Administration of Naked Nucleic Acid Molecules

Abstract

Provided herein is a method of administering a naked nucleic acid molecule to a subject. The method may include injecting the naked nucleic acid molecule into a subject, the injection exhibiting a two-step injection profile including a first step and a second step, the second step being subsequent to the first step, the two-step injection profile having (i) at least two peaks within 15 msec of injection, (ii) a first peak of 2 MPa or more, or (iii) the highest peak of the second step of the two-step injection within 30 msec of injection.

Description

[Background technology]

[0001] 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. However, naked DNA transduction is severely hindered by the barriers posed by the size, shape, and polyanionic charge of DNA, which in turn inhibit DNA cell permeability and susceptibility to serum nucleases. Although naked DNA plasmids can be directly injected intramuscularly, intradermally, or intravenously into mice to deliver genes of interest to muscle, skin, and liver tissues, respectively, the efficiency of naked DNA transduction in vivo is limited by naked DNA's chemical instability, susceptibility to nuclease action, rapid clearance, and inefficient delivery to local lymph nodes. Cationic lipids, which form liposome-DNA complexes to increase transduction, are widely used, and new delivery systems such as transdermal patches can enhance the delivery of DNA plasmids targeted to skin-resident dendritic cells. However, the size, shape, and polyanionic charge of DNA are barriers, which in turn inhibit the cell permeability of DNA, as well as the susceptibility of DNA to serum nucleases, significantly hinder the delivery of naked DNA.In particular, for mRNA-based vaccines, the chemical instability and low delivery efficiency of mRNA remain major barriers to therapeutic efficacy, making the in vivo delivery of naked mRNA still difficult. Summary of the Invention

[0002] To address the problem of instability in naked nucleic acid molecules, the disclosure provides, in one aspect, a method of administering a naked nucleic acid molecule to a subject, comprising injecting the naked nucleic acid molecule into a subject, wherein the injection exhibits a two-stage injection profile having (i) at least two peaks within 15 msec of injection, or (ii) a first peak of 2 MPa or greater.

[0003] In another aspect, the disclosure also provides a method of expressing a gene in a subject, comprising administering to the subject a naked nucleic acid molecule comprising the gene according to the methods described herein.

[0004] In another aspect, the disclosure further provides a method of treating, ameliorating or preventing a disease in a subject in need thereof, comprising expressing a gene in the subject according to the methods described herein, wherein the naked nucleic acid molecule induces an antigen-specific immune response against the disease.

[0005] In another aspect, the disclosure relates to the use of a syringe to administer a naked nucleic acid molecule to a subject according to the methods described herein. In another aspect, the disclosure relates to the use of a syringe to express a gene in a subject according to the methods described herein. In another aspect, the disclosure relates to the use of a syringe to treat, ameliorate or prevent cancer in a subject in need thereof according to the methods described herein. [Brief description of the drawings]

[0006] [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. 13 shows the progression of the combustion pressure associated with powder combustion, the pressure on the sealed dosage liquid, and the injection pressure. [Diagram 3] FIG. 1 shows the gene upregulation effect of naked mRNA encoding GFP by injection as described herein. [Figure 4] FIG. 1 shows the gene upregulation 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

[0007] 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.

[0008] definition 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.

[0009] 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.

[0010] 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.

[0011] 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 includes 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.

[0012] overview 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.

[0013] In one aspect, the disclosure provides a method of administering a naked nucleic acid molecule to a subject, comprising injecting the naked nucleic acid molecule into a subject, wherein the injection exhibits a two-stage injection profile having (i) at least two peaks within 15 msec of injection, or (ii) a first peak of 2 MPa or greater.

[0014] 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. Naked nucleic acid molecules can be produced in the laboratory for use in, or as a result of, genetic engineering.

[0015] In certain embodiments, the naked nucleic acid molecule is DNA. In certain embodiments, the naked nucleic acid molecules described herein do not include DNA. DNA is the common abbreviation for deoxyribonucleic acid. DNA is a polymer of nucleic acid molecules, i.e., 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., 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 the nucleotides of the second strand, for example, by A / T base pairing and G / C base pairing.

[0016] In certain embodiments, the naked nucleic acid molecule 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, polyadenylation, transport from the nucleus or mitochondria, etc. These processes are also referred to together as RNA maturation. Mature messenger RNA usually results in a nucleotide sequence that can be translated into the amino acid sequence of a particular 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 regulating transcription and / or translation. 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. In certain embodiments, the naked nucleic acid molecule is an mRNA. The mRNA may code for any peptide of interest, such as any peptide, naturally or non-naturally occurring or modified. 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 a 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'-stabilization 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).

[0017] In some embodiments, the naked nucleic acid molecule described herein is naked mRNA. In some embodiments, the amount of naked mRNA administered 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 administered 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 administered is about 0.2 μg-150 μg, 50 μg-100 μg, 10 μg-150 μg, 30 μg-100 μg, or 20 μg-110 μg.

[0018] Despite recent advances in vaccine delivery systems, in vivo delivery of naked nucleic acid molecules remains challenging. For example, in the case of mRNA, the ribose (sugar) backbone of RNA, unlike the deoxyribose (sugar) backbone in DNA, is easily hydrolyzed, which reduces the stability of the RNA molecule in the circulation. Mammalian mRNA is about 2,000 nucleotides long on average, and even a single hydrolysis in the backbone of the mRNA can prevent translation. Furthermore, ribonucleases, which are ubiquitous in the body, reduce the stability of RNA and reduce therapeutic efficacy. However, by using the cartridges described herein, personalized vaccines may remain viable without modification of DNA, RNA, or their vaccine compositions.

[0019] In certain embodiments, the naked nucleic acid molecules described herein are administered as a vaccine. The term "vaccine" refers to a biological preparation that induces or enhances immunity against a particular disease. Typically, the 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. Upon introduction into a host, the vaccine can elicit an immune response, including, but not limited to, production of antibodies and / or cytokines, and / or activation of CD8+ T cells, antigen presenting cells, CD4+ T cells, and / or dendritic cells, and / or other cellular responses. In certain embodiments, the method comprises administering a vaccine comprising the naked nucleic acid molecules described herein. In some embodiments, the vaccine does not comprise nanoparticles. In some embodiments, the vaccine does not comprise cationic lipids. In some embodiments, the vaccine does not comprise PEG lipids. In some embodiments, the vaccine does not comprise phospholipids. In some embodiments, the vaccine does not comprise lipids. In some embodiments, the vaccine comprises an adjuvant. In some embodiments, the vaccine does not comprise 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 comprises a nucleic acid molecule and a buffer. In further embodiments, the buffer may be saline. The vaccine may be made according to the methods disclosed, for example, in WO2022112498, WO2022049093, or U.S. Pat. No. 10,913,964, the disclosures of which are incorporated herein by reference.

[0020] In some embodiments, the methods described herein do not include nanoparticles. In some embodiments, the methods do not include cationic lipids. In some embodiments, the methods do not include lipids. In some embodiments, the methods do not include adjuvants. In some embodiments, the methods do not include DNA encoding immunostimulatory genes. In some embodiments, the methods do not include liposomes. In some embodiments, the methods do not include viruses. In some embodiments, the naked nucleic acid molecules are injected with only a buffer solution.

[0021] As described herein, the instability of naked nucleic acid molecules is a challenge in expressing the nucleic acid molecules in a subject upon administration. Naked nucleic acid molecules may be taken up by endocytosis, for example, naked mRNA, which does not have the endosomal escape function of lipid nanoparticles (LNPs), may not be able to escape from endosomes, resulting in low gene expression. The method of injecting naked nucleic acid molecules described herein can increase DNA or RNA expression upon injection into a subject. Naked nucleic acid molecules can be delivered directly to the cytoplasm of cells, resulting in high gene expression. In some embodiments, the injections described herein exhibit 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. "First stage of two-stage injection profile" refers to the first stage measured, and "second stage of two-stage injection profile" refers to the second stage measured immediately after the first stage.

[0022] 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.

[0023] 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 naked nucleic acid molecule or 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. In addition, 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 a 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.

[0024] 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.

[0025] 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 dimensions, size, shape, and especially the surface profile of the gas generating agent can be adjusted to change the combustion completion time of the gas generating agent, and thereby the pressure transition applied to the administered liquid can be adjusted to achieve the desired injection pressure transition of the administered liquid.

[0026] The two-stage injection profile described herein is not limited to the injection pressure profile generated by ignition. The two-stage injection profile described herein can be achieved by other methods, for example, by controlling the gas volume and / or velocity applied to the naked nucleic acid molecule or vaccine thereof.

[0027] 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.

[0028] 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. The term "from injection" herein can mean starting from the time pressure begins to be applied to the naked nucleic acid molecule or vaccine thereof and / or from the time an increase in pressure on the naked nucleic acid molecule or vaccine thereof 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.

[0029] 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.

[0030] 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 naked nucleic acid molecule or its 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 this embodiment, one cycle of the rise and fall of the injection pressure in the pressure vibration is treated as one pressure vibration element.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 lower height peak, 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 naked nucleic acid molecule or its 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, about 1 MPa, about 2 MPa, about 3 MPa, about 4 MPa, 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 about 19 MPa, less than about 18 MPa, less than about 17 MPa, less than about 16 MPa, 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, or less than about 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.

[0036] 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.

[0037] 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.

[0038] In certain embodiments, the pressure fluctuation at a given time Δt may be due to the combustion of the ignition material of the igniter described herein. Also, around the time when the given 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 naked nucleic acid molecule or its vaccine. As a result, in the example shown in FIG. 1A, after the given time Δt has elapsed, the injection pressure rises again, and a peak value Py 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 rate of the gas generating agent may be slower than the burning rate of the ignition material, the rate at which the injection pressure increases 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.

[0039] In some embodiments, the two-stage injection profile may include a second stage having only one peak.

[0040] 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 naked nucleic acid molecule or its 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.

[0041] 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.

[0042] 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.

[0043] In some embodiments, the injection is a transdermal injection. In some embodiments, the injection does not include a transdermal injection.

[0044] 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 naked nucleic acid molecule or a vaccine thereof 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.

[0045] In some embodiments, the naked nucleic acid molecule is injected using a needleless syringe. In some embodiments, the naked nucleic acid molecule is injected using a syringe with an igniter. In some embodiments, the naked nucleic acid molecule is injected using a syringe without a spring. 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 that exhibits pressure characteristics in which the combustion product does not contain gas components when the temperature is then 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, thereby reducing the generated pressure, and a nozzle unit having an outlet through which the naked nucleic acid molecule or its vaccine pressurized by the combustion of the igniter flows and the naked nucleic acid molecule or its vaccine is discharged to the injection target area. In a further embodiment, the temperature of the combustion product produced during pressurization changes to near room temperature within 20 msec after the pressure applied to the naked nucleic acid molecule or its vaccine by the combustion of the ignition agent reaches a first peak ejection force in the pressurization process for ejecting the naked nucleic acid molecule or its 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 ignition agent reaches a first peak ejection force. In a specific embodiment, the syringe may be a syringe that injects the naked nucleic acid molecule or its vaccine into the injection target from the syringe body without injecting the naked nucleic acid molecule or its vaccine into the injection target through a predetermined structure with the predetermined structure inserted into the injection target. In a further embodiment, the syringe includes a cartridge and a nozzle portion including an injection port through which the biomolecule-containing solution flows and is injected into the injection target, and the solution is pressurized by the combustion of the ignition agent in the 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 between 75 m / s and 150 m / s, and the injection speed of the biomolecule-containing solution of between 75 m / s and 150 m / s is sustained for 0.11 ms or more.

[0046] 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.

[0047] 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.

[0048] In one aspect, the disclosure also provides a method of expressing a gene in a subject, comprising administering to the subject a naked nucleic acid molecule comprising the gene according to the methods described herein. In some embodiments, the method of expressing the gene further comprises detecting expression of the gene in the subject within 6 hours, 5 hours, 4 hours, or 3 hours of injection.

[0049] In one aspect, the disclosure further provides a method of treating, ameliorating or preventing a disease in a subject in need thereof, comprising expressing a gene in the subject according to the methods described herein, wherein the naked nucleic acid molecule induces an antigen-specific immune response against the disease.

[0050] 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 a further embodiment, the naked nucleic acid molecule described herein comprises a mutation. In certain embodiments, the naked nucleic acid molecule described herein expresses an antigen. In the context of the present invention, an "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 a 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 molecule described herein expresses an antigen selected from the group consisting of a pathogenic antigen, a tumor antigen, an allergenic antigen and an autoimmune antigen. The antigen may be derived from a pathogen associated with an infectious disease. The antigen may be selected from the group consisting of bacterial, viral, fungal and protozoan pathogens.

[0051] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is a tumor.

[0052] In some embodiments, the subject has a tumor. In some embodiments, the naked nucleic acid molecule induces an antigen-specific immune response in the tumor. In further embodiments, the naked nucleic acid molecule comprises a tumor-specific mutation. In certain embodiments, the antigen naked nucleic acid molecule may be a neoantigen nucleic acid molecule. In some embodiments, the naked nucleic acid molecule is a neoantigen mRNA specific to the tumor. Mutations may occur due to genetic instability in tumor cells, and 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 an immune response, making them promising new targets for tumor immunotherapy. Developments in bioinformatics technology have accelerated the identification of neoantigens, and various neoantigens have been identified.Castle, JCet 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,MMet al.Checkpoint blockade cancer immunotherapy targets tumour-specific mutants 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,PAet 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 naked nucleic acid molecule may be neoantigen mRNA.

[0053] In certain embodiments, the antigen naked nucleic acid molecule may be a neoantigen naked nucleic acid molecule, and the cartridge may further contain an additional vaccine comprising patient-derived dendritic cells (DCs) or synthetic long peptides (SLPs). In some embodiments, the antigen naked nucleic acid molecule may be a 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 infused 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, designed to relieve T cells from immune suppression mediated by the CTLA-4 and PD-1 pathways, can promote a strong and durable T cell response to eliminate tumors and regress cancer.

[0054] In some embodiments, the disease or disorder is a viral infection. In some embodiments, the naked nucleic acid molecule described herein is an mRNA encoding a viral protein. In some embodiments, the viral infection comprises a coronavirus infection. In some embodiments, the naked nucleic acid molecule is an mRNA encoding a coronavirus spike protein.

[0055] In certain embodiments, the subject is in need of a vaccine against an infectious disease, comprising the naked nucleic acid molecule. 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).

[0056] In one aspect, the disclosure relates to the use of a syringe to administer a naked nucleic acid molecule to a subject according to the methods described herein. In another aspect, the disclosure relates to the use of a syringe to express a gene in a subject according to the methods described herein. In another aspect, the disclosure relates to the use of a syringe to treat, ameliorate or prevent cancer in a subject in need thereof according to the methods described herein. EXAMPLES

[0057] 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.

[0058] Equipment used Refrigerated centrifuge (Tomy MDX-300) Autoclave (Tomy, LSX-700) 8mm biopsy pouch (Kai Industries, 8mm biopsy pouch) Luminometer (Kikkoman, C-100N)

[0059] First, whether gene expression of the administered mRNA is enhanced by administering the mRNA to a living body using the device of the present invention was evaluated using mRNA encoding GFP (naked mRNA_GFP). Male BALB / c mice aged 10 weeks were used for administration, and euthanized 6 hours after administration to collect data. 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 for administration. The dosage 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.

[0060] 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.

[0061] 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.

[0062] (2) The device enhances gene expression of the mRNA encoding Luc. 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.

[0063] 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.

[0064] 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.

[0065] (3) Alternative Syringe Injection Pressure Profiles - Example 1 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.

[0066] 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.

[0067] 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.

[0068] (3) Alternative Syringe Injection Pressure Profiles - Example 2 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.

[0069] Below are some exemplary embodiments of the present disclosure.

[0070] Embodiment 1 A method of administering a naked nucleic acid molecule to a subject, comprising injecting the naked nucleic acid molecule into a subject, wherein the injection exhibits a two-stage injection profile comprising a first stage and a second stage, the second stage being subsequent to the first stage, the two-stage injection profile having (i) at least two peaks within 15 msec of injection, (ii) a first peak of 2 MPa or greater, or (iii) the highest peak of the second stage of the two-stage injection within 30 msec of injection.

[0071] Embodiment 2. The method of embodiment 1, wherein the two-stage injection profile has at least two peaks within 15 msec of injection.

[0072] Embodiment 3. The method of embodiment 1 or 2, wherein the two-stage injection profile has at least two peaks within 1.5 msec of injection.

[0073] Embodiment 4. The method of any one of the preceding embodiments, wherein the two-stage injection profile has a first peak within 5 msec.

[0074]

[0023] Embodiment 5. The method of any one of the preceding embodiments, wherein the first stage includes a plurality of vibration elements, each having a vibration peak.

[0075]

[0023] Embodiment 6. The method of embodiment 5, wherein the at least two peaks are vibration peaks of a vibration element.

[0076]

[0023] Embodiment 7. The method of embodiment 5 or 6, wherein the overall amplitude of the vibration element decreases over time.

[0077] Embodiment 8. The method of any one of the preceding embodiments, wherein the first peak is 2 MPa or more.

[0078] Embodiment 9. The method of any one of the preceding embodiments, wherein the first peak is 15 MPa or greater.

[0079] Embodiment 10. The method of any one of the preceding embodiments, wherein the highest peak of the second stage of the two-stage injection profile is within 30 msec of injection.

[0080] Embodiment 11. The method of any one of the preceding embodiments, wherein the highest peak of the second stage of the two-stage injection profile is within 15 msec of injection.

[0081] Embodiment 12. The method of any one of the preceding embodiments, wherein the two-stage injection profile includes a second stage having only one peak.

[0082] Embodiment 13. The method of any one of the preceding embodiments, wherein the highest peak of the second stage of the two-stage profile is 0.1 MPa or more.

[0083] Embodiment 14. The method of any one of the preceding embodiments, wherein the highest peak of the second stage of the two-stage profile is 10 MPa or more.

[0084] Embodiment 15. The method of any one of the preceding embodiments, 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.

[0085] Embodiment 16. The method of any one of the preceding embodiments, wherein the highest peak of the second stage of the two-stage profile is higher than the highest peak of the first stage of the two-stage profile.

[0086] Embodiment 17. The method of any one of the preceding embodiments, wherein the injection is a transdermal injection.

[0087] Embodiment 18. The method of any one of embodiments 1 to 16, wherein the injection does not include a transdermal injection.

[0088] Embodiment 19. The method of any one of embodiments 1-18, wherein the injection is an intramuscular injection, a subcutaneous injection, or an intradermal injection.

[0089] Embodiment 20. The method of any one of embodiments 1-18, wherein the injection is an intralesional injection.

[0090] Embodiment 21. The method of any one of embodiments 1 to 18, wherein the injection is an intratumoral injection.

[0091] Embodiment 22. The method of any one of embodiments 1 to 18, wherein the injection is an intranodal or intralymphatic injection.

[0092] Embodiment 23. The method of any one of embodiments 1 to 21, wherein the injection does not comprise an intranodal injection.

[0093]

[0046] Embodiment 24. The method of any one of the preceding embodiments, wherein the method does not comprise nanoparticles.

[0094]

[0081] Embodiment 25. The method of any one of the preceding embodiments, wherein the method does not comprise a cationic lipid.

[0095]

[0046] Embodiment 26. The method of any one of the preceding embodiments, wherein the method does not include lipid.

[0096] Embodiment 27. The method of any one of the preceding embodiments, wherein the method does not comprise an adjuvant.

[0097]

[0046] Embodiment 28. The method of any one of the preceding embodiments, wherein the method does not include DNA encoding an immunostimulatory gene.

[0098]

[0041] Embodiment 29. The method of any one of the preceding embodiments, wherein the method does not comprise liposomes.

[0099]

[0039] Embodiment 30. The method of any one of the preceding embodiments, wherein the method does not comprise a virus.

[0100]

[0036] Embodiment 31. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is injected with only a buffer solution.

[0101]

[0046] Embodiment 32. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule induces an antigen-specific immune response in the tumor.

[0102]

[0039] Embodiment 33. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is mRNA.

[0103]

[0046] Embodiment 34. The method of embodiment 33, wherein the amount of mRNA injected into the subject is 0.2 μg or more.

[0104]

[0036] Embodiment 35. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is a tumor-specific neoantigen mRNA.

[0105] Embodiment 36. The method of any one of embodiments 1 to 34, wherein the naked nucleic acid molecule is an mRNA encoding a viral protein.

[0106]

[0038] Embodiment 37. The method of embodiment 36, wherein the naked nucleic acid molecule is an mRNA encoding the coronavirus spike protein.

[0107]

[0046] Embodiment 38. The method of any one of embodiments 1 to 32, wherein the naked nucleic acid molecule is DNA.

[0108]

[0041] Embodiment 39. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is injected using a needleless injector.

[0109]

[0046] Embodiment 40. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is injected using a syringe equipped with an igniter.

[0110]

[0046] Embodiment 41. The method of any one of the preceding embodiments, wherein the naked nucleic acid molecule is injected using a syringe without a spring.

[0111] Embodiment 42. A method of expressing a gene in a subject, comprising administering to the subject a naked nucleic acid molecule comprising the gene according to the method of any one of the preceding embodiments.

[0112]

[0081] Embodiment 43. The method of embodiment 42, further comprising detecting expression of the gene in the subject within 6 hours of injection.

[0113] Embodiment 44. A method for treating, ameliorating or preventing a disease or disorder in a subject in need thereof, comprising expressing a gene in the subject according to the method of embodiment 42 or 43, wherein the naked nucleic acid molecule induces an antigen-specific immune response against the disease or disorder.

[0114] Embodiment 45. The method of embodiment 44, wherein the disease or disorder is cancer.

[0115]

[0081] Embodiment 46. The method of embodiment 44, wherein the disease or disorder is a tumor.

[0116]

[0081] Embodiment 47. The method of embodiment 44, wherein the disease or disorder is a viral infection.

[0117]

[0081] Embodiment 48. The method of embodiment 47, wherein the viral infection comprises a coronavirus infection.

[0118] Embodiment 49. Use of a syringe to administer a naked nucleic acid molecule to a subject according to the method of any one of embodiments 1 to 41.

[0119] Embodiment 50. Use of a syringe for expressing a gene in a subject according to the method of embodiment 42 or 43.

[0120] Embodiment 51. Use of a syringe according to the method according to any one of embodiments 44 to 48 for treating, ameliorating or preventing cancer in a subject in need thereof.

Claims

A method of ejecting naked nucleic acid molecules using a needleless injector, comprising: wherein the ejection exhibits a two-stage profile including a first stage and a second stage, the second stage being after the first stage, the two-stage profile having (i) at least two peaks within 15 msec from the ejection, (ii) a first peak of 2 MPa or more, or (iii) the highest peak of the second stage of the two-stage profile within 30 msec from the ejection. **Claim 2** The method according to claim 1, wherein the two-stage profile has at least two peaks within 15 msec from the injection. **Claim 3** The method according to claim 1, wherein the two-stage profile has at least two peaks within 1.5 msec from the injection. **Claim 4** The method according to claim 1, wherein the two-stage profile has the first peak within 5 msec. **Claim 5** The method according to claim 1, wherein the first stage includes a plurality of vibrating elements each having a vibration peak. **Claim 6** The method according to claim 5, wherein the total amplitude of the vibrating elements decreases over time. **Claim 7** The method according to claim 1, wherein the first peak is 2 MPa or more. **Claim 8** The method according to claim 1, wherein the highest peak of the second stage of the two-stage profile is 0.1 MPa or more. **Claim 9** The method according to claim 1, 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. **Claim 10** The method according to claim 1, wherein the highest peak of the second stage of the two-stage profile is higher than the highest peak of the first stage of the two-stage profile. **Claim 11** The method according to claim 1, wherein the naked nucleic acid molecules are injected with only buffer. **Claim 12** The method according to claim 1, wherein the naked nucleic acid molecules are mRNA. **Claim 13** The method according to claim 12, wherein the mRNA is injected in an amount of 0.2 μg or more.

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