-2%, about 1.2-1.8%, or about 1.4-1.6%. In certain embodiments, the polyethylene glycol (PEG) modified lipid molecule may be PEG2000-DMG.

In certain embodiments, the LNP may further comprise cholesterol. In certain embodiments, the cholesterol is present in the lipid nanoparticle in a molar ratio of about 30-50%, for example, about 35-45%, or about 38-42%.

In certain embodiments, the LNP can include cationic lipids, cholesterol, phospholipids, and polyethylene glycol modified lipid molecules. In certain embodiments, the molar ratio of cationic lipid, cholesterol, phospholipid, and polyethylene glycol modified lipid molecule may be 45 to 55: 35-45: 5-15: 0.5 to 2.

The delivery method using the above-mentioned encapsulating carrier is not particularly limited, and any delivery method conventionally used in the art may be employed, for example, the delivery method mentioned in US20160376224A1 or WO2015199952A1 may be employed.

[ example ]

Example 1: production of self-replicating mRNA and non-self-replicating mRNA expressing HER2 antigen variants

[ 1_1. Manufacture of HER2 antigen variants ]

The DNA sequence of wild-type HER2 was obtained from NCBI (https:// www.ncbi.nlm.nih.gov /), wherein the Tyrosine Kinase (TK) active site in the coding sequence of the intracellular domain (ICD) was subjected to inactivating mutation to inhibit the unintended activation of the JAK-STAT signaling pathway, thereby obtaining the DNA sequence of one antigen variant VG-HR1 of HER2 (SEQ ID NO: 1). The VG-HR1 has the amino acid sequence shown in SEQ ID NO:2, the schematic structure of which is shown in fig. 1A.

On the basis of the DNA sequence of VG-HR1 as above, the coding sequence of TMD of HER2 was replaced with the coding sequence of MITD, and the coding sequence of SP of HER2 was further replaced with the coding sequence of SP of MITD to obtain the DNA sequence of VG-HR13, another antigen variant of HER2 (SEQ ID NO: 3). The amino acid sequence of VG-HR13 is shown in SEQ ID NO:4, the schematic structure of which is shown in fig. 1B.

[ 1_2 ] construction of expression cassettes for self-replicating mRNA and expression cassettes for non-self-replicating mRNA derived from VEEV TC-83 strain

Based on the genomic DNA sequence of the VEEV TC-83 strain, the coding sequence of the structural polyprotein (the part behind the promoter of the structural polyprotein) was replaced by "Kozak sequence+SEQ ID NO:1 "or" Kozak sequence+seq ID NO:3, VG-HR 13. Further, a promoter site of T7 RNA polymerase was added to the 5 '-side of the 5' UTR of the above construct. Further, a cleavage site for a poly A and BspQI restriction enzyme was added to the 3 '-side of the 3' UTR of the above construct. The expression cassette of VG-HR 1-expressing self-replicating mRNA from VEEV TC-83 strain (FIG. 2A) and the expression cassette of VG-HR 13-expressing self-replicating mRNA (FIG. 2B) thus constructed were sequentially from 5 'end to 3' end:

expression cassette of self-replicating mRNA expressing VG-HR1 (FIG. 2A) (SEQ ID NO: 5): the promoter site of T7 RNA polymerase, 5' utr, coding sequence for non-structural polyprotein, 26S promoter, "Kozak sequence+seq ID NO:1 ", the DNA sequence of VG-HR1 shown in the specification, the cleavage site of 3' UTR, poly A and BspQI restriction enzyme;

expression cassette of self-replicating mRNA expressing VG-HR13 (FIG. 2B) (SEQ ID NO: 6): the promoter site of T7 RNA polymerase, 5' utr, coding sequence for non-structural polyprotein, 26S promoter, "Kozak sequence+seq ID NO:3, DNA sequence of VG-HR13, cleavage site of 3' UTR, poly A and BspQI restriction enzyme.

Meanwhile, as a control, an expression cassette for non-self-replicating mRNA expressing VG-HR1 (FIG. 3A) and an expression cassette for non-self-replicating mRNA expressing VG-HR13 (FIG. 3B) were constructed in a similar manner, which sequentially included from the 5 'end to the 3' end:

expression cassette for non-self-replicating mRNA expressing VG-HR1 (FIG. 3A): the promoter site of T7 RNA polymerase, 5' utr, "Kozak sequence+seq ID NO:1 ", the DNA sequence of VG-HR1 shown in the specification, the cleavage site of 3' UTR, poly A and BspQI restriction enzyme;

expression cassette for non-self-replicating mRNA expressing VG-HR13 (FIG. 3B): the promoter site of T7 RNA polymerase, 5' utr, "Kozak sequence+seq ID NO:3, DNA sequence of VG-HR13, cleavage site of 3' UTR, poly A and BspQI restriction enzyme.

1_3 construction of plasmids containing expression cassettes for self-replicating mRNA and expression cassettes for non-self-replicating mRNA

Plasmids PSaRNA-VG-HR1 and PSaRNA-VG-HR13 (FIG. 4) containing the expression cassettes for self-replicating mRNA and plasmids pNSaRNA-VG-HR1 and pNSaRNA-VG-HR13 containing the expression cassettes for non-self-replicating mRNA constructed in example 1_2 and the expression cassettes for self-replicating mRNA for VG-HR13 and the expression cassettes for non-self-replicating mRNA for VG-HR1 and the expression cassettes for non-self-replicating mRNA were inserted into pUC57-Kan plasmid, respectively.

[ 1_4 ] linearization of plasmids containing expression cassettes for self-replicating mRNA and expression cassettes for non-self-replicating mRNA ]

50 μl of plasmid linearization reaction system shown in Table 1 below was prepared.

Table 1: linearization System of 50. Mu.L of plasmid containing expression cassettes for self-replicating mRNA and expression cassettes for non-self-replicating mRNA

Reagent(s)	Measuring amount
		Plasmids containing expression cassettes for self-replicating mRNA and expression cassettes for non-self-replicating mRNA	8μg
BspQI restriction enzyme (Nuo Wei Zan, DD 4302)	2μl
		10 Xdigestion buffer (Nuo Wei Zan, DD 4302)	5μl
RNase-free ddH ₂ O (Biyundian, R0022)	To a total volume of 50. Mu.l

Plasmids containing the expression cassette for self-replicating mRNA and the expression cassette for non-self-replicating mRNA obtained in example 1_3 were linearized by incubation at 50℃for 2 hours. After linearization is completed, ddH is further added to the reaction system ₂ O to a total volume of 100. Mu.l. The linearized plasmids containing the expression cassette for self-replicating mRNA and the expression cassette for non-self-replicating mRNA were purified and recovered using agarose gel DNA recovery kits for use as in vitro transcription (in vitro transcription, IVT) templates.

[ 1_5 ] IVT of self-replicating mRNA and non-self-replicating mRNA ]

A20. Mu.L IVT reaction system as shown in Table 2 below was prepared.

Table 2: 20. Mu.L IVT reaction System)

IVT was performed by reaction at 37 ℃ for 2 hours to obtain self-replicating mRNA. After the completion of the reaction, 4U of DNase I (Renzan, EN 401) was added to the reaction system, and the reaction was carried out at 37℃for 15 minutes. Adding RNase-free ddH to the reaction system ₂ O was added to a total volume of 50. Mu.L, and then 25. Mu.L of a 5M LiCl solution was added thereto and mixed well. After standing at-20℃for 30 minutes, the supernatant was discarded after centrifugation at 13000 Xg for 10 minutes at 4 ℃. The precipitate was washed with 70% ethanol, centrifuged at 13000 Xg at 4℃for 2 minutes, and the supernatant was discarded, and the precipitate was dissolved in 30. Mu.L of water.

The concentration was determined by spectrophotometry. 500ng of the in vitro transcribed self-replicating mRNA was mixed with 2. Mu.l of 2 XRNA Loading Dye and incubated at 70℃for 10 minutes, followed by denaturing agarose gel electrophoresis. The electropherogram when linearized PSaRNA-VG-HR1 was used as the IVT template is shown in FIG. 5A. At the same time, the integrity of the in vitro transcribed self-replicating mRNA was checked by capillary electrophoresis. The capillary electrophoresis integrity test data for the use of linearized PSaRNA-VG-HR1 as an IVT template is shown in FIG. 5B. These data indicate that the PSaRNA-VG-HR1 obtained by in vitro transcription has higher integrity, facilitating efficient replication and expression of mRNA.

[ 1_6 ] preparation of lipid nanoparticles encapsulating self-replicating mRNA and non-self-replicating mRNA ]

The self-replicating mRNA and non-self-replicating mRNA obtained in example 1_5 as IVT products were dissolved in citrate buffer and then mixed with a lipid mixture (ionizable lipid ALC-0315: distearoyl phosphatidylcholine: cholesterol: PEG lipid ALC-0159) dissolved in ethanol using a microfluidic device to obtain lipid nanoparticles (lipid nanopartical, LNP) encapsulating the self-replicating mRNA and non-self-replicating mRNA. These lipid nanoparticles were mixed with phosphate buffer salts and residual ethanol was removed by ultrafiltration. The LNP thus obtained was characterized and the results are shown in table 3 below.

Table 3: characterization results of LNP

Items	Value of
		Particle size	89.58±0.34nm
Monodispersity of	0.1265±0.0143
		mRNA encapsulation efficiency	90.63％
Zeta potential	-7.816±0.489mV

This suggests that well purified LNPs were successfully obtained that well encapsulate self-replicating mRNA and non-self-replicating mRNA of the antigen variants VG-HR1 and VG-HR13 that express HER 2. A slightly negative zeta potential is suitable for electrostatic stability and intracellular uptake.

These LNPs were mixed with cryoprotectants and stored at-80 ℃.

Example 2: self-replication efficiency of self-replicating mRNA

[ 2_1 ] cell transfection ]

HEK-293T cells derived from human embryonic kidney cells were inoculated in 75cm containing DMEM high-glucose medium (+10% fetal bovine serum+1% diabody) ² In the cell culture flask, the confluence of cells in the flask is up to 80% or more. The cells were digested with pancreatin and counted. A suitable number of suspended HEK-293T cells were plated in 24-well cell culture plates containing Opti-MEM medium in CO ₂ The cells were incubated overnight at 37 ℃.

After transfecting HEK-293T cells in the above 24-well cell culture plates with 0.5. Mu.g of LNP encapsulating self-replicating mRNA and non-self-replicating mRNA expressing antigen variants VG-HR1 and VG-HR13 of HER2 obtained in example 1 for 24 hours, the transfected cell cultures were used for Western Blot (Western Blot) assay in example 2_2 and detection of amplification efficiency of self-replicating mRNA in example 2_3 as follows.

[ 2_2 ] Western Blot (Western Blot) assay ]

To the cell culture obtained by transfection in example 2_1, 90. Mu.l of the cell lysate containing 1% of the protease inhibitor was added, and then, the mixture was left on ice for 5 minutes. Centrifuge at 14000 Xg for 10 min, transfer the supernatant to a new centrifuge tube, add NuPAGE LDS sample buffer, and metal bath at 95℃for 10 min. The cell lysate thus obtained was stored at-20 ℃.

The cell lysates were subjected to polyacrylamide gel electrophoresis (PAGE) at 200V for 30 minutes. After the electrophoresis, the gel was transferred for 7 minutes at 25V using a transfer kit (Trans-Blot Turbo 0.2. Mu. m PVDF Transfer Kit, bio-rad 1704272). After completion of the transfer, the membrane was blocked with TBSTw blocking solution (Biyun, ST 673) for 15 minutes, and incubated with an anti-HER 2 monoclonal antibody (Abcam, ab 221438) as a primary antibody diluted 1:1000 for 1 hour at room temperature. Wash 5 times with 1×tbst for 5 minutes each. Then incubated with goat anti-rabbit IgG (Abcam, ab 205718) as a secondary antibody, labeled with horseradish peroxide diluted 1:20000 in TBST, for 40 minutes at room temperature. Wash 5 times with 1×tbst for 5 minutes each. The mixed solution of the developer solution a and the developer solution B mixed in a ratio of 1:1 was dropped onto the film, and development was performed in the image forming system.

Western Blot (Western Blot) assay results after transfection of HEK-293T cells with LNP encapsulating VG-HR1 expressing self-replicating mRNA and non-self replicating mRNA are shown in FIG. 6. The results showed that the expression amount of self-replicating mRNA (lane 1 in FIG. 6) was much greater than that of non-self-replicating mRNA (lane 2 in FIG. 6) during the same transfection time.

[ 2_3 ] detection of self-replication efficiency of self-replicating mRNA ]

The supernatant from the cell culture obtained by transfection in example 2_1 was aspirated, and the cells were rinsed once with 500. Mu.l of PBS, and the supernatant was aspirated. After further addition of 500. Mu.l of PBS, the cells were blown down until they shed. The detached cells were collected into an Eppendorf centrifuge tube, centrifuged at 1000rpm for 5 minutes and the supernatant was discarded. The resulting cells were stored at-80 ℃.

Total RNA was extracted from the above cells using RNeasy Mini kit (Qiagen, 74106), and after quantification of the extracted RNA using an ultraviolet spectrophotometer, RNA was reverse transcribed into DNA using HiScript III 1st Strand cDNA Synthesis Kit (Noruzan, R312). Using 2X ChamQ Universal SYBR qPCR Master Mix (Norway, Q711), the following primers (for VG-HR1 expressing self-replicating mRNA) were used at 95℃for 30s → (95℃for 10s → 60℃for 30 s) ₄₀ Fluorescent quantitative PCR was performed under the conditions of (2):

upstream primer F of the coding sequence for the unstructured polyprotein of VEEV TC-83 strain: 5'-AGCAGAGATA GTATTGAAC-3' (SEQ ID NO: 7);

downstream primer R of the coding sequence for the unstructured polyprotein of VEEV TC-83 strain: 5'-TAATGGATAA CGGAACAG-3' (SEQ ID NO: 8);

upstream primer F of the coding sequence of VG-HR 1: 5'-ACTACCTCAG CCTCCTAT-3' (SEQ ID NO: 9);

Downstream primer R of the coding sequence of VG-HR 1: 5'-CACTCGCTGT CAATCATC-3' (SEQ ID NO: 10);

upstream primer F of the coding sequence of GAPDH: 5'-GGTATCGTGG AAGGACTC-3' (SEQ ID NO: 11);

downstream primer R of the coding sequence of GAPDH: 5'-GTAGAGGCAG GGATGATG-3' (SEQ ID NO: 12).

The relative changes in copy number of VG-HR1 expressing self-replicating mRNA (6-48 hours post-transfection/2 hours post-transfection) are shown in FIG. 7. As can be seen from FIG. 7, the copy number of different parts of the self-replicating mRNA expressing VG-HR1 varies with respect to each other. The 24-hour copy number of the coding sequence portion of the nonstructural polyprotein (or the full length of the self-replicating mRNA expressing VG-HR 1) was 98.85 times the 2-hour copy number after transfection (FIG. 7A), while the 24-hour copy number of the coding sequence portion of VG-HR1 was 4666.18 times the 2-hour copy number after transfection (FIG. 7B). This suggests that, especially 24 hours after transfection, self-replicating mRNA was self-replicating efficiently in cells transfected with it, and that self-replication occurred predominantly in the VG-HR1 coding sequence portion (the schematic representation of which is shown in FIG. 7C).

Example 3: safety and efficacy of LNP encapsulating self-replicating mRNA and non-self-replicating mRNA of antigen variants VG-HR1 and VG-HR13 expressing HER2

[ 3_1 ] immunization with LNP ]

Female BALB/c mice of 6-8 weeks of age were divided into 8 groups of 5 each, and vaccination was achieved by initial intramuscular injection (Prime) administration of LNP prepared in example 1, encapsulating self-replicating mRNA and non-self-replicating mRNA expressing antigen variants VG-HR1 and VG-HR13 of HER2 on day 0, and by additional intramuscular injection (Boost) administration of the corresponding LNP on day 14, after 3 days of adaptive feeding observation. Group 1 (control group) was administered with physiological saline. The properties of the LNP administered in each group are shown in table 4 below.

Table 4: attribute of LNP administered in each group ]

Group of	mRNA	Uridine triphosphate	Dosage of
				1 (control group)	-	-	-
2	Self-replicating mRNA	UTP						1 mug/g only
				3	Self-replicating mRNA	UTP			10 mug/g only
4	Self-replicating mRNA	UTP						44 mug/just
				5	Self-replicating mRNA	N1-Me-pUTP	1 mug/g only
6	Self-replicating mRNA	N1-Me-pUTP	10 mug/g only
				7	Self-replicating mRNA	N1-Me-pUTP	44 mug/just
8	Non-self-replicating mRNA	N1-Me-pUTP	44 mug/just

[ 3_2 ] changes in body weight after immunization with LNP ]

For each group of mice treated in example 3_1, body weights were measured on

days

1, 4, 6, 8, 10, 16, 19, 20, 22 and 25.

The results are shown in fig. 8A and 8B, and mice in the group (groups 2 to 8) to which the LNP encapsulating the self-replicating mRNA expressing VG-HR13 and the non-self-replicating mRNA were administered exhibited weight loss within 10% compared to group 1 (control group) to which physiological saline was administered, but the weight was restored to normal level within 4 days. This indicates that whether mRNA in LNP is self-replicating mRNA or non-self-replicating mRNA, and whether uridine triphosphate in mRNA is UTP or N1-Me-pUTP, is safe enough to vaccinate subjects without significant side effects.

[ 3_3 ] specific anti-HER 2 antibodies generated after immunization with LNP

For each group of mice treated in example 3_1, serum was collected on day 13 (week 2) and 28 (week 4), and the levels of specific anti-HER 2 antibodies therein were detected.

ELISA Plate Coating buffer (Elabscience, E-ELIR-001) was diluted with deionized water to working concentration (1X), then formulated as a coating solution (Yeasen, 93098ES 20) containing 1.5. Mu.g/ml HER2, after which 100. Mu.l of HER2 coating solution was added to high binding EIA/RIA plate (Corning costar, 42592), covered with a sealing film and incubated overnight at 4 ℃.

The coated detection plate was removed, the coating solution was removed by pipetting, and the plate was dried on filter paper. Adding 300 μl of washing buffer, standing for more than 30s, removing liquid, drying on absorbent paper, and repeating washing three times. After completion, 100. Mu.l ELISA Plate blocking buffer was added to each well and incubated at room temperature for 2h. After completion, the liquid in the wells was discarded and the wells were dried on filter paper. Adding 300 μl of washing buffer, standing for more than 30s, removing liquid, drying on filter paper, and repeating washing for three times.

Mice serum obtained on the indicated date after immunization was diluted 100-fold for detection, diluted samples were added to the blocked elisa plate, 100 μl per well, and incubated at room temperature for 2h. After completion, the supernatant was discarded, and the mixture was washed 3 times with a washing buffer. Simultaneously, HRP-conjugated Affinipure goat anti-mouse IgG (H+L) was diluted 1:10000 times with sample reagent, then added to the ELISA plate and incubated at room temperature for 1H. The supernatant was discarded after the incubation was completed, and the mixture was washed 3 times with a washing buffer. After completion of the washing, 0.1ml of TMB substrate (Solarbio, PR 1200) solution was added to each reaction well, and the reaction was developed at room temperature for about 10 minutes, followed by adding 0.1ml of stop solution to each reaction well. The concentration of specific anti-HER 2 antibodies therein was detected by measuring the absorbance at 450nm (OD 450) using a microplate reader.

As a result, as shown in fig. 9, the concentration of the specific anti-HER 2 antibody in the 4 th week serum was significantly higher in the mice of the group (groups 2 to 8) to which the self-replicating mRNA expressing VG-HR13 and the LNP that did not self-replicating mRNA were administered, as compared to the group 1 (control group) to which physiological saline was administered. Furthermore, serum antibody levels equivalent to and even higher than 44 μg of non-self-replicating mRNA per dose can be achieved by administering only 10 μg of self-replicating mRNA per dose. This suggests that self-replicating mRNA has significantly better antibody induction than non-self-replicating mRNA.

[ 3_4 ] specific cellular immune response against HER2 following immunization with LNP

For each group of mice treated in example 3_1, mice were sacrificed on day 28, spleens were collected, and spleen cells were obtained.

Spleen cells of each group were resuspended in 1640 medium, inoculated in U-bottom 96-well plates in a uniform manner, cultured for 24 hours with HER2 protein at a final concentration of 1. Mu.g/ml, after 18 hours of culture, the cells were centrifuged and the medium was removed, 100. Mu.l of fresh medium containing Golgilug (BD, 555029) was added, and the positive control group was added with medium containing T cell stimulator and monesin, and culture was continued for 4 to 6 hours. After stimulation, the cells were centrifuged and washed once with PBS, 50. Mu.l of cell surface antibody prepared with PBS was added and incubated at 4℃for 30 minutes in the absence of light. After that, the cells were centrifuged and washed once with 1640 medium containing 2% FBS, 50. Mu.l of a fixing solution was added thereto, and the mixture was fixed at 4℃for 1 hour. After that, the cells were centrifuged and rinsed once with wash buffer, and 50. Mu.l of cytokine antibody was added and incubated at 4℃for 1 hour. Finally, the cells were centrifuged and rinsed once with wash buffer, and then resuspended in PBS for detection on a flow cytometer.

As a result, as shown in FIG. 10, after 4 weeks of immunization, the remaining respective replication mRNA administration groups (groups 2 to 4,6 to 7) produced significantly higher levels of granzyme B, i.e., induced significantly higher levels of CD8, than the non-self-replication mRNA administration group (

group

8, 44. Mu.g/only), except for group 5 (self-replication mRNA, N1-Me-pUTP, 1. Mu.g/only) ⁺ T cell immunity. This suggests that self-replicating mRNA has significantly better cellular immune-inducing effects than non-self-replicating mRNA.

Claims

1. A messenger ribonucleic acid (mRNA) molecule comprising:

expression cassette 1 comprising coding sequence for RNA replicase, and

expression cassette 2 comprising coding sequences for antigens.

2. The mRNA molecule of claim 1, wherein the RNA replicase is a viral RNA replicase.

3. The mRNA molecule of claim 1, wherein the expression cassette 1 is derived from a coding sequence of a viral non-structural polyprotein.

4. The mRNA molecule of claim 2 or 3, wherein the virus is a virus of the Togaviridae family (Togaviridae).

5. The mRNA molecule of claim 4, wherein the virus of the Togaviridae family (Togaviridae) is a virus of the Alphavirus genus (Alphavirus).

6. The mRNA molecule of claim 4, wherein the virus of the Alphavirus genus is venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV).

7. The mRNA molecule of claim 1, wherein the antigen is a HER2 antigen or variant thereof.

8. The mRNA molecule of claim 7, wherein the HER2 antigen variant has an engineered intracellular domain (ICD) in which a Tyrosine Kinase (TK) active site is inactivated mutated.

9. The mRNA molecule according to claim 7 or 8, wherein the HER2 antigen variant has a major histocompatibility complex 1 (MHC 1) transport domain (MITD) which replaces the transmembrane domain (TMD) of HER2 antigen.

10. The mRNA molecule of claim 9, wherein the HER2 antigen variant has a Signal Peptide (SP) of MITD that replaces the SP of HER2 antigen.

11. The mRNA molecule according to claim 8 or 10, wherein the HER2 antigen variant has the amino acid sequence of SEQ id no:2 or 4.

12. The mRNA molecule of claim 11, wherein the coding sequence of the HER2 antigen variant has the sequence of SEQ ID NO:1 or 3.

13. The mRNA molecule of claim 7, wherein the 5' side of the coding sequence of the HER2 antigen or variant thereof has a Kozak sequence.

14. The mRNA molecule of claim 13, wherein the Kozak sequence is GCCACCAUGG.

15. The mRNA molecule of claim 1, wherein the expression cassette 2 has a promoter, the coding sequence for the antigen being located downstream of the promoter.

16. The mRNA molecule of claim 15, wherein the promoter is derived from the species from which the expression cassette 1 is derived.

17. The mRNA molecule according to claim 15 or 16, wherein the promoter is a 26S promoter.

18. The mRNA molecule of claim 1, wherein

The expression cassette 2 also has a 3' UTR and a poly A downstream.

19. The mRNA molecule of claim 18, wherein the RNA polymerase is a T7 RNA polymerase.

20. The mRNA molecule of claim 18, wherein the 5'utr and 3' utr are derived from a species from which the expression cassette 1 is derived.

21. The mRNA molecule according to claim 19 or 20, whose corresponding DNA sequence has the sequence of SEQ ID NO:5 or 6.

22. The mRNA molecule of claim 1, wherein uridine triphosphate is natural Uridine Triphosphate (UTP) or N1-methyl pseudouridine triphosphate (N1-Me-pucp).

23. Messenger ribonucleic acid (mRNA) vaccine obtained by encapsulating an mRNA molecule according to any one of claims 1 to 22 into Lipid Nanoparticles (LNP).

A dna molecule transcribed into an mRNA molecule according to any one of claims 1 to 22.

25. Use of an mRNA molecule according to any one of claims 1 to 22 in the manufacture of a messenger ribonucleic acid (mRNA) vaccine for eliciting an anti-HER 2 immune response.

26. A method of making a messenger ribonucleic acid (mRNA) molecule comprising:

(2) A promoter site for RNA polymerase is added to the 5 'side of the 5' UTR;

(3) Adding poly-A to the 3 'side of the 3' UTR; and

27. The method of claim 26, wherein the virus is a virus of the Togaviridae family (Togaviridae).

28. The method of claim 27, wherein the virus of the Togaviridae family (Togaviridae) is a virus of the Alphavirus genus (Alphavirus).

29. The method of claim 28, wherein the virus of the genus Alphavirus (Alphavirus) is venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV).

30. The method of claim 26, wherein the antigen is a HER2 antigen or variant thereof.

31. The method of claim 30, wherein the HER2 antigen variant has an engineered intracellular domain (ICD) in which a Tyrosine Kinase (TK) active site is inactivated mutated.

32. The method of claim 30 or 31, wherein the HER2 antigen variant has a major histocompatibility complex 1 (MHC 1) transport domain (MITD) that replaces the transmembrane domain (TMD) of the HER2 antigen.

33. The method of claim 32, wherein the HER2 antigen variant has a Signal Peptide (SP) of MITD that replaces the SP of HER2 antigen.

34. The method of claim 31 or 33, wherein the HER2 antigen variant has the amino acid sequence of SEQ ID NO:2 or 4.

35. The method of claim 34, wherein the coding sequence of the HER2 antigen variant has the sequence of SEQ ID NO:1 or 3.

36. The method of claim 30, wherein the 5' side of the coding sequence of the HER2 antigen or variant thereof has a Kozak sequence.

37. The method of claim 36, wherein the Kozak sequence is GCCACCATGG.

38. The method of claim 26, wherein the DNA molecule constructed by steps (1) - (3) has the sequence of SEQ ID NO:5 or 6.

39. The method of claim 26, wherein the transcription is in vitro transcription.

40. The method of claim 39, wherein the in vitro transcription uses:

RNA polymerase.

41. A method according to claim 40, wherein the uridine triphosphate is natural Uridine Triphosphate (UTP) or N1-methyl pseudouridine triphosphate (N1-Me-pUTP).

42. The method of claim 40, wherein the RNA polymerase is T7 RNA polymerase.