Preparation of novel mRNA vaccine and application of novel mRNA vaccine in treatment of neurodegenerative diseases

Abstract

The invention is suitable for the technical fields of molecular biology and immunology, and provides preparation of a novel mRNA vaccine and application of the mRNA vaccine in treatment of neurodegenerative diseases. The invention provides mRNA (messenger Ribonucleic Acid), which is formed by fusing three copies of an A beta 1-6 or A beta 1-14 antigen epitope, a Tau294-305 or Tau325-336 antigen epitope and a Th auxiliary epitope. The invention provides an amino acid sequence coded by mRNA and a vaccine composition obtained by mixing the mRNA and a lipid phase through a microfluidic system, and the mRNA or the vaccine composition is applied to preparation of drugs for treating or preventing Alzheimer's disease (AD). The multifunctional AD vaccine provided by the invention is controllable in number of inserted epitopes, quick to prepare, stable in vaccine property, uniform in particle size, good in safety, high in immunogenicity and high in titer of an antibody generated by induction, and can efficiently target and eliminate A beta deposition and Tau entanglement and effectively block the pathological process of AD.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and immunology, and in particular relates to the preparation of a novel mRNA vaccine and its application in the treatment of neurodegenerative diseases. Background Art

[0002] As the global population ages, the number of patients with neurodegenerative diseases has increased significantly, placing a serious burden and impact on my country's public health system. Alzheimer's disease (AD) is the most common chronic neurodegenerative disease in the elderly and the most widespread globally. Its main clinical features are progressive and irreversible damage to memory and cognitive functions. In the pathological process of AD, amyloid-β (hereinafter referred to as Aβ) and Tau protein play important roles. Aβ is a large molecular protein derived from the fatty membrane of nerve cells. Certain types of Aβ (such as Aβ38 / 40 / 42) are prone to coagulation to form plaques, infecting normal Aβ and accelerating plaque formation. At the same time, Tau protein, as a microtubule-binding protein in nerve cells, undergoes abnormal phosphorylation in the brains of AD patients. Hyperphosphorylated Tau protein can detach from axonal microtubules, destroying microtubule structure and affecting axonal transport. pTau protein aggregates into oligomers in neuronal cell bodies and eventually forms neurofibrillary tangles (NFTs). Therefore, the typical molecular pathological characteristics of AD are the formation of Aβ plaques and the formation of neurofibrillary tangles by Tau protein, which together endanger the health of neurons.

[0003] Currently, the main treatment strategies for AD in clinical practice include the following: neuroprotective agents (donepezil, galantamine, rivastigmine, and memantine), Aβ scavengers (Aβ monoclonal antibodies, Aβ vaccines), Tau protein targeted therapy (Tau protein inhibitors, Tau monoclonal antibodies, Tau vaccines), anti-inflammatory and immunomodulatory (anti-inflammatory drugs, immunomodulators), metabolic regulators (glucose metabolism regulators), and gene therapy (APOE gene regulation). Immunotherapy is the most promising approach and is divided into active and passive immunization. However, in the field of AD immunotherapy, although Aβ antibodies have been approved for marketing, their limited therapeutic effect and serious safety issues have made them controversial. Active vaccines containing Aβ or Tau short peptides, although well tolerated, have been declared clinically ineffective due to weak immunogenicity and insufficient efficacy. The main reasons include: (1) the antibody injection dose is too large and lacks safety; (2) the vaccine uses highly safe epitope peptides as immunogens, which makes it difficult to achieve a cure level in terms of antibody levels and durability; (3) Aβ vaccines or Tau vaccines alone act on only one AD pathogenic protein, resulting in poor efficacy. Therefore, it is urgent to develop a new strategy for AD immunotherapy that is multi-targeted, efficient, and safe.

[0004] Compared with the second-generation recombinant protein vaccine with weaker immunogenicity, the third-generation nucleic acid vaccine is widely used in the prevention and treatment of infectious diseases and chronic diseases because it can induce stronger and longer-lasting immune responses. Among them, mRNA vaccine, as a type of vaccine that has attracted much attention in recent years, is a new type of vaccine that introduces mRNA containing encoding antigen proteins into the human body through a delivery system such as lipid nanoparticles (LNP), directly translates it into the corresponding antigen protein, and thus induces the body to produce a specific immune response to prevent and treat the disease. After mRNA vaccination, the antigen peptide is mainly taken up and presented at the injection site and draining lymph nodes. At the same time, a small amount of mRNA or lipids can also be detected in the tissues of the liver, lungs, spleen and non-draining lymph nodes. After the mRNA vaccine enters the cell, the antigen protein is expressed in the cytoplasm, and after being hydrolyzed by the proteasome, it is transported to the cell surface through MHC class I molecules for presentation to CD8 + T cells. Some antigen proteins are secreted outside the cell and taken up by antigen-presenting cells such as dendritic cells or macrophages, and transported to the cell surface through MHC class II molecules for presentation to CD4 + T cells activate it, and then assist B cells in activation. The activated B cells produce antibodies to promote humoral immune response. Compared with traditional AD polypeptide vaccines and protein vaccines, mRNA vaccines have many advantages: (1) Smaller doses can induce strong CD4 + (1) It can stimulate T cells, germinal center (GC) B cells, and long-lived plasma cells (LLPC) immune responses to produce long-lasting protective antibodies; (2) It can design antigens more quickly, flexibly, and diversely, which is conducive to carrying both Aβ and Tau epitopes and inducing the production of mixed antibodies, making it very suitable for the development of dual-target AD vaccines. However, to date, there have been no relevant research reports on the application of mRNA vaccines in the prevention and treatment of neurodegenerative diseases, mainly AD. Therefore, the design and development of new AD vaccines using the mRNA vaccine platform is expected to solve the core problems in the field of AD treatment and show broad application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide the preparation of a novel mRNA vaccine and its application in the treatment of neurodegenerative diseases, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] mRNA, which is composed of three copies of Aβ1-6 or Aβ1-14 antigen epitopes, Tau294-305 or Tau325-336 antigen epitopes, and Th helper epitopes; wherein the three copies of Aβ1-6, Aβ1-14, Tau294-305 or Tau325-336 antigen epitopes and the Th helper epitopes are connected by a linker.

[0008] Furthermore, the following six types are included:

[0009] 3copy Aβ1-6-Th mRNA: composed of three copies of Aβ1-6 antigen epitopes and Th helper epitopes connected by a linker;

[0010] 3copy Tau294-305-Th mRNA: composed of three copies of the Tau294-305 antigen epitope and the Th helper epitope connected by a linker;

[0011] 3copyAβ1-6+3copyTau294-305-Th mRNA: composed of three copies of Aβ1-6 antigen epitopes and three copies of Tau294-305 antigen epitopes connected to Th helper epitopes through a linker;

[0012] 3copy Aβ1-14-Th mRNA: composed of three copies of Aβ1-14 antigen epitopes and Th helper epitopes connected by a linker;

[0013] 3copy Tau325-336-Th mRNA: composed of three copies of the Tau325-336 antigen epitope and the Th helper epitope connected by a linker;

[0014] 3copyAβ1-14+3copyTau325-336-Th mRNA: composed of three copies of Aβ1-14 antigen epitope and three copies of Tau325-336 antigen epitope connected to Th auxiliary epitope through linker.

[0015] Preferably, the Aβ1-6 / 1-14 sequence can be the Aβ1-6 / 1-14 sequence of humans, mice, primates, clawed frogs, and guinea pigs, that is, a sequence consisting of amino acids 1-6 / 1-14 of the complete Aβ42 protein; more preferably, the Aβ1-6 / 1-14 sequence has an amino acid sequence as shown in SEQ ID NO:13 and SEQ ID NO:14, and is encoded by the mRNA sequence corresponding to SEQ ID NO:1 and SEQ ID NO:2.

[0016] The Tau sequence can be the Tau294-305 / 325-336 sequence of human, mouse, primate, rabbit, smooth clawed frog, rat, or guinea pig; more preferably, the Tau294-305 / 325-336 sequence has an amino acid sequence as shown in SEQ ID NO: 15 and SEQ ID NO: 16, and is encoded by the mRNA sequence corresponding to SEQ ID NO: 3 and SEQ ID NO: 4.

[0017] The dual-targeting sequences include Aβ1-6 and Tau294-305 sequences, and the Aβ1-14 and Tau325-336 sequences can be human, mouse, primate, rabbit, clawed frog, rat, or guinea pig Aβ1-6 and Tau294-305 sequences, Aβ1-14 and Tau325-336 sequences. More preferably, the Aβ1-6 and Tau294-305 sequences have the amino acid sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15, and are encoded by the mRNA sequences corresponding to SEQ ID NO: 1 and SEQ ID NO: 3. The Aβ1-14 and Tau325-336 sequences have the amino acid sequences shown in SEQ ID NO: 14 and SEQ ID NO: 16, and are encoded by the mRNA sequences corresponding to SEQ ID NO: 2 and SEQ ID NO: 4.

[0018] The Aβ1-6 / 1-14 sequence and the Tau294-305 / 325-336 sequence can be connected via an amino acid linker; more preferably, the amino acid linker is (Gly)n(Ser)n or (Lys)n, wherein n is 1-10, preferably n=4.

[0019] Furthermore, the Th helper epitopes are composed of permutations and combinations of various Th epitopes. Preferably, the Th helper epitopes are sequences with optimal secondary structures. Th epitopes include norovirus epitope P1, norovirus epitope P2, tetanus epitope T1, tetanus epitope T2, tetanus epitope T3, pan-HLA DR binding epitope PADRE, measles epitope MVF5, and hepatitis B epitope HBsAg.

[0020] The coding region amino acid sequence is translated and encoded by the mRNA as described in any one of the above items, and comprises any one of the amino acid sequences of SEQ ID NO:31 to SEQ ID NO:36.

[0021] The vaccine composition is obtained by extruding and mixing the mRNA described above with a mixed lipid phase of SM102, DSPC, cholesterol and PEG2000 in a NanoAssemblr microfluidic system.

[0022] Use of the mRNA or vaccine composition described above in the preparation of a medicament for treating or preventing Alzheimer's disease in a subject. Preferably, the subject is a mammal; more preferably, the subject is a human. Preferably, the medicament is in the form of mRNA; more preferably, the vaccine composition is administered intramuscularly.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention utilizes the mRNA vaccine platform to design and develop a multifunctional Alzheimer's disease (AD) vaccine that can carry Aβ (β-amyloid protein) and Tau antigen sequences alone or simultaneously and is supplemented with Th epitopes. The number of inserted epitopes is controllable, the preparation is rapid, the vaccine properties are stable, the particle size is uniform, the safety is good, the immunogenicity is high, and the antibody titer induced is high. It can efficiently target and eliminate Aβ deposits and Tau tangles, thereby effectively blocking the pathological process of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Schematic diagram of mRNA vaccine plasmid; A is a schematic diagram of pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Aβ1-6 plasmid obtained by splicing 3 copies of Aβ1-6 with Th auxiliary epitopes and carrying them in pVAX1 vector; B is a schematic diagram of pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Aβ1-14 plasmid obtained by splicing 3 copies of Aβ1-14 with Th auxiliary epitopes and carrying them in pVAX1 vector; C is a schematic diagram of pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Tau294-305 plasmid obtained by splicing 3 copies of Tau294-305 with Th auxiliary epitopes and carrying them in pVAX vector; D is a schematic diagram of 3 copies of Schematic diagram of the pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Tau325-336 plasmid obtained by splicing Tau325-336 with Th auxiliary epitopes and carrying them into the pVAX1 vector; E is a schematic diagram of the pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Dual1-6+294-305 mRNA plasmid obtained by splicing 3 copies of Aβ1-6 and 3 copies of Tau294-305 with Th auxiliary epitopes and carrying them into the pVAX vector; F is a schematic diagram of the pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Dual1-6+294-305 mRNA plasmid obtained by splicing 3 copies of Aβ1-14 and 3 copies of Tau294-305 with Th auxiliary epitopes and carrying them into the pVAX vector. Schematic diagram of the pVAX1-BsaI-SpeI-free-Terminator-3171A-BsaI-Dual1-14+325-336 mRNA plasmid obtained by splicing Tau325-336 and Th helper epitope and carrying them into the pVAX vector.

[0026] Figure 2 Schematic diagram of the secondary structure prediction after optimization of mRNA vaccine.

[0027] Figure 3 The nucleic acid electrophoresis diagram after mRNA synthesis; A is the nucleic acid electrophoresis of the DNA template encoding six mRNAs; B is the RNA nucleic acid electrophoresis of the six mRNAs.

[0028] Figure 4Figure 1 is a graph showing the verification of in vitro mRNA expression; A is a WB verification of intracellular Aβ expression of Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine and Dual1-14 + 325-336 mRNA vaccine; B is an ELISA verification of Aβ supernatant secretion of Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine and Dual1-14 + 325-336 mRNA vaccine; C is a WB verification of intracellular Tau expression of Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine and Dual1-14 + 325-336 mRNA vaccine; D is an ELISA verification of Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine Tau supernatant secretion of mRNA vaccine, Dual1-6+294-305mRNA vaccine and Dual1-14+325-336mRNA vaccine; E is immunofluorescence verification of intracellular Tau expression of Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305mRNA vaccine and Dual1-14+325-336mRNA vaccine; F is immunofluorescence verification of intracellular Aβ expression of Aβ1-6mRNA vaccine, Aβ1-14mRNA vaccine, Dual1-6+294-305mRNA vaccine and Dual1-14+325-336mRNA vaccine.

[0029] Figure 5 The figure shows the particle size / electron microscopy characterization of six mRNAs after coating LNP; A is the particle size result of LNP; B is the particle size result of Aβ1-6 mRNA vaccine; C is the particle size result of Tau294-305 mRNA vaccine; D is the particle size result of Dual1-6 + 294-305 mRNA vaccine; E is the electron microscopy result of LNP; F is the electron microscopy result of Aβ1-6 mRNA vaccine; G is the electron microscopy result of Tau294-305 mRNA vaccine; H is the electron microscopy result of Dual1-6 + 294-305 mRNA vaccine; I is the particle size result of Aβ1-14 mRNA vaccine; J is the particle size result of Tau325-336 mRNA vaccine; K is the particle size result of Dual1-14 + 325-336 mRNA vaccine; L is the electron microscopy result of Aβ1-14 mRNA vaccine; M is the electron microscopy result of Tau325-336 mRNA vaccine. Electron microscopy results of mRNA vaccine; N is the electron microscopy results of Dual1-14+325-336 mRNA vaccine.

[0030] Figure 6Figure 6 shows the results of ELISA detection of mouse serum antibody titers and ELISpot safety of the mRNA vaccines (i.e., Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, and Dual1-14 + 325-336 mRNA vaccine) in Example 6, the LNP control group, and the PBS control group after two immunizations in C57BL / 6 mice; wherein AC are the antibody titers against Aβ4 produced by Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, the PBS control group, and the LNP control group 14 days after the first immunization (A), 14 days after the second immunization (B), and 28 days after the second immunization (C). 2; D is the persistence of specific antibodies against Aβ42 produced by Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, and Dual1-14 + 325-336 mRNA vaccine; E is the typing of specific antibodies against Aβ42 produced by Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, PBS control group, and LNP control group; FH are Tau294-305 14 days after the first immunization (F), 7 days after the second immunization (G), and 14 days after the second immunization (H). The levels of specific antibodies against the Tau epitope produced by the mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine, Dual1-14+325-336 mRNA vaccine, PBS control group, and LNP control group; I is the level of specific antibodies against the Tau epitope produced by the Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine, Dual1-14+325-336 mRNA vaccine, PBS control group, and LNP control group 28 days after the second immunization; J is the typing of specific antibodies against the Tau epitope produced by the Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine, Dual1-14+325-336 mRNA vaccine, PBS control group, and LNP control group;K is the ELISpot detection of IFN-γ secretion of spleen cells against Aβ42 in mice with Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, PBS control group and LNP control group; L is the Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, IFN-γ secretion against Tau protein by splenocytes of mice receiving mRNA vaccine, Dual1-6+294-305mRNA vaccine, Dual1-14+325-336mRNA vaccine, PBS control group, and LNP control group; M is IL-4 secretion against Aβ42 by splenocytes of mice receiving Aβ1-6mRNA vaccine, Aβ1-14mRNA vaccine, Dual1-6+294-305mRNA vaccine, Dual1-14+325-336mRNA vaccine, PBS control group, and LNP control group; N is IL-4 secretion against Tau protein by splenocytes of mice receiving Tau294-305mRNA vaccine, Tau325-336mRNA vaccine, Dual1-6+294-305mRNA vaccine, Dual1-14+325-336mRNA vaccine, PBS control group, and LNP control group.

[0031] Figure 7 The results of flow cytometry analysis of the changes in GC B cells, Plasma B cells and Tfh cells in the germinal center after immunization with the mRNA vaccine in Example 6 are shown; A represents the level of Aβ42-specific GC B cells in the Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, LNP control group and PBS control group; B represents the level of Aβ42-specific plasma B cells in the Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, LNP control group and PBS control group; C represents the level of Tau294-305 mRNA vaccine, Aβ1-14 mRNA vaccine, Dual1-6 + 294-305 mRNA vaccine, Dual1-14 + 325-336 mRNA vaccine, LNP control group and PBS control group; The levels of Tau epitope-specific plasma B cells were obtained from the mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine, Dual1-14+325-336 mRNA vaccine, LNP control group, and PBS control group; D is the levels of Tfh cells obtained from the Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine, Dual1-14+325-336 mRNA vaccine, LNP control group, and PBS control group.

[0032] Figure 8 Figure 7 shows the results of ELISA detection of serum antibody concentrations after immunization with APP / PS1 transgenic model, Tau transgenic model or 3×Tg transgenic model mice using mRNA vaccines (i.e., Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine, Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine, Dual1-6+294-305 mRNA vaccine and Dual1-14+325-336 mRNA vaccine) in Example 7; AD represents the serum antibody concentrations of Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine and LNP control group APP / PS1 transgenic model mice 7 days after the first immunization (A), 7 days after the second immunization (B), 14 days after the second immunization (C), and 14 days after the booster immunization (D), respectively. The specific antibody levels against Aβ42 produced by the model; E is the persistence of specific antibodies against Aβ42 produced by the APP / PS1 transgenic model with Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine and LNP control group; F is the typing of specific antibodies against Aβ42 produced by the APP / PS1 transgenic model with Aβ1-6 mRNA vaccine, Aβ1-14 mRNA vaccine and LNP control group; GI is 7 days after the first immunization (G), 7 days after the second immunization (H), 14 days after the second immunization (I), and 14 days after the booster immunization (J) Tau294-305 The levels of specific antibodies against Tau epitopes produced by the Tau transgenic models of the mRNA vaccine, Tau325-336 mRNA vaccine and LNP control group; K is the persistence of specific antibodies against Tau epitopes produced by the Tau transgenic models of the Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine and LNP control group; L is the typing of specific antibodies against Tau epitopes produced by the Tau transgenic models of the Tau294-305 mRNA vaccine, Tau325-336 mRNA vaccine and LNP control group.

[0033] Figure 9 These are the behavioral experimental results of mice after immunotherapy of APP / PS1 transgenic model mice with Aβ1-6 mRNA vaccine and Aβ1-14 mRNA vaccine in Example 7; A is the nesting score result of APP / PS1 transgenic mice; B is the open field test result of APP / PS1 transgenic mice; C is the novel object recognition test result of APP / PS1 transgenic mice; D is the water maze test result of APP / PS1 transgenic mice.

[0034] Figure 10These are the behavioral experimental results of Tau transgenic model mice after immunotherapy with Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine in Example 7; wherein A is the open field test result of Tau transgenic mice; B is the novel object recognition test result of Tau transgenic mice; C is the water maze test result of Tau transgenic mice; D is the hind limb score of Tau transgenic mice; E is the stride test result of 8.5-month-old Tau transgenic mice; F is the stride test result of 9-month-old surviving mice.

[0035] Figure 11 These are the behavioral experiment results of 3×Tg transgenic model mice after immunotherapy with Dual1-6+294-305 mRNA vaccine and Dual1-14+325-336 mRNA in Example 7; A is the nesting score result of 3×Tg mice; B is the open field test result of 3×Tg mice; C is the novel object recognition test result of 3×Tg mice; D is the novel object recognition test result of 3×Tg mice; E is the hind limb clamping test result.

[0036] Figure 12 These are the research results on the clearance of pathological proteins in AD model mice by mRNA vaccines in Example 7; wherein A is the Aβ pathology in the hippocampus of APP / PS1 transgenic mice after immunotherapy with Aβ1-6 mRNA vaccine and Aβ1-14 mRNA vaccine; B is the Tau pathology in the hippocampus of Tau transgenic mice after immunotherapy with Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine; C is the Aβ pathology in the hippocampus of 3×Tg transgenic mice after immunotherapy with Dual1-6+294-305 mRNA vaccine and Dual1-14+325-336 mRNA; D is the Tau pathology in the hippocampus of 3×Tg transgenic mice after immunotherapy with Dual1-6+294-305 mRNA vaccine and Dual1-14+325-336 mRNA. DETAILED DESCRIPTION

[0037] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0038] This invention utilizes an mRNA vaccine platform to design and develop novel AD-targeted vaccines that deliver Aβ and Tau antigen peptides, either singly or simultaneously, supplemented with Th epitopes. The novel mRNA vaccine is composed of an antigen sequence and multiple auxiliary epitopes. The antigen sequences include Aβ1-6, Aβ1-14, Tau294-305, or Tau325-336, with the amino termini of the antigen epitopes and Th helper epitopes connected via a linker.

[0039] Th helper epitopes: Regardless of the vaccine format, stimulating the patient's own immune system to produce sufficient and long-lasting specific antibodies is the primary principle of vaccine design. An immune response requires a synergistic interaction between antigen-presenting cells (APCs) and T helper (Th) cells. The elicitation of an effective antibody response requires both recognition of the target antigenic site of the test immunogen by APCs and recognition of the Th epitope by T helper cells. Disclosed artificial T helper (Th) epitopes can be linked to B cell epitopes and / or effector T cell epitopes ("target antigenic sites") via optional spacers to generate immunogenic constructs. An example of a Th peptide is the pan-DR epitope (PADRE), which has been shown to enhance the immune system's anti-tumor effects. Several clinical trials are underway to evaluate the efficacy of multi-epitope DNA vaccines against breast, cervical, ovarian, and pancreatic cancers. Furthermore, studies are underway to enhance the immunogenicity of HBsAg by inserting helper T cell epitopes from tetanus toxoid. Based on this, in order to enhance the immunogenicity and therapeutic effect of the vaccine, in addition to increasing the immunogen to three copies, we screened a series of Th helper epitopes from the sequence library and spliced ​​them behind the antigen epitope, including norovirus epitopes P1 and P2; tetanus epitopes T1, T2, T3; pan-HLA DR binding epitope PADRE, measles epitope MVF5 and hepatitis B epitope HBsAg.

[0040] The mRNA sequences and corresponding amino acid sequences of the antigenic epitopes and Th helper epitopes are shown in Table 1:

[0041] Table 1 mRNA sequences and corresponding amino acid sequences of antigenic epitopes and Th helper epitopes

[0042]

[0043]

[0044] Regarding the epitope selection of the AβmRNA vaccine, in order to avoid inducing T cell responses against Aβ42, the present invention selects the Aβ1-6 / 1-14 fragment of the B cell recognition epitope located at the N-terminus (i.e., a polypeptide consisting of the first 6 or 14 amino acids of the complete Aβ1-42 protein starting from the amino terminus) with higher safety as the immunogen of the targeted Aβ vaccine, and in order to improve the immunogenicity of the fragment and increase its copy number, 3 copies of Aβ1-6 / 1-14 are finally used as the immunogen fragment. We found that for the Aβ1-6 / 1-14 peptide, the Aβ1-6 / 1-14 sequence is connected to the screened Th epitope (permutation combination) with a linker, and the vaccine is finally presented in the form of mRNA, forming an orderly and repetitive linear antigen array, which is conducive to maximally stimulating the body to produce a specific immune response against the Aβ42 protein.

[0045] For epitope selection in the tau mRNA vaccine, we chose to target a common sequence in pathological tau, specifically the region necessary for pathological tau-tau interactions to form tau tangles. Tau294-305 / 325-336 was selected as the immunogenic fragment for the tau protein-targeted vaccine. Similar to the Aβ mRNA vaccine, to enhance immunogenicity, the copy number was increased, ultimately using three copies of Tau294-305 / 325-336 as the immunogenic fragment.

[0046] Considering that Aβ and Tau proteins play an important synergistic role in the pathogenesis of AD, and that current treatments targeting Aβ or Tau alone have not achieved good clinical results, further development of immunotherapies targeting both Aβ and Tau for the treatment of AD is very necessary. Based on this, the present invention also designed two dual-targeted vaccines targeting both Aβ and Tau, respectively linking 3 copies of Aβ1-6 and 3 copies of Tau294-305, and 3 copies of Aβ1-14 and 3 copies of Tau325-336 as immunogens.

[0047] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0048] Example 1: Construction of recombinant mRNA plasmid;

[0049] 1. Construction of 3copy-Aβ1-6 / 1-14-Th-pVAX recombinant plasmid;

[0050] XbaI was selected as the restriction site for fusion expression, and three copies of the nucleotide sequence of human Aβ1-6 / 1-14 were fused to the nucleotide sequence encoding the Th helper epitope. A nucleotide sequence encoding five glycine amino acids (GGGGS) was used as a linker between the nucleotide sequence of each copy of Aβ1-6 / 1-14, and a nucleotide sequence encoding five glycine amino acids (GGGGS) was used as a linker between the Aβ1-6 / 1-14 nucleotide sequence and the Th epitope sequence. The fused sequence was connected to the commercial vector pVAX1(+) to obtain a recombinant plasmid, as shown in the schematic diagram. Figure 1 As shown in A and B.

[0051] 2. Construction of 3copy-Tau294-305 / 325-336-Th-pVAX recombinant plasmid;

[0052] XbaI was selected as the restriction site for fusion expression, and three copies of the nucleotide sequence of human Tau294-305 / 325-336 were fused to the nucleotide sequence encoding the Th auxiliary epitope. A nucleotide sequence encoding five glycine (GGGGS) was used as a linker between the nucleotide sequence of each Tau294-305 / 325-336 copy, and a nucleotide sequence encoding five glycine (GGGGS) was used as a linker between the Tau294-305 / 325-336 nucleotide sequence and the Th epitope sequence. The fused sequence was connected to the commercial vector pVAX1(+) vector to obtain a recombinant plasmid, as shown in the schematic diagram. Figure 1 As shown in C and D.

[0053] 3. Construction of 3copy Aβ1-6-3copy Tau294-305-Th-pVAX recombinant plasmid and 3copy Aβ1-14-3copy Tau325-336-Th-pVAX recombinant plasmid;

[0054] XbaI was selected as the restriction site for fusion expression, and three copies of the nucleotide sequence of human Aβ1-6 / 14 and three copies of the nucleotide sequence of human Tau294-305 / 325-336 were fused to the nucleotide sequence encoding the Th helper epitope. A nucleotide sequence encoding five glycine residues (GGGGS) was used as a linker between each copy of the nucleotide sequence of Aβ1-6 / 1-14, and a nucleotide sequence encoding five glycine residues (GGGGS) was used as a linker between the nucleotide sequence of Aβ1-6 / 1-14 and the nucleotide sequence of Tau294-305 / 325-336. A nucleotide sequence encoding five glycine residues (GGGGS) was used as a linker between each copy of the nucleotide sequence of Tau294-305 / 325-336, and a nucleotide sequence encoding five glycine residues (GGGGS) was used as a linker between the nucleotide sequence of Aβ1-6 / 1-14 and the Th epitope sequence. The fused sequence was connected to the commercial vector pVAX1(+) to obtain a recombinant plasmid, as shown in the schematic diagram. Figure 1 As shown in E and F.

[0055] Table 2 shows the mRNA sequence of the mRNA vaccine and the corresponding amino acid sequence encoded:

[0056] Table 2 mRNA sequences of mRNA vaccines and the corresponding amino acid sequences

[0057]

[0058]

[0059]

[0060]

[0061] Example 2: Structural optimization and synthesis of mRNA vaccines;

[0062] The mRNA sequence corresponding to the plasmid constructed in Example 1, its optimization degree determines the final protein expression, and mRNA secondary structure becomes another important direction of sequence optimization, which is closely related to the minimum free energy of folding. The fewer the secondary structures formed by the first ten codons in the mRNA 5'UTR+CDS region, the higher the protein expression level encoded by it, and the more secondary structures formed by the remaining CDS region+3'UTR, the higher the protein expression level encoded by it. Based on these principles, the designed mRNA sequence is subjected to codon optimization, GC ratio optimization and RNA secondary structure prediction. After completing the depth optimization, the recombinant mRNA plasmid secondary structure diagram ( Figure 2 ). We then commissioned a company to perform full gene synthesis and insert the synthesized gene into the in vitro transcribed mRNA plasmid.

[0063] Example 3: Preparation and characterization of mRNA vaccines;

[0064] The mRNA recombinant plasmid constructed in Example 1 was transformed into Escherichia coli cloning competent Stable3 cells for large-scale amplification. High-purity and high-concentration recombinant mRNA template DNA plasmid was obtained by large-scale extraction, linearized by enzyme digestion, and subjected to in vitro transcription, capping reaction, and multi-step purification. The integrity of the RNA molecule was verified by nucleic acid electrophoresis (new TAE, 3% gel, 140-160V 20min) to obtain mature mRNA. The sample was diluted 20 times, the concentration was determined by Nano assay, and the sample was frozen at -80°C.

[0065] The results are as follows Figure 3 As shown in Figures A and B, DNA electrophoresis revealed that the linear DNA sizes of the six mRNAs were all between 4 and 5 kb, consistent with expectations. RNA electrophoresis revealed that the six mRNAs were approximately 1 kb in size, also consistent with expectations. These results demonstrate that the designed sequences can successfully generate mRNAs of the correct size through in vitro transcription and capping reactions.

[0066] Example 4: Verification of in vitro expression of mRNA antigen protein;

[0067] Taking 3 copies of Aβ1-6-Th as an example, mRNA was transfected into eukaryotic HEK293T cells, and immunofluorescence, Western Blot, and ELISA were used to verify that the antigen protein encoded by the mRNA could be expressed in the cells and in the cell supernatant.

[0068] The specific steps of the immunofluorescence experiment are as follows: fix the cells with 4% paraformaldehyde, add blocking solution and incubate at room temperature for 1 hour. Add primary antibody and incubate at 4°C overnight, then wash 3 times with PBS, 10 minutes each time. Add fluorescent secondary antibody and incubate in the dark for 2-3 hours. After incubation, wash 3 times with PBS. After staining, move the brain slices to a slide, dry in the dark, seal the slides, and observe and photograph using a fluorescence microscope. The results are as follows Figure 4 As shown in E and F, it can be seen that the cells transfected with mRNA exhibit green fluorescence, indicating that the target protein encoded by the mRNA is successfully expressed in the cells.

[0069] The specific steps of the Western Blot experiment are as follows: collect the sample 48 hours after transfection, lyse it, heat it at 100℃ for 15 minutes, centrifuge it at 5000rpm for 10 minutes, and take the supernatant as the sample. Take the protein marker and sample for SDS-PAGE electrophoresis. After the electrophoresis, wet transfer the protein gel. After the transfer, place the membrane in 5% skim milk powder for blocking at room temperature for 1 hour. Immerse in the primary antibody and incubate at 4℃ overnight. After washing the membrane with PBST and PBS the next day, immerse the membrane in HRP-labeled secondary antibody for incubation for 45 minutes, and then wash the membrane and develop color. The results are as follows Figure 4 As shown in A and C, a protein band with a molecular weight of approximately 20 kD can be detected in the cell sample transfected with mRNA, indicating that the target protein encoded by the mRNA is successfully expressed in the cell. The proteins expressed by the two dual-targeting mRNAs can be recognized by both Aβ and Tau antibodies.

[0070] The specific steps of the Elisa experiment are as follows: 48 hours after transfection, the cell culture supernatant was collected, centrifuged at 5000rpm for 10 minutes, the residual cells were discarded, and the supernatant was taken as the sample. The sample was replaced with antigen coating solution and concentrated to a final volume of 200μL (2mL original cell supernatant), and this was used as the antigen to coat a 96-well plate, 100μL per well, and incubated at 4°C overnight. After washing three times with PBST, blocking solution was added and blocked at 37°C for 1h. After washing again, 0.5μg / mL of Aβ1-6 / Tau antibody was added, 100μL per well, and incubated at 37°C for 2h. After washing, 0.3μg / mL HRP (horseradish peroxidase)-labeled goat anti-mouse secondary antibody (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was added, 100μL per well, and incubated at 37°C for 1h. After washing again, add the substrate tetramethylbenzidine, 100 μL per well, color development for 20 min in the dark, add 50 μL of 2M sulfuric acid per well to terminate the reaction, and measure the absorbance at 450 nm using a microplate reader. Figure 4As shown in B and D, Aβ can be detected in the supernatant of the culture medium of cells transfected with Aβ1-6 mRNA, Aβ1-14 mRNA vaccine, Dual1-6+294-305 mRNA and Dual1-14+325-336 mRNA, and Tau can be detected in cells transfected with Tau294-305 mRNA, Tau325-336 mRNA, Dual1-6+294-305 mRNA and Dual1-14+325-336 mRNA, indicating that the target protein encoded by the mRNA can be successfully secreted extracellularly.

[0071] Example 5: Particle size, potential and electron microscopy characterization of mRNA coated LNPs;

[0072] First, mRNA was coated, that is, mRNA and lipid phase were mixed through a microfluidic system to achieve mRNA-LNP encapsulation. Then, a nanoparticle size analyzer (purchased from Malvern) was used according to the manufacturer's instructions to measure the particle size and potential of the coated mRNA-LNP, and its morphology was observed by electron microscopy. The particle size test results are shown in Figure 2. Figure 5 As shown in Figures AD and IK, the particle size and potential results of the mRNA-coated particles of the present invention are shown in Table 3. The particle sizes of the six mRNA vaccines and the LNP control group are around 100-200 nm and present a single peak shape, indicating that the prepared lipid nanoparticles have good uniformity and suitable size. Figure 5 As shown in EH and LN, it can be seen that the six mRNAs are spherical in shape after LNP coating, indicating that the coating is successful.

[0073] Table 3 mRNA particle size and potential characterization

[0074]

[0075]

[0076] The above results show that after the mRNA of the present invention is coated with LNP, the particle size is correct, the structure is complete and the state is stable.

[0077] Example 6: mRNA vaccine immunization test;

[0078] The following experimental and control groups were set up (as shown in Table 4):

[0079] Experimental group: 6-8 week old female C57BL / 6 mice, 8 per group, received 6 mRNA vaccines by intramuscular injection, with each injection dose of 10 μg per mouse.

[0080] Control group: 6-8 week old female C57BL / 6 mice, 8 mice / group, received intramuscular injection of 100 μL PBS.

[0081] LNP control group: 6-8 week old female C57BL / 6 mice, 8 mice / group, were intramuscularly injected with 100 μL of LNP (empty LNP required to encapsulate 10 μg mRNA).

[0082] The immunization process was once every two weeks (on the 1st day and the 14th day, respectively), for a total of 2 immunizations. The mice were killed 10 days after the second immunization.

[0083] Table 4 Immunogenicity exploration plan

[0084]

[0085]

[0086] The concentrations of Aβ42 antibodies and Tau294-305 / 325-336 in the serum after immunization with the recombinant mRNA vaccine were determined by ELISA. The results showed that all mRNA vaccines could induce the production of high concentrations of antibodies, that is, whether it was an AβmRNA or Tau mRNA vaccine targeting a single target or a dual mRNA vaccine targeting two targets at the same time, the vaccine stimulated mice to produce antibodies against their respective targets 14 days after the first immunization, and the dilution ratio of 1:3200 could still show positive results; 14 days after the second immunization, the antibody level rose sharply, and the maximum dilution ratio of the positive well reached 1:51200 ( Figure 6 AD and FI).

[0087] Antibody typing ELISA kits were used to type and identify serum antibodies. A 1-42 peptide / Tau294-305 peptide / Tau325-336 peptide stock solution (1 mg / mL) was diluted to 1 μg / mL with coating buffer and added to a 96-well ELISA plate at 100 μL / well. The plate was incubated overnight at 4°C. After washing and blocking for 1 hour, 100 μL of the serum sample to be tested (1 / 800 dilution) was added and incubated at 37°C for 2 hours. After washing again, 100 μL of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM antibodies (all diluted 1 / 1000) were added and incubated at room temperature for 1 hour. The plate was then washed and 100 μL of HRP-labeled secondary antibody (1 / 1000) was added and incubated at room temperature for 30 minutes. The plate was washed and color development was stopped after 15 minutes. The OD value was read at 450 nm to determine the changes in serum antibody types before and after immunization. The results showed that the immune response induced by the mRNA vaccine was IGg1 / IGg2a>1, which is inclined to the humoral immune response type. This shows that the vaccine can indeed induce a high level of specific humoral immunity in mice ( Figure 6 (E and J).

[0088] The ELISpot assay was used to detect the secretion of IFN-γ and IL-4 in mouse spleen cells to determine the type and level of T cell immune response induced by the vaccine against different epitopes and to investigate the safety of the vaccine. The specific experimental steps are as follows:

[0089] A 96-well plate was coated with a monoclonal antibody against cytokines IFN-γ / IL-4 (from an ELISpot kit, purchased from BD Biosciences) at a concentration of 5 μg / mL, with 50 μL per well, at 4°C overnight. After washing once with complete medium containing 10% fetal bovine serum, 200 μL of complete medium was added, and the plate was blocked at 37°C for 1 hour before the medium was discarded. Ten days after the second immunization, mice were sacrificed by cervical dislocation, and spleen cells were harvested to prepare a cell culture plate with a cell concentration of 1×10 7 100 μL of the cell suspension (1 μg / mL) was added to the 96-well plate described above. 100 μL of the specific antigen Aβ42 / Tau294-305 / Tau325-336 (1 μg / mL) was then added to each well. The cells were stimulated and activated by incubation at 37°C in a 5% CO2 incubator for 24 hours. After washing, 50 μL of an IFN-γ antibody was added to each well and incubated at room temperature for 2 hours. After washing, 50 μL of an HRP-conjugated biotin secondary antibody was added to each well and incubated at room temperature for 2 hours. After washing, 50 μL of Elispot colorimetric solution was added to each well and incubated at room temperature for 40 minutes in the dark. The staining solution was discarded, and the cells were washed with distilled water. After drying overnight, the number of activated cells in the sample was counted using a microscope.

[0090] ELISpot assay results Figure 6 As shown in KL, the spleen cells of mice immunized with mRNA stimulated with Aβ42 or Tau294-305 or Tau325-336 produced very few IFN-γ positive spots (the difference in the number of spots was between -4 and 6), that is, each group of vaccines did not produce a Th1 type cellular immune response against Aβ and Tau. For interleukin-4 (IL-4), the number of ELISpot spots produced by the immunized group was significantly different from the number of spots produced by the unstimulated spleen cells of each group, that is, each group of vaccines could produce an enhanced Th2 type cellular immune response that secreted IL-4 against Aβ and Tau. Thus, the types and levels of T cell immune responses induced by the vaccine against different epitopes have been determined, and the safety of the vaccine has been preliminarily clarified ( Figure 6 M and N).

[0091] The steps for flow cytometry evaluation of germinal center activation are as follows:

[0092] Ten days after the second immunization, the inguinal lymph nodes of mice were isolated, and the changes in GC B cells, Plasma B cells and Tfh cells in the germinal center were detected by flow cytometry. After the inguinal lymph nodes were minced with scissors, collagenase was added to a final concentration of 0.04 mg / mL, and DNaseI was added to 10 U / mL. After digestion at 37°C for 1 hour, the suspension was suspended by blowing, and the supernatant was taken at 400g, centrifuged for 5 minutes, and the precipitate was resuspended with R10 to obtain a cell suspension, passed through a 200-mesh sieve into a centrifuge bucket, and centrifuged at 350g for 5 minutes before flow staining. The changes in GC B cells in the inguinal lymph nodes of mice were evaluated by flow cytometry, and the ratios of antigen-specific GC B cells and Plasma B cells in each mRNA vaccine group were analyzed and compared. The results are shown in Figure 2. Figure 7 As shown in Figures AD, the proportion of GC B cells targeting Aβ was significantly higher in the Aβ1-6 mRNA, Aβ1-14 mRNA, Dual1-6 + 294-305 mRNA, and Dual1-14 + 325-336 mRNA vaccine groups than in the PBS control group. The proportion of plasma B cells targeting Aβ and / or Tau was significantly higher in all six vaccine groups than in the PBS control group. Furthermore, in the germinal center (GC) reaction, a specific differentiated CD4 T cell subset, follicular helper T (Tfh) cells, is required to help B cells produce high-affinity antibodies against the antigen. Analysis of changes in Tfh cells in the lymph nodes after immunization revealed that the proportion of follicular helper T cells (Tfh) was significantly increased in all six vaccine groups compared to the PBS control group.

[0093] Example 7: Therapeutic evaluation of mRNA vaccines in AD transgenic mice;

[0094] The six mRNA vaccines of the present invention were applied to three transgenic mouse models for therapeutic research. The specific immunization schemes are as follows:

[0095] Aβ mRNA vaccines: The experimental group consisted of 3-4 month old APPswe / PS1dE9 mice (8 males and 8 females per group) who were injected intramuscularly with two Aβ mRNA vaccines (Aβ1-6 mRNA vaccine and Aβ1-14 mRNA vaccine) at a dose of 10 μg per mouse. The control group consisted of 3-4 month old APPswe / PS1dE9 mice (8 males and 8 females per group) who were injected intramuscularly with 100 μL of LNP (empty LNPs required to encapsulate 10 μg of mRNA).

[0096] Tau mRNA Vaccine: The experimental group consisted of 8 male and 8 female Tau P301S mice aged 3-4 months who were injected intramuscularly with two tau mRNA vaccines (Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine) at a dose of 10 μg per mouse. The control group consisted of 8 male and 8 female Tau P301S mice aged 3-4 months who were injected intramuscularly with 100 μL of LNP (empty LNPs required to encapsulate 10 μg of mRNA).

[0097] Dual mRNA Vaccine: The experimental group consisted of 3×Tg mice, 8 males and 8 females, aged 3-4 months. Two dual mRNA vaccines (Dual1-6 + 294-305 mRNA vaccine and Dual1-14 + 325-336 mRNA vaccine) were injected intramuscularly at a dose of 10 μg per mouse. The control group consisted of 3×Tg mice, 8 males and 8 females, aged 3-4 months. They were injected intramuscularly with 100 μL of LNP (empty LNPs required to encapsulate 10 μg of mRNA).

[0098] The immunization process is once every two weeks, for a total of 2 times, and then continuous booster immunization is performed based on the antibody level. The immunization schedule for the above groups is shown in Table 5.

[0099] Table 5 Immunization strategy of AD transgenic mice mRNA vaccine

[0100]

[0101] 1. ELISA was used to determine the titer of Aβ42, Tau294-305, or Tau325-336 antibodies in the serum of immunized AD mice;

[0102] ELISA was used to measure serum Aβ42, Tau294-305, or Tau325-336 antibody concentrations in the corresponding Alzheimer's disease transgenic mice immunized with the Aβ1-6 mRNA vaccine, the Aβ1-14 mRNA vaccine, the Tau294-305 mRNA vaccine, the Tau325-336 mRNA vaccine, the Dual1-6 + 294-305 mRNA vaccine, and the Dual1-14 + 325-336 mRNA vaccine. The results showed that all six mRNA vaccines were highly efficacious, stimulating the production of antibodies against their respective targets seven days after the first immunization. The second immunization induced high concentrations of antibodies, and seven days after the booster immunization, all three vaccines induced a highly potent immune response in the corresponding transgenic mouse models, with the maximum dilution ratio of positive wells reaching 1:204,800. This indicates that the first two immunizations effectively generated immune memory cells in the mice, which is of great significance for the prevention and treatment of Alzheimer's disease. Moreover, the antibody level of AβmRNA vaccine continued to fluctuate at a high level within 3 months after booster immunization, showing good persistence ( Figure 8 The dual-target vaccine stably and efficiently produces antibodies against two antigens. Antibody typing kits were used to detect antibody types, and the results showed that the six mRNA vaccines induced antibodies with an IGg1 / IGg2a ratio greater than 1, indicating that the induced immune response tended to be humoral immunity ( Figure 8 F, L and R).

[0103] 2. Behavioral evaluation of mRNA vaccines in AD model mice;

[0104] After the mRNA vaccine treatment, a series of behavioral experiments were conducted to comprehensively and carefully analyze the cognitive and behavioral improvement effects of the vaccine. The behavioral experiments mainly included nesting experiments, novel object recognition experiments, water maze experiments, and movement-related experiments such as hind limb clamping / tension / stride.

[0105] (1) Behavioral evaluation of an mRNA vaccine targeting Aβ in APP / PS1 transgenic mice;

[0106] The nesting experiment is an indicator for evaluating the cognitive behavior of mice. Before the experiment began, two square pieces of cotton with a side length of 5 cm and a weight of 1.2±0.2 grams were placed side by side in the middle of the long side of the cage. Each mouse was placed in a cage individually for nesting, and the experiment was usually carried out between 16:00 and 18:00. A nesting experiment was conducted on 7-month-old APP / PS1 transgenic mice and wild-type mice born in the same litter, and nesting scores were performed at 16h and 24h to evaluate the cognitive ability of mice. The scoring criteria are: 1 point if the cotton is still square and there are no obvious bite marks; 2 points if the cotton is torn into large pieces and spread in the cage or stacked on the underside of the mouse's body; 3 points if the cotton is torn into pieces and mixed with wood shavings to form a nest, but there are obvious gaps; 4 points if the cotton is completely torn into pieces and mixed with wood shavings to form a perfect nest. The higher the score, the lighter the damage to the mouse hippocampus. The results are as follows Figure 9 As shown in Figure A, the untreated control group (LNP group) had a relatively low nesting score, while mice treated with the Aβ1-6 mRNA vaccine and the Aβ1-14 mRNA vaccine were able to build nests better, with relatively higher scores. This result demonstrates that mRNA vaccine immunization can enhance the nesting ability of mice, delay damage to the hippocampus, and have a good therapeutic effect.

[0107] At 9 months of age, an open field test was performed to evaluate the motor status and anxiety level of mice. There was no significant difference between the vaccine group and the control group in this experiment, indicating that the Aβ1-6 mRNA vaccine and the Aβ1-14 mRNA vaccine had no positive effect on alleviating the anxiety of transgenic mice ( Figure 9 (B) A novel object recognition experiment was then conducted to assess the mice's short-term memory. The experiment was divided into three phases: adaptation, training, and testing. During the adaptation phase, the mice moved freely in the experimental apparatus (without objects) for 10 minutes, which was equivalent to the open field test. During the training phase, the mice explored two identical objects. During the testing phase, one hour after the completion of the second phase, one of the previously explored objects was replaced with a new object and the mice were asked to explore it. The results are shown in Figure 2. Figure 9 As shown in middle C, during the test period, APP / PS1 mice vaccinated with Aβ1-6 mRNA vaccine and Aβ1-14 mRNA vaccine spent more time exploring novel objects and showed a higher frequency of exploring novel objects compared with mice in the LNP control group, indicating that the vaccine rescued the short-term memory recognition of APP / PS1 mice.

[0108] Finally, we used a water maze experiment to test whether the mRNA vaccine has a positive effect on spatial learning and memory ability. The Morris water maze experiment is divided into two parts: a four-day hidden platform acquisition training experiment and an exploration experiment. The circular pool is divided into four quadrants, and four different patterns are posted on the pool wall. The platform is hidden 2 cm underwater in the middle of any platform. During the training period, each mouse was trained four times a day, entering the water from a different quadrant, and allowed to find the underwater hidden platform within 60 seconds. If it is not found within 60 seconds, it will be guided to swim onto the platform. The entire process was monitored by a video recording system to monitor the activities of the mice, and the time to find the platform, that is, the latency, was recorded. On the fifth day, the hidden platform was removed, and the mice were allowed to explore the entire pool area, recording the number of times they crossed the location where the platform was once located, as well as the time they stayed in the quadrant where the platform was located and the swimming distance. The results are as follows Figure 9 As shown in Figure D, the latency for mice in each group to find the platform gradually decreased with increasing training time. On the third and fourth days of training, the latency of transgenic mice in the LNP control group was longer than that in the Aβ1-6 mRNA vaccine and Aβ1-14 mRNA vaccine groups. During the test period, mice in the vaccine group spent longer in the target quadrant (the quadrant where the hidden platform was previously located) than those in the control group, indicating that the long-term memory of these mice improved to some extent.

[0109] The above behavioral results indicate that the two AβmRNA vaccines can effectively improve cognitive impairment in APP / PS1 transgenic mice.

[0110] (2) Behavioral evaluation of mRNA vaccines targeting Tau in Tau transgenic mice;

[0111] A series of behavioral experiments were conducted on Tau transgenic mice and wild-type mice from the same litter starting at 8 months of age to verify the effectiveness of vaccine treatment. The results showed that in the open field test, the movement distance of female mice in the vaccine group in the central area was significantly longer than that in the LNP control group ( Figure 10 In the novel object recognition experiment, the exploration time and movement distance of female mice in the vaccine group were significantly longer than those in the LNP control group ( Figure 10 Middle B); In the water maze experiment, the latency of mice treated with Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine was always shorter than that of the LNP control group during the training period, and both were able to reach the platform faster. There was a significant difference on the fourth day, and during the test, the number of times the mice in the treatment group crossed the platform tended to be more than that in the control group ( Figure 10 These behavioral results indicate that both Tau mRNA vaccines effectively alleviated anxiety and stress in Tau transgenic mice, had a positive impact on their short-term and long-term memory, and effectively improved cognitive impairment in these mice.

[0112] Studies have confirmed that as the AD course of Tau P301S mice progresses, the degree of muscle atrophy gradually worsens, which directly affects their motor ability. Therefore, when the mice in the control group and vaccine treatment group were 8 to 9 months old, stride length, tension test, hind limb clamping and other experiments were used to evaluate the effect of Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine treatment on the motor ability of Tau transgenic mice. In the hind limb clamping experiment, the experimenter grabbed the mouse's tail and hung it in the air (avoiding any objects around it), and scored the degree of hind limb contraction after observing it for 10 seconds. The scoring criteria are: 0 points, both sides of the hind limbs are always in an open state; 1 point, the time of unilateral hind limb contraction toward the abdomen exceeds 50%; 2 points, the total time of bilateral hind limbs contracting toward the abdomen at intervals exceeds 50%; 3 points, the mouse's hind limbs are completely retracted to the abdomen for more than 50% of the total time. The results are as follows Figure 10 As shown in Figure D, the hind limb scores of mice treated with the Tau294-305mRNA vaccine and the Tau325-336mRNA vaccine were significantly lower than those of the LNP control group, and the scores were similar to those of wild-type mice, indicating that mRNA vaccine treatment significantly improved the hind limb muscle atrophy of Tau transgenic mice and almost restored it to the level of wild-type mice. This improvement was more obvious in male mice. At 8.5 months of age, a stride test (inkblot method) was performed on Tau transgenic mice and wild-type mice. The results are shown in Figure 3. Figure 10 As shown in E, the stride length of the mice in the vaccine treatment group was significantly longer than that of the control group and similar to that of the wild-type mice, indicating that the mRNA vaccine treatment significantly restored the motor ability of the Tau transgenic mice to the level of wild-type mice, and one female mouse in the control group had extreme motor loss, with a stride length of only 1.35 cm, and died before the age of 9 months due to severe muscle atrophy and difficulty eating. The stride length experiment was conducted again on the surviving mice at 9 months of age, showing that the motor ability of the male mice in the control group was still significantly worse than that of the treatment group, and there was one male mouse in the control group with extreme motor loss, with an average stride length of only 1 cm. In the grip strength test, a grip strength tester was used to objectively quantify the muscle strength of the mice, and the results are shown as follows: Figure 10 As shown in Figure F, the grip strength of mice in the Tau294-305 mRNA vaccine and Tau325-336 mRNA vaccine treatment groups was significantly greater than that in the control group, and this was more obvious in male mice, indicating that mRNA vaccine treatment effectively improved the muscle strength loss of Tau transgenic mice.

[0113] The above results show that the two Tau mRNA vaccine treatments effectively alleviated the muscle atrophy and motor ability of Tau transgenic mice, had a certain degree of positive effect on their short-term and long-term memory, and could effectively improve the cognitive impairment of the model mice.

[0114] (3) Behavioral evaluation of two dual mRNA vaccines in 3×Tg mice;

[0115] A series of behavioral experiments were conducted on 3×Tg mice and wild-type mice born in the same litter from 8 months of age to verify the effectiveness of vaccine treatment. The results showed that the nesting ability of transgenic mice in the two dual mRNA vaccine treatment groups was significantly stronger than that of the LNP control group ( Figure 11 In the open field test, the movement distance of the vaccine group mice in the central area was significantly longer than that of the LNP control group ( Figure 11 In the novel object recognition experiment, the vaccine group mice explored novel objects significantly more times than the LNP control group ( Figure 11 In the water maze experiment, the latency of the mice in the treatment group was always shorter than that in the LNP control group during the training period, and a significant difference was shown on the 4th day. During the test, the mice in the treatment group spent significantly longer time exploring the quadrant where the hidden platform was located than those in the control group ( Figure 11 Because tau pathology negatively impacts motor function and muscle strength in mice, we evaluated muscle atrophy in treated and control mice using a hindlimb clamp test. Figure 11 As shown in Figure E, hind limb strength was restored in mice treated with the Dual1-6+294-305mRNA vaccine and the Dual1-14+325-336mRNA vaccine. These behavioral results indicate that treatment with the Dual1-6+294-305mRNA and Dual1-14+325-336mRNA vaccines effectively alleviated stress and anxiety in 3×Tg mice, significantly improved their memory and cognitive impairment, and had a positive impact on muscle atrophy.

[0116] 3. Study on the clearance of pathological proteins in AD model mice by mRNA vaccines;

[0117] (1) Study on the clearance of pathological proteins by mRNA vaccines targeting Aβ in APP / PS1 transgenic mice;

[0118] APP / PS1 transgenic mice were anesthetized at 10.5 months of age and whole brains were harvested via perfusion. After dehydration, paraffin embedding, and sectioning, immunohistochemistry was used to assess Aβ pathological protein levels in the brains of mice in different immunization groups. The experimental procedures included the following: 1. Deparaffinization: Paraffin sections were immersed in xylene I and II for 30 minutes each. Subsequently, sections were immersed in different concentrations of ethanol for 5 minutes each: two times in absolute ethanol, two times in 95% ethanol, then in 85% ethanol, and finally in distilled water. 2. Antigen Retrieval: Sections were boiled in citrate antigen retrieval solution to retrieve antigen epitopes. After cooling to room temperature, sections were washed three times with TBS (5 minutes each). 3. Blocking: 100 μL of 3% H₂O₂ was added to each tissue sample for 10 minutes at room temperature, followed by washing. 4. Serum Blocking: 100 μL of 10% goat serum was added to each tissue sample for 20 minutes at room temperature. 5. Primary antibody incubation: Use 1% goat serum to prepare the antibody, discard the blocking solution and add 100μL of antibody, and incubate at 4℃ overnight. 6. Washing: Wash 3 times with TBST, 5min / time, and wash 3 times with TBS, 5min / time. 7. Primary antibody amplifier: Add 100μL of primary antibody amplifier, incubate at room temperature in the dark for 40min, and wash. 8. Secondary antibody incubation: Add 100μL of secondary antibody, incubate at room temperature in the dark for 40min, and wash. 9. Color development: After DAB color development and hematoxylin staining, rinse with tap water. 10. Dehydration and sealing: Put in 70% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene for 3min each, and finally seal with neutral gum. The results are as follows Figure 12 As shown in center A, the hippocampal Aβ plaque area in mice treated with the Aβ1-6 and Aβ1-14 mRNA vaccines was significantly smaller than that in the LNP control group. This indicates that the vaccine effectively eliminated Aβ pathological proteins in the brains of APP / PS1 transgenic mice, preliminarily confirming the vaccine's potent therapeutic effect.

[0119] (2) Study on the clearance of pathological proteins by mRNA vaccines targeting Tau in Tau transgenic mice;

[0120] Tau transgenic mice were anesthetized at 9.5 months of age and the whole brains of the mice were obtained by perfusion. After dehydration, paraffin embedding, and sectioning, the levels of Tau pathological protein in the brains of mice in different immune groups were detected by immunohistochemistry. The main component of neurofibrillary lesions in AD brains is composed of paired helical filaments (PHFs) of abnormally hyperphosphorylated tau. We used antibodies specifically targeting PHFs to perform immunohistochemistry on mouse brain sections, and the results are shown in Figure 2. Figure 12 As shown in Figure B, treatment with the Tau294-305 mRNA vaccine and the Tau325-336 mRNA vaccine significantly reduced the PHF-positive area in the hippocampus of transgenic mice. This demonstrates that vaccine treatment significantly improves tau pathology in transgenic mice.

[0121] (3) Study on the clearance of pathological proteins by two dual mRNA vaccines in 3×Tg mice;

[0122] At 11 months of age, 3×Tg transgenic mice were anesthetized and perfused to obtain the whole brain of the mice. After dehydration, paraffin embedding, and sectioning, the levels of Aβ and Tau pathological proteins in the brains of mice in different immune groups were detected by immunohistochemistry. Figure 12 As shown in Figures C and D, treatment with the Dual1-6 + 294-305 mRNA vaccine and the Dual1-14 + 325-336 mRNA vaccine significantly reduced Aβ plaques and PHF-positive areas in the hippocampus of transgenic mice. This demonstrates that dual-targeted mRNA vaccine treatment effectively ameliorates Aβ and tau pathology in transgenic mice.

[0123] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. mRNA, characterized in that It is composed of three copies of Aβ1-6 or Aβ1-14 antigen epitopes, Tau294-305 or Tau325-336 antigen epitopes, and Th helper epitopes; the three copies of Aβ1-6, Aβ1-14, Tau294-305 or Tau325-336 antigen epitopes and Th helper epitopes are connected by a linker.

2. The mRNA according to claim 1, wherein There are six types: 3copy Aβ1-6-Th mRNA: composed of three copies of Aβ1-6 antigen epitopes and Th helper epitopes connected by a linker; 3copy Tau294-305-Th mRNA: composed of three copies of the Tau294-305 antigen epitope and the Th helper epitope connected by a linker; 3copyAβ1-6+3copyTau294-305-Th mRNA: composed of three copies of Aβ1-6 antigen epitopes and three copies of Tau294-305 antigen epitopes connected to Th helper epitopes through a linker; 3copy Aβ1-14-Th mRNA: composed of three copies of Aβ1-14 antigen epitopes and Th helper epitopes connected by a linker; 3copy Tau325-336-Th mRNA: composed of three copies of the Tau325-336 antigen epitope and the Th helper epitope connected by a linker; 3copyAβ1-14+3copyTau325-336-Th mRNA: composed of three copies of Aβ1-14 antigen epitope and three copies of Tau325-336 antigen epitope connected to Th auxiliary epitope through linker.

3. The mRNA according to claim 1, wherein The Th helper epitopes are composed of permutations and combinations of Th epitopes; the Th epitopes include norovirus epitope P1, norovirus epitope P2, tetanus epitope T1, tetanus epitope T2, tetanus epitope T3, pan-HLA DR binding epitope PADRE, measles epitope MVF5 and hepatitis B epitope HBsAg.

4. The coding region amino acid sequence is characterized in that The method is encoded by translation of the mRNA according to any one of claims 1 to 3.

5. A vaccine composition, characterized in that The mRNA is obtained by mixing the mRNA according to any one of claims 1 to 3 with a lipid phase via a microfluidic system.

6. Use of the mRNA according to any one of claims 1 to 3 or the vaccine composition according to claim 5 in the preparation of a drug for treating or preventing Alzheimer's disease.

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