Personalized vaccine administration

Abstract

Provided herein are methods of producing a personalized packaged vaccine for a subject. Also provided are methods of administering the personalized vaccine to a subject. Additionally provided are syringes having an igniter and a removable cartridge.

Description

[Background technology]

[0001] Current medical practice in vaccinology is to uniformly administer the same set of vaccines to everyone in a population unless there are contraindications, an approach based on several assumptions. One assumption is that in principle everyone will develop a similar level of antibody protection or cell-mediated immunity and will respond to the immune system in a similar way with few relevant side effects. A major drawback of this approach is that it does not take into account individual differences in disease risk, immune response, any genetic predisposition to reactogenicity, or even differences in the dosage required for immunity generation. On the other hand, advances in immunology, genetics, molecular biology, and bioinformatics have demonstrated the value of an individualized approach to therapeutic drug selection and administration.

[0002] Recent advances in genomics and proteomics have also focused attention on disease-associated mutations in patients. For example, tumor mutanomes have revealed that every tumor harbors a unique set of “driver” and “passenger” mutations. These observations provide unique opportunities for personalized therapy. Tumor cells expressing mutant proteins such as neoantigens present new epitopes to major histocompatibility complex (MHC) molecules. In contrast to tumor-associated antigens, whose expression is found in both healthy and tumor cells, neoantigens arise from mutations in tumors and are therefore entirely restricted to tumor cells. Thus, immunotherapy that leverages the wealth of genomic and proteomic data to develop personalized strategies based on disease-associated mutations allows for highly specific targeting of disease-involved cells without compromising healthy tissues and without being limited by immune tolerance mechanisms.

[0003] Non-viral gene delivery, as exemplified by the reports of DNA-based gene delivery systems developed and optimized over the past decades, offers a promising solution to the limitations associated with viral vector-based vaccines. Although naked DNA plasmids can be directly injected into mice via intramuscular, intradermal, or intravenous routes to introduce genes of interest into muscle, skin, and liver tissues, respectively, the efficiency of in vivo delivery of naked DNA is limited by its chemical instability, susceptibility to nuclease action, rapid clearance, and inefficient delivery to local lymph nodes. Cationic lipids that form liposome-DNA complexes to increase delivery have been widely used, and new delivery systems such as transdermal patches can enhance the delivery of DNA plasmids targeted to skin-resident dendritic cells. In particular, for mRNA-based vaccines, the chemical instability and low delivery efficiency of mRNA remain major barriers to therapeutic efficacy, making in vivo delivery of naked mRNA still challenging. Summary of the Invention

[0004] To address the issue of instability in DNA, RNA, peptide or vaccines thereof, the present disclosure provides, in one aspect, a method for producing a packaged vaccine personalized for a subject, comprising synthesizing a vaccine comprising DNA, RNA or peptide, and placing the vaccine in a cartridge configured to be loaded into a syringe.

[0005] In another aspect, the present disclosure also provides a method of administering an individualized vaccine to a subject, comprising manufacturing an individualized packaged vaccine for a subject according to the methods disclosed herein, loading the cartridge into a syringe, and injecting the individualized vaccine from the syringe into the subject.

[0006] In another aspect, the present disclosure further provides a method of treating or ameliorating a disease associated with a mutation in a subject, comprising administering to the subject a personalized vaccine according to the methods disclosed herein.

[0007] In another aspect, the present disclosure further provides a syringe comprising an igniter and a removable cartridge. In some embodiments, the removable cartridge is configured to contain a vaccine comprising DNA, RNA, or peptide. In another aspect, the present disclosure provides a use of the syringe to administer a personalized vaccine to a subject according to the methods described herein. In another aspect, the present disclosure provides a use of the syringe to treat or ameliorate a tumor in a subject according to the methods described herein. [Brief description of the drawings]

[0008] [Figure 1A] FIG. 13 is a diagram showing an exemplary injection pressure progression. [Figure 1B] FIG. 13 is a diagram showing an exemplary injection pressure progression. [Diagram 2] FIG. 1 shows the progression of the combustion pressure associated with powder combustion, the pressure on the sealed dosing liquid, and the injection pressure. [Diagram 3] FIG. 1 shows the gene expression enhancing effect of naked mRNA encoding GFP by injection as described herein. [Figure 4] FIG. 1 shows the gene expression enhancing effect of naked mRNA encoding Luc by injection as described herein. [Figure 5A] FIG. 13 is a diagram showing the progress of injection pressure in the first modified example. [Figure 5B] FIG. 13 is a diagram showing the progress of injection pressure in the first modified example. [Figure 6A] FIG. 13 is a diagram showing the progress of the injection pressure in the second modified example. [Figure 6B] FIG. 13 is a diagram showing the progress of the injection pressure in the second modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Hereinafter, exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the embodiments disclosed below, and can be implemented in various forms. The following embodiments are described to enable those skilled in the art to embody and implement the embodiments of the present disclosure.

[0010] definition

[0011] Terms such as first, second, etc. may be used to describe various elements, but these elements are not limited by these terms. These terms are used only to distinguish one element from another. For example, a first element can be referred to as a second element, and similarly, a second element can be referred to as a first element, without departing from the scope of the exemplary embodiments. The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0012] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. The singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0013] The terms "comprises", "comprising", "includes" and / or "including" as used above specify the presence of stated features, integers, steps, operations, elements, components and / or groups thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. All terms used herein, including technical and scientific terms, may have the same meaning as commonly understood by those skilled in the art of the present invention. Predefined, commonly used terms may have the same or similar meaning as the contextual meaning of the relevant art, and are not to be construed in an idealized or overly formal sense unless the context clearly indicates otherwise.

[0014] As used herein, the term "about" is meant to modify values ​​such as, for example, the length of a nucleotide sequence, the degree of error, dimensions, the amount of raw materials in a composition, concentration, volume, process temperature, process time, yield, flow rate, and pressure, and ranges thereof, and refers to the variations in quantities that may occur due to, for example, typical measuring and handling procedures used to make a compound, composition, concentrate, or formulation used; inadvertent errors in these procedures; differences in the manufacture, source, or purity of starting materials or raw materials used to carry out these methods; and similar considerations. The term "about" also encompasses amounts that differ due to, for example, aging of a composition, formulation, or cell culture having a particular initial concentration or mixture, and amounts that differ due to mixing or processing of a composition or formulation having a particular initial concentration or mixture. The claims appended hereto include equivalents of these amounts, whether modified by the term "about". The term "about" may further refer to a range of values ​​similar to the stated reference value. In certain embodiments, the term "about" refers to a range of values ​​that fall within a range of 50% or less, 25% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of a stated reference value.

[0015] overview

[0016] Exemplary embodiments of the present disclosure are described in detail below with reference to the accompanying drawings: To facilitate understanding of the present disclosure, like reference numerals refer to like elements throughout the description of the figures and descriptions of the same elements are not repeated.

[0017] In one aspect, the disclosure provides a method of producing a personalized packaged vaccine for a subject, the method comprising synthesizing a vaccine comprising DNA, RNA or a peptide, and placing the vaccine in a cartridge configured to be loaded into a syringe.

[0018] The term "vaccine" refers to a biological preparation that induces or enhances immunity against a particular disease. Typically, a vaccine comprises a conventional saline or buffered aqueous medium in which the composition of the invention is suspended or dissolved. In this form, the composition of the invention can be conveniently used to prevent, ameliorate, or otherwise treat a disease or disorder, such as an infectious disease. When introduced into a host, the vaccine can elicit an immune response, including, but not limited to, the production of antibodies and / or cytokines, and / or the activation of CD8+ T cells, antigen-presenting cells, CD4+ T cells, and / or dendritic cells, and / or other cellular responses.

[0019] In certain embodiments, packaged vaccine may mean that the vaccine is contained in a container, which may be a sealed cartridge that can be subsequently connected to a syringe that injects the vaccine into a subject. In certain embodiments, "individualized" herein may mean that the vaccine is tailored to a specific subject and that the cartridge is labeled or configured to deliver the vaccine therein to the specific subject. The term "cartridge" refers to an element of the syringe that is configured to be removably attached to a syringe. In certain embodiments, the mechanical movement of the movable part of the syringe controls the operation of the cartridge outside, i.e., externally, of the cartridge. In further embodiments, the cartridge comprises a linear arrangement of valves with an opening geometry that allows another part of the syringe to be attached, either by penetrating a needle through a membrane separation element of the cartridge or via an airtight connection. The valves may have a male-male part connection that mates with a corresponding movable handle of the syringe. External rotation of the handle can control the opening and closing of the valve when the cartridge is attached to the syringe.

[0020] In certain embodiments, the packaged vaccines herein may be sealed and only opened at the time of administration. In some embodiments, the vaccine is packaged directly into the cartridge after synthesis. In some embodiments, the cartridge is depressurized.

[0021] In certain embodiments, the DNA, RNA or peptide in the vaccines described herein is DNA. In certain embodiments, the vaccines described herein do not comprise DNA, and the vaccines are not DNA solutions.

[0022] DNA is the usual abbreviation for deoxyribonucleic acid. DNA is a nucleic acid molecule, i.e. a polymer made up of nucleotides. Nucleotides are usually monomers of deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate and deoxycytidine monophosphate, which themselves consist of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, which polymerize to form a characteristic backbone structure. The backbone structure is typically formed by a phosphodiester bond between the sugar moiety, i.e. the deoxyribose, of a first nucleotide and the phosphate moiety of a second adjacent monomer. The specific order of the monomers, i.e. the order of the bases linked to the sugar / phosphate backbone, is called the DNA sequence. DNA may be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with those of the second strand, for example by A / T base pairing and G / C base pairing.

[0023] In certain embodiments, the DNA, RNA or peptide described herein is RNA. The RNA may be selected from the group consisting of small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), short hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

[0024] In certain embodiments, the DNA, RNA or peptide described herein is a peptide. A peptide or polypeptide is typically a polymer of amino acid monomers linked by peptide bonds. A peptide typically contains less than 50 monomer units. However, the term peptide does not exclude molecules having more than 50 monomer units. Long peptides, also called polypeptides, typically have 50-600 monomer units.

[0025] In certain embodiments, the DNA, RNA or peptide described herein is RNA. RNA is the usual abbreviation for ribonucleic acid. RNA is a nucleic acid molecule, i.e. a polymer of nucleotides. The nucleotides of RNA are usually adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate monomers, which are linked together in a so-called backbone. The backbone is formed by a phosphodiester bond between the first sugar, i.e. the ribose, and the phosphate moiety of the adjacent second monomer. A particular sequence of monomers is called an RNA sequence. Usually, RNA can be obtained, for example, by transcription of a DNA sequence in a cell. In eukaryotic cells, transcription typically takes place in the nucleus or mitochondria. In vivo, transcription of DNA usually produces a so-called premature RNA, which is processed into a so-called messenger RNA, usually abbreviated as mRNA. The processing of premature RNA, for example in eukaryotic organisms, includes various different post-transcriptional modifications, such as splicing, formation of a 5' cap, and polyadenylation, transport from the nucleus or mitochondria, etc. These processes are collectively referred to as RNA maturation. Mature messenger RNA usually results in a nucleotide sequence that can be translated into the amino acid sequence of a specific peptide or protein. Typically, mature mRNA contains a 5' cap, a 5'-UTR, an open reading frame, a 3'-UTR and a poly(A) sequence. Apart from messenger RNA, there are several non-coding RNAs that can be involved in the regulation of transcription and / or translation.

[0026] In certain embodiments, the DNA, RNA, or peptide described herein is an mRNA. The mRNA may encode any peptide of interest, such as any naturally occurring or non-naturally occurring or modified peptide. The peptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In further embodiments, the peptide encoded by the mRNA may have a therapeutic effect when expressed in a cell. The RNA or mRNA described herein may include a first region (e.g., a coding region) linked with nucleosides encoding the peptide of interest, a first flanking region (e.g., a 5'-UTR) located at the 5' end of the first region, a second flanking region (e.g., a 3'-UTR) located at the 3' end of the first region, at least one 5' cap region, and a 3'-stabilizing region. In certain embodiments, the RNA or mRNA further includes a poly-A region or a Kozak sequence (e.g., in the 5'-UTR). In certain embodiments, the RNA or mRNA may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal. Any one of the regions of the RNA or mRNA may include one or more alternative moieties (e.g., alternative nucleosides). For example, the 3'-stabilizing region may contain alternative nucleosides such as L-nucleosides, inverted thymidines, or 2'-O-methyl nucleosides, and / or the coding region, 5'-UTR, 3'-UTR, or cap region may include alternative nucleosides such as 5-substituted uridines (e.g., 5-methoxyuridine), 1-substituted pseudouridines (e.g., 1-methyl-pseudouridine or 1-ethyl-pseudouridine), and / or 5-substituted cytidines (e.g., 5-methyl-cytidine).

[0027] In some embodiments, the DNA, RNA, or peptide described herein is a naked nucleic acid molecule. A "naked" nucleic acid molecule refers to a nucleic acid molecule that is not associated with proteins, lipids, or any other molecules that serve to protect the nucleic acid molecule. A naked nucleic acid molecule may be produced in a laboratory for use in genetic engineering or as a result of genetic engineering. In some embodiments, the DNA, RNA, or peptide described herein is naked mRNA. In some embodiments, the amount of naked mRNA in the cartridge is about 0.1 μg or more, about 0.2 μg or more, about 0.3 μg or more, about 0.4 μg or more, about 0.5 μg or more, about 0.6 μg or more, about 0.7 μg or more, about 0.8 μg or more, about 0.9 μg or more, about 1 μg or more, about 5 μg or more, about 10 μg or more, about 20 μg or more, about 30 μg or more, about 40 μg or more, about 50 μg or more, or about 60 μg or more. In further embodiments, the amount of naked mRNA in the cartridge is about 200 μg or less, about 190 μg or less, about 180 μg or less, about 170 μg or less, about 160 μg or less, about 150 μg or less, about 140 μg or less, about 130 μg or less, about 120 μg or less, about 110 μg or less, about 100 μg or less, about 90 μg or less, about 80 μg or less, about 70 μg or less, about 60 μg or less, about 50 μg or less, about 40 μg or less, about 30 μg or less, about 20 μg or less, about 10 μg or less, about 9 μg or less, about 8 μg or less, about 7 μg or less, about 6 μg or less, about 5 μg or less, about 4 μg or less, about 3 μg or less, about 2 μg or less, or about 1 μg or less. In further embodiments, the amount of naked mRNA in the cartridge is about 0.2 μg to 150 μg, about 50 μg to 100 μg, about 10 μg to 150 μg, about 30 μg to 100 μg, or about 20 μg to 110 μg.

[0028] Despite recent advances in vaccine delivery systems, in vivo delivery of vaccines remains challenging. For example, in the case of mRNA vaccines, the ribose (sugar) backbone of RNA, unlike the deoxyribose (sugar) backbone in DNA, is easily hydrolyzed, reducing the stability of the RNA molecule in the circulation. Mammalian mRNA is on average about 2,000 nucleotides long, and a single hydrolysis in the backbone of the mRNA can prevent translation. Furthermore, ribonucleases, which are ubiquitous in the body, can reduce the stability of RNA and also reduce therapeutic efficacy. However, by using the cartridges described herein, personalized vaccines may remain viable without modification of the DNA, RNA or peptide, or vaccine composition. In some embodiments, the vaccine does not include nanoparticles. In some embodiments, the vaccine does not include cationic lipids. In some embodiments, the vaccine does not include PEG lipids. In some embodiments, the vaccine does not include phospholipids. In some embodiments, the vaccine does not include lipids. In some embodiments, the vaccine includes an adjuvant. In some embodiments, the vaccine does not include an adjuvant. In certain embodiments, the adjuvant may be polyinosinic:polycytidylic acid (poly(IC)). In some embodiments, the vaccine does not comprise DNA encoding an immunostimulatory gene. In some embodiments, the vaccine does not comprise liposomes. In some embodiments, the vaccine is non-viral. In some embodiments, the vaccine is comprised of DNA, RNA or a peptide and a buffer. In further embodiments, the buffer may be saline.

[0029] In certain embodiments, the subject has a disease associated with the mutation. A disease herein may include a disorder caused by a genetic mutation. In further embodiments, the DNA, RNA, or peptide described herein comprises a mutation.

[0030] The vaccines described herein may be therapeutic vaccines, comprising antigen DNA, antigen RNA or antigen peptides. In certain embodiments, the antigen DNA or antigen RNA described herein expresses an antigen. In the context of the present invention, "antigen" typically refers to a substance that can be recognized by the immune system, preferably the adaptive immune system, and that can induce an antigen-specific immune response, for example by the formation of antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, an antigen may be or include a peptide or protein that can be presented to T cells by MHC. In the sense of the present invention, an antigen may be the translation product of a nucleic acid molecule provided as defined herein. In this context, fragments, variants and derivatives of peptides and proteins that comprise at least one epitope are also understood as antigens. In certain embodiments, the naked nucleic acid molecules described herein express an antigen selected from the group consisting of pathogenic antigens, tumor antigens, allergenic antigens and autoimmune antigens. The antigen may be from a pathogen associated with an infectious disease. The antigen may be selected from the group consisting of bacterial, viral, fungal and protozoan pathogens. Vaccines can be made, for example, according to the methods disclosed in WO2022112498, WO2022049093 or US 15 / 304701, the disclosures of which are incorporated herein by reference.

[0031] In some embodiments, the subject has a tumor. In further embodiments, the DNA, RNA, or peptide comprises a tumor-specific mutation.

[0032] In certain embodiments, the antigen DNA, antigen RNA, or antigen peptide may be neoantigen DNA, neoantigen RNA, or neoantigen peptide. Mutations may occur due to genetic instability in tumor cells, and the expression of nonsynonymous mutations may produce tumor-specific antigens called neoantigens. Neoantigens are not expressed in normal tissues and are therefore highly immunogenic. Neoantigens have the ability to activate CD4+ and CD8+ T cells to generate immune responses, making them promising new targets for tumor immunotherapy. The development of bioinformatics technology has accelerated the identification of neoantigens, and various neoantigens have been identified. Castle, JC et al. Exploiting the Mutanome for Tumor Vaccination, Cancer Res. 72, 1081-1091 (2012); Yadav, M. et al. Predicting immunogenic tumor mutations by combining mass spectrometry and exome sequencing, Nature 515, 572-576 (2014); Gubin, MM et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens, Nature 515, 577-581 (2014); Kreiter, S. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer, Nature 520, 692-696 (2015); Ott, PA et al. An immunogenic personal neoantigen vaccine for patients with melanoma, Nature 547, 217-221 (2017); Sahin, U. et al.Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer, Nature 547, 222-226 (2017); Keskin, DB et al. Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial, Nature 565, 234-239 (2019); Hilf, N. et al. Actively personalized vaccination trial for newly diagnosed glioblastoma, Nature 565, 240-245 (2019). In some embodiments, the antigen DNA, antigen mRNA, or antigen peptide may be neoantigen mRNA.

[0033] In certain embodiments, the antigen DNA, RNA or peptide may be neoantigen DNA, RNA or peptide, and the cartridge may further contain additional vaccines including patient-derived dendritic cells (DCs) or synthetic long peptides (SLPs). In some embodiments, the antigen DNA, RNA or peptide may be neoantigen mRNA. Cell therapy based on patient-derived DCs (e.g., obtained from ex vivo differentiation of peripheral blood monocytes) exposed to tumor-associated antigens (TAAs) can be enhanced by infusing the DCs back into the patient to enhance T cell activation and tumor cell killing. In some embodiments, the cartridge further comprises a blocking antibody specific for an immune checkpoint protein. In some embodiments, the immune checkpoint protein comprises cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and / or programmed death receptor-1 (PD-1). These antibodies, which are designed to relieve T cells from immune suppression mediated by the CTLA-4 and PD-1 pathways, can promote strong and durable T cell responses that can eliminate tumors and drive cancer regression.

[0034] In some embodiments, the method of producing a personalized packaged vaccine for a subject described herein further comprises detecting a mutation from a sample taken from the subject. In further embodiments, the detecting may comprise whole exome sequencing of the subject's genomic DNA and / or genomic RNA. In further embodiments, the detecting may comprise comparing the subject's genomic sequence to a reference sequence of a somatic genome. In yet further embodiments, the detecting may comprise whole exome sequencing of the subject's tumor DNA and / or RNA. In yet further embodiments, the detecting may comprise comparing the subject's tumor genomic sequence to a reference sequence of a somatic genome. In some embodiments, the sample is a tumor biopsy sample.

[0035] In some embodiments, the method of producing a personalized packaged vaccine for a subject described herein further comprises identifying disease-associated DNA, RNA, or peptides based on mutations detected in a sample taken from the subject. In further embodiments, the identification comprises predicting proteasomal processing. In further embodiments, the identification comprises predicting binding affinity to MHC class I and class II molecules. In further embodiments, the identification comprises mass spectrometry of immunoprecipitated peptides. In some embodiments, the sample is a tumor biopsy.

[0036] Exemplary detection and identification methods are described in Ott, PA et al. An immunogenic personal neoantigen vaccine for patients with melanoma, Nature 547, 217-221 (2017), and other references cited herein.

[0037] In certain embodiments, the subject is in need of a vaccine against an infectious disease. In some embodiments, the vaccine induces an antigen-specific immune response against a coronavirus, including but not limited to Sars-CoV2. In certain embodiments, the vaccine is a cytomegalovirus (CMV) vaccine, including but not limited to, mRNA, for example, as described in John, S. et al. Multi-antigenic human cytomegalovirus mRNA vaccines that elicit potent humoral and cell-mediated immunity, Vaccine 36(12), 1689-1699 (2018).

[0038] In certain embodiments, the packaged vaccine is stored at room temperature. In further embodiments, the packaged vaccine is stored at a temperature between 2° C. and 8° C. In further embodiments, the packaged vaccine is stored at a temperature below −10° C., below −20° C., below −30° C., below −40° C., below −50° C., below −60° C., or below −70° C. In certain embodiments, the packaged vaccine may be diluted before administration to a subject, either before or after storage.

[0039] In one aspect, the present disclosure provides a method of administering an individualized vaccine to a subject, comprising manufacturing a packaged vaccine individualized for a subject according to the methods disclosed herein, loading the cartridge into a syringe, and injecting the individualized vaccine from the syringe into the subject.

[0040] In some embodiments, the syringes described herein may comprise a cartridge and an igniter, whereby when the igniter is ignited, the contents of the cartridge are injected into a subject.

[0041] In some embodiments, the syringe is needleless. In certain embodiments, the syringe may be a needleless syringe, which includes a cartridge as described herein, an igniter including an igniter that generates plasma during combustion immediately after ignition, and then has a pressure characteristic in which the combustion product does not contain gas components when the temperature is reduced to room temperature and the combustion product is condensed, or the amount of gas components contained in the combustion product is reduced compared to before condensation, resulting in a decrease in the generated pressure, and a nozzle portion having an outlet through which the vaccine pressurized by the combustion of the igniter flows and is discharged to the injection target area. In a further embodiment, the temperature of the combustion product during pressurization changes to near room temperature within 20 msec after the pressure applied to the vaccine by the combustion of the igniter reaches a first peak discharge force in the pressurization process for discharging the vaccine. In a further embodiment, the temperature of the combustion product during pressurization changes to near room temperature within 10 msec after the pressure applied to the DNA solution by the combustion of the igniter reaches a first peak discharge force. In a specific embodiment, the syringe may be a syringe that injects a vaccine into an injection target from a syringe body without injection through a predetermined structure in a state where the predetermined structure is inserted into the injection target. In a further embodiment, the syringe includes a cartridge and a nozzle part including an injection port through which the biomolecule-containing solution flows and is injected into the injection target, and the solution is pressurized by combustion of an ignition charge in an igniter. In a further embodiment, the maximum injection speed of the biomolecule-containing solution from the start of injection of the biomolecule-containing solution to a time of 0.20 ms is 75 m / s to 150 m / s, and the injection speed of the biomolecule-containing solution of 75 m / s to 150 m / s is sustained for 0.11 ms or more.

[0042] In certain embodiments, exemplary syringes and methods of using the syringes may be those described in U.S. Patent Application Publication Nos. 2018 / 0168789, 2018 / 0369484, and / or 2021 / 0023302, all of which are incorporated by reference herein.

[0043] In certain embodiments, the subject described herein is a human. In certain embodiments, the subject described herein is a non-human. In certain embodiments, the subject described herein is a rodent. In certain embodiments, the subject described herein is a mammal, a bird, a reptile, a fish, an amphibian, or an invertebrate.

[0044] As described herein, vaccine instability is a challenge in expressing the DNA or RNA of the vaccine in a subject. The method of injecting the vaccine can increase the expression of the DNA or RNA upon injection into a subject. In some embodiments, the injection described herein exhibits a two-stage injection profile. By "two-stage injection profile" herein is meant that at least two stages of injection pressure are measured over time during injection. The "first stage of the two-stage injection profile" refers to the first stage measured, and the "second stage of the two-stage injection profile" refers to the second stage measured immediately after the first stage.

[0045] A two-stage injection profile may be obtained, for example, by different pressure sources, an exemplary two-stage injection profile is as shown in Figures 1A and 1B.

[0046] 1A and 1B are injection profiles showing an exemplary progression of pressure (hereinafter simply referred to as "injection pressure") that can be applied to the vaccine described herein. In FIG. 1A and FIG. 1B, the horizontal axis represents elapsed time in milliseconds (msec), and the vertical axis represents injection pressure in MPa. The injection pressure can be measured using conventional techniques. For example, similar to the measurement method described in JP 2005-21640 A, the injection force can be measured by distributing and applying an injection force to the diaphragm of a load cell arranged downstream of the nozzle, sampling the output from the load cell with a data sampling device via a detection amplifier, and storing the sampled output as an injection force (N) per unit time. The injection pressure is calculated by dividing the injection force measured in this way by the area of ​​the injection port of the syringe.

[0047] In certain embodiments, the progression of the injection pressure, and therefore the injection profile, can be altered by employing different ignition charge materials in the igniter. For example, the ignition charge materials can include zirconium and potassium perchlorate (ZPP), titanium hydride and potassium perchlorate (THPP), titanium and potassium perchlorate (TiPP), aluminum and potassium perchlorate (APP), aluminum and bismuth oxide (ABO), aluminum and molybdenum oxide (AMO), aluminum and copper oxide (ACO), aluminum and iron oxide (AFO), and combinations of these explosives. These explosives generate high-temperature and high-pressure plasma during combustion immediately after ignition, and when the combustion products reach room temperature and condense, the combustion products do not have gas components, so the generated pressure can be immediately reduced. Other explosives may be used as ignition charge materials as long as they are appropriately administered.

[0048] In a particular embodiment, the injection pressure and therefore the injection profile can be adjusted by employing various gas generating agents that are burned by an igniter into combustion products to generate gas. The gas generating agent may be exposed to the combustion products from the igniter. The gas generating agent disposed inside the igniter is already known, as disclosed in International Publication No. 2001 / 031282 and Japanese Patent Application Laid-Open No. 2003-25950. An example of the gas generating agent is a single-base smokeless powder consisting of 98% by mass of nitrocellulose, 0.8% by mass of diphenylamine, and 1.2% by mass of potassium sulfate. In addition, various gas generating agents used in gas generators for airbags and gas generators for seat belt pretensioners can also be used. When the gas generating agent is disposed, the combustion completion time of the gas generating agent can be changed by adjusting the dimensions, size, shape, and especially the surface profile of the gas generating agent, and thus the pressure transition applied to the administered liquid can be adjusted to achieve a desired injection pressure transition of the administered liquid.

[0049] The two-stage injection profile described herein is not limited to injection pressure profiles generated by ignition, the two-stage injection profile described herein may be achieved by other methods, for example, by controlling the gas volume and / or rate applied to the vaccine.

[0050] For example, the injection profile shown in Figures 1A and 2B shows a first stage based on the initial ignition of the ignition charge material and a second stage with one peak based on the gas generating agent described above. The first stage in this example includes four vibration elements (i.e., S1-S4), each vibration element having two minima before and after the vibration peak. One vibration element ends with a rear minimum after the vibration peak.

[0051] In some embodiments, the two-stage injection profile has at least two peaks within about 15 msec, about 14 msec, about 13 msec, about 12 msec, about 11 msec, about 10 msec, about 9 msec, about 8 msec, about 7 msec, about 6 msec, about 5 msec, about 4.5 msec, about 4 msec, about 3.5 msec, about 3 msec, about 2.5 msec, about 2 msec, about 1.5 msec, about 1 msec, or about 0.5 msec of injection. As used herein, the term "from injection" can mean starting from the time pressure begins to be applied to the vaccine and / or from the time an increase in pressure on the vaccine is detected. In certain embodiments, the two-stage injection profile has a first peak within about 5 msec, within about 4 msec, within about 3 msec, within about 2 msec, within about 1 msec, within about 0.9 msec, within about 0.8 msec, within about 0.7 msec, within about 0.6 msec, within about 0.5 msec, within about 0.4 msec, within about 0.3 msec, within about 0.2 msec, or within about 0.1 msec of injection.

[0052] In some embodiments, the two-stage injection profile described herein may include a first stage including a plurality of vibration elements, each vibration element having an oscillation peak and two local minima before and after the oscillation peak. In some embodiments, the total amplitude of the vibration element decreases with time. In some embodiments, the at least two peaks are oscillation peaks. In some embodiments, the first peak of the two-stage injection profile described above is an oscillation peak.

[0053] FIG. 1A shows an exemplary injection profile showing the transition of the injection pressure from the start of combustion at the time when the start button of the syringe is pressed for about 40 msec, and FIG. 1B shows an enlarged view of the initial transition of the injection pressure (about 10 msec from the origin) among the pressure transitions shown in FIG. 1A. The rise of the injection pressure does not occur at the origin but at about 5 msec. This is because it takes a certain time for the ignition material to burn, the piston to be pushed out by the combustion energy of the ignition charge, and the vaccine to be pressurized. In the exemplary transition of the injection pressure shown in FIG. 1A and FIG. 1B, there are multiple pressure vibration elements S1 to S4 in a predetermined time Δt from the rise timing T0 to about 2 msec thereafter, and the pressure vibrations generally converge after the predetermined time Δt has passed. In the present embodiment, one cycle of the rise and fall of the injection pressure in the pressure vibration is treated as one pressure vibration element.

[0054] In certain embodiments, the two-stage injection profile is within about 15 msec, within about 14 msec, within about 13 msec, within about 12 msec, within about 11 msec, within about 10 msec, within about 9 msec, within about 8 msec, within about 7 msec, within about 6 msec, within about 5 msec, within about 4 msec, within about 3 msec, within about 2 msec, within about 1.9 msec, within about 1.8 msec, within about 1.7 msec, within about 1.6 msec, within about 1.5 msec, within about 1. The first stage is completed within 4 msec, within about 1.3 msec, within about 1.2 msec, within about 1.1 msec, within about 1.0 msec, within about 0.9 msec, within about 0.8 msec, within about 0.7 msec, within about 0.6 msec, within about 0.5 msec, within about 0.4 msec, within about 0.3 msec, within about 0.2 msec, or within about 0.1 msec (e.g., the first stage is completed, for example, at the minimum after the last vibration element and / or at the beginning of the second stage).

[0055] In FIG. 1A and FIG. 1B, a pressure vibration element S1 (hereinafter referred to as "first vibration element S1") may occur first during a predetermined time Δt from the rising timing T0. The first vibration element S1 is an injection pressure transition in a period including a peak value Px1 (about 45 MPa in this embodiment) from the injection pressure at the rising timing T0 (about 0 MPa in this embodiment) until the next minimum value arrives. The total amplitude of the first vibration element S1 is about 45 MPa in this embodiment. The first vibration element S1 is followed by a second vibration element S2, a third vibration element S3, and a fourth vibration element S4. The period from the rising timing T0 until the last minimum value of the vibration element (for example, the rear minimum value at the end of the last vibration element) is called the "first stage". The second vibration element S2 is an injection pressure transition in a period including a peak value Px2 (about 37 MPa in this embodiment) from the timing when the first vibration element S1 ends until the next minimum value arrives. The period including the peak value Px2 from the end of the minimum value at the end of the first vibration element to the arrival of the next minimum value is called the "second vibration element". In addition, the total amplitude from the smallest minimum value of the second vibration element S2 to the peak of the second element is about 10 MPa in this embodiment. As for the third vibration element S3 and the fourth vibration element S4, the period defining each vibration element and the total amplitude of each vibration element are similar to those of the second vibration element S2, and detailed explanations are omitted, but the total amplitude of the third vibration element S3 and the total amplitude of the fourth vibration element S4 decrease with the passage of time. That is, during a predetermined time Δt, the pressure transition becomes a damped oscillation with the passage of time, and after the predetermined time Δt has passed, the pressure transition becomes a state in which the oscillation is somewhat converged.

[0056] In some embodiments, the total amplitude of the vibrational element in at least one stage of the two-stage injection profile decreases over time. In some embodiments, the total amplitude of the vibrational element in a first stage of the two-stage injection profile decreases over time.

[0057] In some embodiments, the first peak of the two-stage injection profiles described herein is about 0.5 MPa or more, about 1 MPa or more, about 2 MPa or more, about 3 MPa or more, about 4 MPa or more, about 5 MPa or more, about 6 MPa or more, about 7 MPa or more, about 8 MPa or more, about 9 MPa or more, about 10 MPa or more, about 11 MPa or more, about 12 MPa or more, about 13 MPa or more, about 14 MPa or more, about 15 MPa or more, or about 16 MPa or more. In some embodiments, the first peak of the two-stage injection profiles described herein is less than about 50 MPa, less than about 49 MPa, less than about 48 MPa, less than about 47 MPa, less than about 46 MPa, less than about 45 MPa, less than about 44 MPa, less than about 43 MPa, less than about 42 MPa, less than about 41 MPa, less than about 40 MPa, less than about 39 MPa, less than about 38 MPa, less than about 37 MPa, less than about 36 MPa, or less than about 35 MPa.

[0058] The first peak of the two-stage injection profile may be the highest peak in the first stage of the two-stage injection. The first peak of the two-stage injection profile may be the highest vibration peak in the first stage of the two-stage injection, and the vibration element after the first stage may have a peak with a lower height, for example, as shown in Figures 1A and 1B. The height of the highest peak of the first stage and / or the height of the first peak in the two-stage profile can be predetermined or adjusted depending on the target tissue to which the vaccine is administered. In the case of direct administration to organs and vulnerable lesions, for example, in the two-stage injection profile, the highest peak of the first stage and / or the first peak may be about 0.5 MPa or more, about 1 MPa or more, about 2 MPa or more, about 3 MPa or more, about 4 MPa or more, or about 5 MPa or more. In further embodiments, in a two-stage injection profile, the highest peak and / or the first peak of the first stage is less than about 20 MPa, less than 19 MPa, less than 18 MPa, less than 17 MPa, less than 16 MPa, less than 15 MPa, less than 14 MPa, less than 13 MPa, less than 12 MPa, less than 11 MPa, less than 10 MPa, less than 9 MPa, less than 8 MPa, less than 7 MPa, less than 6 MPa, or less than 5 MPa. In further embodiments, in a two-stage injection profile, the highest peak and / or the first peak of the first stage is between 0.5 MPa and 20 MPa, between 0.5 MPa and 15 MPa, or between 0.5 MPa and 5 MPa. In the case of transdermal injection, for example, in a two-stage injection profile, the highest peak and / or the first peak is about 15 MPa or more, about 16 MPa or more, about 17 MPa or more, about 18 MPa or more, about 19 MPa or more, about 20 MPa or more, about 21 MPa or more, about 22 MPa or more, about 23 MPa or more, about 24 MPa or more, about 25 MPa or more, about 26 MPa or more, about 27 MPa or more, about 28 MPa or more, about 29 MPa or more, about 30 MPa or more, about 31 MPa or more, about 32 MPa or more, about 33 MPa or more, about 34 MPa or more, about 35 MPa or more, about 36 MPa or more, about 37 MPa or more, about 38 MPa or more, about 39 MPa or more, about 40 MPa or more, about 41 MPa or more, about 42 MPa or more, about 43 MPa or more, about 44 MPa or more, or about 45 MPa or more.In further embodiments, in a two-stage injection profile, the highest peak and / or the first peak of the first stage is less than about 50 MPa, less than about 49 MPa, less than about 48 MPa, less than about 47 MPa, less than about 46 MPa, less than about 45 MPa, less than about 44 MPa, less than about 43 MPa, less than about 42 MPa, less than about 41 MPa, less than about 40 MPa, less than about 39 MPa, less than about 38 MPa, less than about 37 MPa, less than about 36 MPa, or less than about 35 MPa. In further embodiments, in a two-stage injection profile, the highest peak and / or the first peak of the first stage is between 15 MPa and 50 MPa, between 30 MPa and 36 MPa, or between 20 MPa and 36 MPa.

[0059] Alternatively, as shown in Figures 5A, 5B, 6A and 6B, the highest peak in the two-step injection profile may be in the second step of the two-step injection. The height of the highest peak of the first and second steps in the two-step profile may be predetermined or adjusted depending on the target tissue to which the vaccine is administered. For example, in the two-step injection profile, the highest peak of the first step may be about 0.5 MPa or more, about 1 MPa or more, about 2 MPa or more, about 3 MPa or more, about 4 MPa or more, about 5 MPa or more, about 6 MPa or more, about 7 MPa or more, about 8 MPa or more, about 9 MPa or more, about 10 MPa or more, about 11 MPa or more, about 12 MPa or more, about 12 MPa or more, about 14 MPa or more, or about 15 MPa or more. Also, the highest peak of the first step may be less than 50 MPa, less than 45 MPa, less than 40 MPa, less than 39 MPa, less than 38 MPa, less than 37 MPa, less than 36 MPa, or less than 35 MPa. Further, in a two-stage injection profile, the highest peak of the second stage may be about 10 MPa or more, about 12 MPa or more, about 14 MPa or more, about 16 MPa or more, about 20 MPa or more, about 21 MPa or more, about 22 MPa or more, about 23 MPa or more, about 24 MPa or more, about 25 MPa or more, about 26 MPa or more, or about 27 MPa or more, and the highest peak of the first stage may be less than 80 MPa, less than 75 MPa, less than 70 MPa, less than 68 MPa, less than 66 MPa, less than 65 MPa, less than 64 MPa, less than 63 MPa, less than 62 MPa, less than 61 MPa, or less than 60 MPa.

[0060] In certain embodiments, the calculated period from the peak value of the first vibration element S1 to the peak value of the second vibration element S2 is within about 1 msec, within about 0.9 msec, within about 0.8 msec, within about 0.7 msec, within about 0.6 msec, within about 0.5 msec, within about 0.4 msec, or within about 0.3 msec. In certain embodiments, the calculated period from the peak value of the second vibration element S2 to the peak value of the third vibration element S3 is within about 1.1 msec, within about 1 msec, within about 0.9 msec, within about 0.8 msec, within about 0.7 msec, within about 0.6 msec, within about 0.5 msec, within about 0.4 msec, or within about 0.3 msec. The period just before the convergence state is reached may be slightly shorter, but the transition of the injection pressure may occur at a generally constant period within a predetermined time Δt. In certain embodiments, the progression of the injection pressure at a given time Δt may be a pressure oscillation at a frequency of about 2200 Hz or less, about 2100 Hz or less, about 2000 Hz or less, about 1900 Hz or less, about 1800 Hz or less, or about 1700 Hz or less. In certain embodiments, the pressure oscillation may be an oscillation at a frequency of about 1500 Hz or more, about 1600 Hz or more, about 1700 Hz or more, about 1800 Hz or more, about 1900 Hz or more, or about 2000 Hz or more. If the second stage of the two-stage injection profile is higher than the first stage, there may not be a need for injection pressure oscillation during the first stage of the two-stage injection profile. For example, if the pressure at the highest peak of the second stage is 2, 3, 4, 5, 6, or 7 times the pressure at the highest peak of the first stage, there may not be a need for a first stage oscillation to inject the vaccine. Similarly, if the first stage has a higher peak than the second stage, there may not be a need for injection pressure oscillation during the first stage of the two-stage injection profile.

[0061] In a particular embodiment, the pressure fluctuation at the predetermined time Δt may be due to the combustion of the ignition material of the igniter described herein. Also, near the timing when the predetermined time Δt has elapsed, the combustion product of the ignition material may start the combustion of the gas generating agent in the syringe, and the combustion energy may further start to act on the vaccine. As a result, in the example shown in FIG. 1A, after the predetermined time Δt has elapsed, the injection pressure rises again, and a peak value Pv called the "highest peak of the second stage" arrives at a timing of about 18 msec. Also, thereafter, the injection pressure gradually decreases over time. Since the burning speed of the gas generating agent may be slower than the burning speed of the ignition material, the rate of increase in the injection pressure due to the burning of the gas generating agent may also be relatively slow. In certain embodiments, combustion of the gas generant may commence about 8 msec, about 7.5 msec, about 7 msec, about 6.5 msec, about 6 msec, about 5.5 msec, about 5 msec, about 4.5 msec, about 3 msec, about 3.5 msec, about 3 msec, about 2.5 msec, about 2 msec, about 1.5 msec, or about 1 msec prior to injection. In certain embodiments, the peak Py of the gas generant combustion or the highest peak of the second stage may occur about 30 msec, about 29 msec, about 28 msec, about 27 msec, about 26 msec, about 25 msec, about 24 msec, about 23 msec, about 22 msec, about 21 msec, about 20 msec, about 19 msec, about 18 msec, about 17 msec, about 16 msec, about 15 msec, about 14 msec, about 13 msec, about 12 msec, about 11 msec, or about 10 msec prior to injection. In certain embodiments, the peak Py of the gas generant combustion or the highest peak of the second stage may occur at about 7 msec, about 8 msec, about 9 msec, about 10 msec, about 11 msec, about 12 msec, about 13 msec, about 14 msec, about 15 msec, about 16 msec, about 17 msec, about 18 msec, about 19 msec, or about 20 msec after injection.In certain embodiments, the two-stage injection profile has at least one peak of the second stage at about 30 msec, about 29 msec, about 28 msec, about 27 msec, about 26 msec, about 25 msec, about 24 msec, about 23 msec, about 22 msec, about 21 msec, about 20 msec, about 19 msec, about 18 msec, about 17 msec, about 16 msec, about 15 msec, about 14 msec, about 13 msec, about 12 msec, about 11 msec, about 10 msec, about 9 msec, about 8 msec, about 7 msec, about 6 msec, about 5 msec, or about 4 msec prior to injection. In certain embodiments, the two-stage injection profile has at least one peak of the second stage at about 7 msec, about 8 msec, about 9 msec, about 10 msec, about 11 msec, about 12 msec, about 13 msec, about 14 msec, about 15 msec, about 16 msec, about 17 msec, about 18 msec, about 19 msec, or about 20 msec after injection.

[0062] In some embodiments, the two-stage injection profile may include a second stage having only one peak.

[0063] The height of the highest peak of the second stage of the two-stage profile can be predetermined or adjusted depending on the target tissue to which the vaccine is administered. In certain embodiments, in the case of direct administration to organs and vulnerable lesions, in the two-stage injection profile, the highest peak of the second stage is about 0.1 MPa or more, about 0.2 MPa or more, about 0.3 MPa or more, about 0.4 MPa or more, about 0.5 MPa or more, about 0.6 MPa or more, about 0.7 MPa or more, about 0.8 MPa or more, about 0.9 MPa or more, about 1 MPa or more, about 2 MPa or more, about 3 MPa or more, about 4 MPa or more, about 5 MPa or more, about 6 MPa or more, about 7 MPa or more, about 8 MPa or more, about 9 MPa or more, or about 10 MPa or more. In further embodiments, in a two-stage injection profile, the highest peak of the second stage is less than about 15 MPa, less than about 14 MPa, less than about 13 MPa, less than about 12 MPa, less than about 11 MPa, less than about 10 MPa, less than about 9 MPa, less than about 8 MPa, less than about 7 MPa, less than about 6 MPa, less than about 5 MPa, less than about 4 MPa, less than about 3 MPa, less than 2 MPa, or less than about 1 MPa. In further embodiments, in a two-stage injection profile, the second peak is between about 0.1 MPa and about 15 MPa, between about 1 MPa and about 10 MPa, or between about 3 MPa and about 6 MPa. In certain embodiments, for transdermal injection, in a two-stage injection profile, the highest peak of the second stage is about 20 MPa or more, about 21 MPa or more, about 22 MPa or more, about 23 MPa or more, about 24 MPa or more, about 25 MPa or more, about 26 MPa or more, about 27 MPa or more, about 28 MPa or more, about 29 MPa or more, about 30 MPa or more, about 31 MPa or more, about 32 MPa or more, about 33 MPa or more, about 34 MPa or more, or about 35 MPa or more. In further embodiments, in a two-stage injection profile, the highest peak of the second stage is less than about 45 MPa, less than about 44 MPa, less than about 43 MPa, less than about 42 MPa, less than about 41 MPa, less than about 40 MPa, less than about 39 MPa, less than about 38 MPa, less than about 37 MPa, less than about 36 MPa, less than about 35 MPa, less than about 34 MPa, less than about 33 MPa, less than about 32 MPa, less than about 31 MPa, less than about 30 MPa, or less than about 29 MPa.In further embodiments, in a two stage injection profile, the highest peak of the second stage is 30 MPa to 40 MPa, 30 MPa to 36 MPa, or 20 MPa to 36 MPa. In further embodiments, the highest peak of the first stage may be in the range of 10.0 MPa to 38.0 MPa and the highest peak of the second stage may be in the range of 25.0 MPa to 64.0 MPa.

[0064] In some embodiments, the highest peak of the second stage of the two-stage profile is lower than the highest peak of the first stage of the two-stage profile. In certain embodiments, the highest peak of the second stage of the two-stage profile is lower than the first peak of the first stage of the two-stage profile. In some embodiments, the highest peak of the first stage of the two-stage profile is lower than the highest peak of the second stage of the two-stage profile. In certain embodiments, the highest peak of the second stage of the two-stage profile is higher than the first peak of the first stage of the two-stage profile.

[0065] In certain embodiments, the injection is completed within about 400 msec, within about 450 msec, within about 300 msec, within about 250 msec, within about 200 msec, within about 150 msec, or within about 100 msec of injection.

[0066] In some embodiments, the injection is a transdermal injection. In some embodiments, the injection does not include a transdermal injection.

[0067] In some embodiments, the injection is an intramuscular injection. In some embodiments, the injection is a subcutaneous injection. In some embodiments, the injection is an intradermal injection. In some embodiments, the injection is an intralesional injection. In certain embodiments, the vaccine may be injected into a specific organ of interest, for example, during surgery. In some embodiments, the injection is an intratumoral injection. In some embodiments, the injection is an intranodal injection. Some embodiments do not include an intranodal injection. In some embodiments, the injection is an intralymphatic injection.

[0068] In some embodiments, the amount of mRNA in the personalized vaccine injected into a subject is about 0.1 μg or more, about 0.2 μg or more, about 0.3 μg, about 0.4 μg or more, about 0.5 μg or more, about 0.6 μg or more, about 0.7 μg or more, about 0.8 μg or more, about 0.9 μg or more, about 1 μg or more, about 5 μg or more, about 10 μg or more, about 20 μg or more, about 30 μg or more, about 40 μg or more, about 50 μg or more, or about 60 μg or more. In further embodiments, the amount of mRNA in the personalized vaccine injected into a subject is about 200 μg or less, about 190 μg or less, about 180 μg or less, about 170 μg or less, about 160 μg or less, about 150 μg or less, about 140 μg or less, about 130 μg or less, about 120 μg or less, about 110 μg or less, about 100 μg or less, about 90 μg or less, about 80 μg or less, about 70 μg or less, about 60 μg or less, about 50 μg or less, about 40 μg or less, about 30 μg or less, about 20 μg or less, about 10 μg or less, about 9 μg or less, about 8 μg or less, about 7 μg or less, about 6 μg or less, about 5 μg or less, about 4 μg or less, about 3 μg or less, about 2 μg or less, or about 1 μg or less. In further embodiments, the amount of mRNA in the personalized vaccine injected into a subject is about 0.2 μg to about 150 μg, about 50 μg to about 100 μg, about 10 μg to about 150 μg, about 30 μg to about 100 μg, or about 20 μg to about 110 μg.

[0069] In one aspect, the present disclosure further provides a method of treating or ameliorating a disease associated with a mutation in a subject, comprising administering to the subject a personalized vaccine according to the methods disclosed herein. In some embodiments, the disease is a tumor (cancer).

[0070] In one aspect, the present disclosure further provides a syringe comprising an igniter and a removable cartridge. The syringe may be a syringe as described above. In some embodiments, the removable cartridge is configured to contain a vaccine as described herein, or DNA, RNA, or peptide. In some embodiments, the syringe does not comprise a needle. In some embodiments, the syringe does not comprise a spring. In some embodiments, the syringe is configured for delivery into a cell.

[0071] In another aspect, the present disclosure provides a use of the syringe to administer a personalized vaccine to a subject according to the methods described herein.In another aspect, the present disclosure provides a use of the syringe to treat or ameliorate a tumor in a subject according to the methods described herein. EXAMPLES

[0072] material Dulbecco's phosphate buffered saline (Nacalai Tesque, D-PBS) TE buffer pH 8.0 (Nacalai Tesque) PBS-Tablet (Takara Bio Inc.) Water (Nacalai Tesque) Naked mRNA GFP (CleanCap® EGFP mRNA, TriLink) Naked mRNA_Luc (TriLink, CleanCap® FLuc mRNA) Naked mRNA_U modified_Luc (TriLink, CleanCap® FLuc mRNA (5 MoU)) Passive Lysis Buffer 5X (Promega) Luciferase assay (Promega, Luciferase Assay System) C57BL / 6 and BALB / c mice were purchased from Claire Japan.

[0073] Equipment used Refrigerated centrifuge (Tomy MDX-300) Autoclave (Tomy, LSX-700) 8mm biopsy pouch (Kai Industries, 8mm biopsy pouch) Luminometer (Kikkoman, C-100N)

[0074] First, the device of the present invention was used to administer mRNA to a living body, and whether gene expression of the administered mRNA was enhanced was evaluated using mRNA encoding GFP (naked mRNA GFP). For administration, 10-week-old male BALB / c mice were used, and euthanized 6 hours after administration to collect data. For administration, a device containing 30 mg of ZPP ignition material and 30 mg of GG gas generating material in a container with a nozzle diameter of 0.1 mm was used. The dose per administration was 20 μL, and the mRNA was 0.01 to 0.5 mg / mL (0.2 to 10 μg / shot). The skin at the administration site was attached to a glass bottom dish manufactured by Matsunami Glass Industry Co., Ltd., and gene expression was observed using a fluorescent microscope BZ-X710 manufactured by KEYENCE Corporation.

[0075] As a result, when the mRNA was administered using a 30G injection needle, only a small amount of gene expression was obtained for each amount of mRNA. On the other hand, when the mRNA was administered using the device, gene expression was confirmed in the administered skin for all amounts of mRNA from 0.01 to 0.5 mg / mL (Figure 3). The fluorescence intensity corresponding to the gene expression level increased depending on the amount of mRNA used, confirming that the administered mRNA was expressing genes. With the device, gene expression of naked mRNA was confirmed even with a small amount of only 0.01 mg / mL, and therefore efficient mRNA cytoplasmic transfer and subsequent gene expression were obtained using the device.

[0076] When comparing the GFP fluorescence intensity between the 30G injection needle and the device, the device showed clearly stronger fluorescence at all mRNA amounts from 0.01 to 0.5 mg / mL (Figure 3). These results confirmed that the device is easier to induce gene expression of naked mRNA than the injection needle, and confirmed the effect of enhancing gene expression using the device.

[0077] (2) The device enhances gene expression of the mRNA encoding Luc.

[0078] The gene expression enhancing effect of the device was evaluated for mRNA encoding a different protein, Luc (naked mRNA Luc). Ten-week-old male BALB / c mice were used for administration, and euthanized 6 hours after administration to collect data. For administration, a device containing 30 mg of ZPP ignition material and 30 mg of GG gas generator in a container with a nozzle diameter of 0.1 mm was used. The dose was 20 μL, and the mRNA was 0.01 to 0.1 mg / mL (0.2 to 2 μg / shot). For gene expression, the skin at the administration site was sampled using an 8 mm biopsy trephine, and a lysate was prepared using 5-fold diluted Passive Lysis Buffer 5X. Next, the amount of luciferase emitted in 10 seconds was measured using a Promega luciferase assay system and a Kikkoman luminometer C-100N to evaluate gene expression.

[0079] As a result, as in the case of GFP, when the 30G needle was used for administration, only a small amount of gene expression was obtained for both amounts of mRNA. On the other hand, when the device was used for administration, high gene expression was confirmed (Figure 4). To evaluate the effect of the device in enhancing gene expression, the gene expression levels were compared between the 30G needle and the device; at 0.01 mg / mL mRNA, the device was approximately 2,300 times higher, and at 0.1 mg / mL mRNA, the device was approximately 300 times higher (Figure 4). The effect of using the device in enhancing gene expression was also confirmed for mRNA encoding Luc.

[0080] As described above, the gene expression enhancement effect of the device was confirmed for multiple reporter proteins such as GFP and Luc, which demonstrated that gene expression by the device does not depend on the gene sequence encoded in the mRNA. This suggests that the gene expression enhancement effect by the device can be obtained for mRNA encoding any gene.

[0081] (3) Alternative Syringe Injection Pressure Profiles - Example 1

[0082] A syringe with a nozzle diameter of 0.5 mm was filled with 150 μL of water, and the injection pressure in the syringe was evaluated from the time when the water was pressurized by the combustion of the ignition charge until after injection. 55 mg of explosive containing zirconium and potassium perchlorate (ZPP) was used as the explosive, and 40 mg of single-base smokeless explosive (hereinafter sometimes referred to as "GG") was used as the gas generating agent.

[0083] The injection pressure was measured by distributing the injection force to the diaphragm of a load cell placed downstream of the nozzle as described in JP 2005-21640 A, and the output from the load cell was collected by a data collection and display device via a detection amplifier and displayed and stored as the injection force (N) per time. The injection pressure was calculated by dividing the injection force (N) by the area of ​​the nozzle port. The measurements were taken using a CLS-2NA manufactured by Tokyo Keiki Kenkyusho. A total of 30 measurements were taken.

[0084] Of the 30 measurements, the two measurements where the highest and lowest peaks of the second stage of the two-stage profile were detected are shown in Figures 5A and 5B. The second stage peak was higher in all 30 measurements, and the average peak pressures of the first and second stages were 4.574 MPa and 9.598 MPa, respectively. On average, the first and second stage peaks were detected at 5.230 msec and 24.150 msec after ignition.

[0085] (3) Alternative Syringe Injection Pressure Profiles - Example 2

[0086] The same conditions used in Example 1 above were repeated, except that the amounts of ZPP and GG were both increased from 55 mg to 65 mg. A total of 30 measurements were made, and the two measurements where the highest and lowest peaks of the second stage of the two-stage profile were detected are shown in Figures 6A and 6B. The second stage peak was higher in all 30 measurements, and the average peak pressures of the first and second stages were 6.102 MPa and 12.562 MPa, respectively. On average, the first and second stage peaks were detected at 5.243 msec and 21.957 msec after ignition.

[0087] Below are some exemplary embodiments of the present disclosure. Embodiment 1. A method of producing a personalized packaged vaccine for a subject, comprising synthesizing a vaccine comprising DNA, mRNA or a peptide, and placing the vaccine in a cartridge configured to be loaded into a syringe.

[0023] Embodiment 2. The method of embodiment 1, wherein the subject has a tumor and the DNA, mRNA or peptide comprises a tumor-specific mutation.

[0023] Embodiment 3. The method of embodiment 1 or 2, wherein the subject has a tumor and the DNA, mRNA or peptide is neoantigen DNA, neoantigen mRNA or neoantigen peptide. Embodiment 4. The method of any one of embodiments 1 to 3, further comprising detecting a mutation from a sample taken from the subject.

[0023] Embodiment 5. The method of embodiment 4, wherein the detection comprises whole-exome sequencing of the subject's genomic DNA and / or genomic RNA.

[0023] Embodiment 6. The method of embodiment 4 or 5, wherein detecting comprises comparing the subject's genomic sequence to a reference sequence of a somatic genome. Embodiment 7. The method of any one of embodiments 4 to 6, wherein the detection comprises whole exome sequencing of DNA and / or RNA of the subject's tumor.

[0023] Embodiment 8. The method of any one of embodiments 4 to 7, wherein detecting comprises comparing the subject's tumor genomic sequence to a reference sequence of a somatic genome.

[0021] Embodiment 9. The method of any one of the preceding embodiments, further comprising identifying DNA, mRNA or peptides associated with the disease based on the mutations detected in the sample obtained from the subject. Embodiment 10. The method of embodiment 9, wherein the identifying comprises predicting proteasomal processing and binding affinity to MHC class I and class II molecules.

[0031] Embodiment 11. The method of embodiment 9 or 10, wherein the identification comprises mass spectrometry of the immunoprecipitated peptides. Embodiment 12. The method of any one of embodiments 3 to 11, wherein the sample is a tumor biopsy sample. Embodiment 13. The method of any one of the preceding embodiments, wherein the vaccine does not comprise nanoparticles. Embodiment 14 The method of any one of the preceding embodiments, wherein the vaccine does not comprise a cationic lipid. Embodiment 15. The method of any one of the preceding embodiments, wherein the vaccine does not comprise a lipid. Embodiment 16. The method of any one of the preceding embodiments, wherein the vaccine does not comprise an adjuvant. Embodiment 17. The method of any one of the preceding embodiments, wherein the vaccine does not comprise DNA encoding an immunostimulatory gene.

[0046] Embodiment 18. The method of any one of the preceding embodiments, wherein the vaccine does not comprise a liposome. Embodiment 19. The method of any one of the preceding embodiments, wherein the vaccine is non-viral.

[0023] Embodiment 20. The method of any one of the preceding embodiments, wherein the vaccine consists of neoantigen DNA, neoantigen mRNA or neoantigen peptide and a buffer.

[0036] Embodiment 21. The method of any one of the preceding embodiments, wherein the DNA, mRNA or peptide is a naked nucleic acid molecule.

[0046] Embodiment 22. The method of any one of the preceding embodiments, wherein the DNA, mRNA or peptide is naked mRNA.

[0036] Embodiment 23. The method of embodiment 22, wherein the amount of naked mRNA in the cartridge is at least 0.2 μg. Embodiment 24. The method of any one of the preceding embodiments, wherein the vaccine is packaged directly into the cartridge after synthesis. Embodiment 25. The method of any one of the preceding embodiments, wherein the cartridge is depressurized. Embodiment 26. The method of any one of the preceding embodiments, wherein the vaccine induces an antigen-specific immune response against coronavirus.

[0046] Embodiment 27. The method of any one of the preceding embodiments, wherein the cartridge further comprises a blocking antibody specific for an immune checkpoint protein.

[0081] Embodiment 28. The method of embodiment 27, wherein the immune checkpoint protein comprises cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and / or programmed death receptor-1 (PD-1). Embodiment 29. The method of any one of the preceding embodiments, wherein the syringe further comprises an igniter, and when the igniter is ignited, the contents of the cartridge are injected into the subject. Embodiment 30. A method of administering an individualized vaccine to a subject, comprising manufacturing a packaged vaccine individualized for the subject according to the method of any one of the preceding embodiments, loading the cartridge into a syringe, and injecting the individualized vaccine from the syringe into the subject. Embodiment 31. The method of embodiment 30, wherein the infusion exhibits a two-stage infusion profile comprising a first stage and a second stage, the second stage being after the first stage. Embodiment 32. The method of embodiment 31, wherein the two-stage injection profile has at least two peaks within 15 msec of injection. Embodiment 33. The method of embodiment 31 or 32, wherein the two-stage injection profile has at least two peaks within 1.5 msec of injection. Embodiment 34. The method of any one of embodiments 31-33, wherein the two-stage injection profile has a first peak within 5 msec. Embodiment 35. The method of any one of embodiments 31 to 34, wherein the first stage includes a plurality of vibration elements, each having a vibration peak.

[0046] Embodiment 36. The method of embodiment 35, wherein the at least two peaks are vibration peaks of a vibration element.

[0046] Embodiment 37. The method of embodiment 35 or 36, wherein the overall amplitude of the vibration element decreases over time. Embodiment 38. The method of any one of embodiments 31-37, wherein the first peak is at least 2 MPa. Embodiment 39. The method of any one of embodiments 31-38, wherein the first peak is at least 15 MPa. Embodiment 40. The method of any one of embodiments 31-39, wherein the highest peak of the second stage of the two-stage injection profile is within 30 msec of injection. Embodiment 41. The method of any one of embodiments 31 to 40, wherein the highest peak of the second stage of the two-stage injection profile is within 15 msec of injection. Embodiment 42. The method of any one of embodiments 31 to 41, wherein the two-stage injection profile comprises a second stage having only one peak. Embodiment 43. The method of any one of embodiments 31 to 42, wherein the highest peak of the second stage of the two-stage profile is at least 0.1 MPa. Embodiment 44. The method of any one of embodiments 31 to 43, wherein the highest peak of the second stage of the two-stage profile is at least 10 MPa. Embodiment 45. The method of any one of embodiments 31 to 44, wherein the highest peak of the second stage of the two-stage profile is lower than the highest peak of the first stage of the two-stage profile. Embodiment 46. The method of any one of embodiments 30 to 45, wherein the injection is a transdermal injection. Embodiment 47. The method of any one of embodiments 30-45, wherein the injection does not include a transdermal injection. Embodiment 48. The method of any one of embodiments 30-47, wherein the injection is an intramuscular injection, a subcutaneous injection, or an intradermal injection. Embodiment 49. The method of any one of embodiments 30-47, wherein the injection is an intralesional injection. Embodiment 50. The method of any one of embodiments 30 to 47, wherein the injection is an intratumoral injection. Embodiment 51. The method of any one of embodiments 30 to 47, wherein the injection is an intranodal or intralymphatic injection. Embodiment 52. The method of any one of embodiments 30 to 50, wherein the injection does not include an intranodal injection. Embodiment 53. The method of any one of embodiments 30 to 52, wherein the amount of mRNA in the personalized vaccine injected into the subject is at least 0.2 μg. Embodiment 54. The method of any one of embodiments 30 to 53, wherein the syringe is needleless. Embodiment 55. The method of any one of embodiments 30 to 54, wherein the injector further comprises an igniter, and when the igniter is ignited, the contents of the cartridge are injected into the subject. Embodiment 56. A method for treating, ameliorating or preventing a disease associated with a mutation in a subject, comprising administering to the subject a personalized vaccine according to the method of any one of embodiments 30 to 55. Embodiment 57 The method of embodiment 56, wherein the disease is a tumor (cancer). Embodiment 58. A syringe comprising an igniter and a removable cartridge, the removable cartridge configured to contain a vaccine comprising DNA, mRNA or a peptide. Embodiment 59. The syringe of embodiment 58, wherein the syringe does not include a needle. Embodiment 60. A syringe as described in embodiment 58 or 59, wherein the syringe does not include a spring. Embodiment 61. A syringe described in any one of embodiments 58 to 60, wherein the vaccine does not contain nanoparticles. Embodiment 62. A syringe according to any one of embodiments 58 to 61, wherein the vaccine does not contain a cationic lipid. Embodiment 63. A syringe described in any one of embodiments 58 to 62, wherein the vaccine does not contain lipids. Embodiment 64. A syringe according to any one of embodiments 58 to 63, wherein the vaccine does not contain an adjuvant. Embodiment 65. A syringe according to any one of embodiments 58 to 64, wherein the vaccine does not contain DNA encoding an immunostimulatory gene. Embodiment 66. A syringe according to any one of embodiments 58 to 65, wherein the vaccine does not contain liposomes. Embodiment 67. A syringe according to any one of embodiments 58 to 66, wherein the vaccine is non-viral. Embodiment 68. A syringe described in any one of embodiments 58 to 67, wherein the vaccine consists of neoantigen DNA, neoantigen mRNA or neoantigen peptide and a buffer solution. Embodiment 69. A syringe according to any one of embodiments 58 to 68, wherein the DNA, mRNA or peptide is a naked nucleic acid molecule. Embodiment 70. A syringe according to any one of embodiments 58 to 69, wherein the DNA, mRNA or peptide is naked mRNA. Embodiment 71. The syringe of embodiment 70, wherein the amount of naked mRNA in the cartridge is at least 0.2 μg. Embodiment 72. A syringe described in any one of embodiments 58 to 71, wherein the syringe is configured for delivery into a cell. Embodiment 73. Use of a syringe according to any one of embodiments 30 to 55 for administering a personalized vaccine to a subject according to the method according to any one of embodiments 30 to 45. Embodiment 74. Use of a syringe according to any one of embodiments 55 to 57 for treating or ameliorating a tumor in a subject according to the method according to embodiment 46 or 47.

Claims

**Claim 1** A method for manufacturing an individualized packaged vaccine for a subject, comprising: synthesizing a vaccine comprising DNA, RNA, or a peptide; and placing the vaccine into a cartridge configured to be loaded into a syringe. **Claim 2** The method according to claim 1, wherein the vaccine consists of a neoantigen DNA, a neoantigen RNA, or a neoantigen peptide and a buffer solution. **Claim 3** The method according to claim 1, wherein the DNA, RNA, or peptide is a naked nucleic acid molecule. **Claim 4** The method according to claim 1, wherein the DNA, RNA, or peptide is naked RNA. **Claim 5** The method according to claim 1, wherein the vaccine is directly packaged into the cartridge after synthesis. **Claim 6** The method according to claim 1, wherein the syringe further comprises an igniter, and when the igniter is ignited, the content of the cartridge is injected into the subject. **Claim 7** A syringe comprising an igniter and a removable cartridge, wherein the removable cartridge is configured to contain a vaccine comprising DNA, RNA, or a peptide. **Claim 8** The syringe according to claim 7, wherein the vaccine does not contain nanoparticles, cationic lipids, lipids, adjuvants, DNA encoding an immune activation gene, and liposomes. **Claim 9** The syringe according to claim 7, wherein the DNA, RNA, or peptide is a naked nucleic acid molecule. **Claim 10** The syringe according to claim 7, wherein the DNA, RNA, or peptide is naked RNA. **Claim 11** The syringe according to claim 7, wherein the igniter is for generating a two-stage injection profile of injection pressure. **Claim 12** comprising an ignition powder material and a gas generating agent, The syringe according to claim 11, wherein the two-stage injection profile includes a first stage based on the combustion of the ignition powder material and a second stage based on the combustion of the gas generating agent.

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