Nucleic acid composition for coding collagen

Abstract

The nucleic acid composition containing the coded collagen is characterized in that the nucleic acid composition comprises mRNA (messenger ribonucleic acid) of the coded collagen.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to a nucleic acid composition encoding collagen. Background Art

[0002] Collagen is the main protein involved in human wound repair and regulates the formation of various tissues, such as tendons, ligaments, skin, cornea, etc. Collagen peptides are a mixture of small molecule peptides with an average molecular weight below 10,000a formed by the hydrolysis of macromolecular collagen, also known as hydrolyzed collagen or collagen hydrolysate. The raw materials for preparing collagen peptides are mainly the skin and bones of terrestrial livestock and poultry (pigs, cattle, chickens, etc.) and the skin, scales and bones of aquatic animals (fish, jellyfish, etc.).

[0003] Currently, the main methods for producing collagen include traditional extraction, chemical synthesis and genetic engineering. All three methods have defects such as complicated processes, long preparation processes, low purity and poor drug safety. Summary of the invention

[0004] In one aspect, the present disclosure provides a nucleic acid composition encoding collagen, wherein the nucleic acid composition comprises mRNA encoding collagen.

[0005] In another aspect, the present disclosure provides use of the above-mentioned nucleic acid composition encoding collagen in the preparation of a drug for promoting wound healing.

[0006] In another aspect, the present disclosure provides use of the above-mentioned nucleic acid composition encoding collagen in the preparation of a drug for promoting keratinocyte migration.

[0007] In another aspect, the present disclosure provides an isolated nucleic acid encoding collagen, wherein the isolated nucleic acid is at least one of a linear DNA molecule, a circular DNA molecule, an mRNA, or a circular RNA.

[0008] In some embodiments, the collagen is selected from at least one of collagen COL1A2 and collagen COL1A1.

[0009] In some embodiments, the collagen is collagen COL1A2, and the amino acid sequence of the collagen COL1A2 is shown in Seq ID NO.7.

[0010] In some embodiments, the nucleic acid composition comprises mRNA encoding collagen COL1A2, and the nucleotide sequence of collagen COL1A2 is shown in Seq ID NO.8.

[0011] In some embodiments, the collagen is collagen COL1A1, and the amino acid sequence of the collagen COL1A1 is shown in Seq ID NO.9.

[0012] In some embodiments, the nucleic acid composition comprises mRNA encoding collagen COL1A1, and the nucleotide sequence of collagen COL1A2 is shown in Seq ID NO.10.

[0013] In some embodiments, the mRNA includes a 5'UTR, an ORF region, and a 3'UTR.

[0014] In some embodiments, the nucleotide sequence of the 5'UTR is shown as SEQ ID NOs. 1-3; and / or, the nucleotide sequence of the 3'UTR is shown as SEQ ID NOs. 4-6.

[0015] In some embodiments, the mRNA further contains a 3'-end poly (A), and the length of the poly (A) is 50-200, preferably 80-200.

[0016] In some embodiments, one or more uridines in the mRNA molecule are replaced with modified nucleosides.

[0017] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U) (m5U).

[0018] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0019] In some embodiments, the isolated nucleic acid is DNA, and the DNA is obtained by reverse transcription of the aforementioned mRNA, or the DNA is a sequence that can transcribe the aforementioned mRNA.

[0020] On the other hand, the present invention relates to a biological material, which comprises any one of an expression cassette, a vector, an engineered bacterium or a cell line, and the biological material contains or expresses the aforementioned mRNA or the aforementioned isolated DNA.

[0021] The advantages of the present invention are: (1) administration by mRNA encoding collagen avoids the shortcomings of collagen protein production such as complicated process, long preparation process and low purity, thereby improving production efficiency; (2) A new mechanism of action was discovered: low-concentration administration of mRNA encoding collagen can promote keratinocyte migration and promote skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The figure shows the Western Blot detection result of the nucleic acid composition containing mRNA encoding collagen.

[0024] Figure 2 The figure shows the experimental results of the effect of a nucleic acid composition comprising mRNA encoding collagen on the cell migration of HaCaT cells.

[0025] Figure 3 This figure shows the experimental results of the effect of a nucleic acid composition containing mRNA encoding collagen on wound healing in mice.

[0026] Figure 4 This is a graph showing the results of detecting the viability of HaCaT cells cck-8 using a nucleic acid composition containing mRNA encoding collagen. DETAILED DESCRIPTION

[0027] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0028] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0029] The present disclosure discloses a "nucleic acid composition", which comprises a delivery vector and an isolated nucleic acid encapsulated by the delivery vector. The "delivery vector" refers to a vector that provides the isolated nucleic acid to a subject. In some embodiments, the delivery vector is selected from a lipid nanoparticle (LNP), a lipid polymer complex (LPP), a liposome, a lipid vesicle, a plasmid vector or a viral vector.

[0030] The present disclosure "isolated nucleic acid" can be understood as a non-natural nucleic acid molecule. Such a nucleic acid molecule can be non-natural due to its individual sequence (which does not exist naturally) and / or due to other modifications (e.g., structural modifications of nucleotides that do not exist naturally). An artificial nucleic acid molecule can be a DNA molecule, an RNA molecule, or a hybrid molecule comprising a DNA and RNA portion. Typically, an artificial nucleic acid molecule can be designed and / or produced by genetic engineering methods. In this case, the artificial nucleic acid molecule comprises an artificial sequence that is generally not naturally present, and the artificial sequence is different from the wild-type sequence by at least one nucleotide. The term "wild-type sequence" can be understood as a naturally occurring sequence.

[0031] As used herein, "modified" refers to non-natural. For example, RNA can be modified RNA. That is, RNA can include one or more non-naturally occurring nucleobases, nucleosides, nucleotides or linking groups. "Modified" groups can also be referred to as "altered" groups in this article. Groups can be modified or altered chemically, structurally or functionally. For example, a modified nucleobase can include one or more non-naturally occurring substitutions.

[0032] In some embodiments, the isolated nucleic acid can optimize the mRNA sequence by sequence optimization means to improve the properties related to the expression efficacy after in vivo administration: for example, improving mRNA stability, increasing translation efficacy in target tissues, reducing the number of truncated proteins expressed, improving the folding of expressed proteins or preventing their misfolding, reducing the toxicity of expression products, reducing cell death caused by expression products, increasing and / or reducing protein aggregation, and obtaining mRNA with improved properties. The purpose of sequence optimization also includes: optimizing the formulation and delivery characteristics of nucleic acid-based therapeutic agents while maintaining structural and functional integrity; overcoming the threshold of expression; increasing expression rate; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization methods include: (1) codon optimization based on codon frequencies in specific organs and / or host organisms to ensure proper folding and appropriate expression; (2) adjustment of G / C content to increase mRNA stability or reduce secondary structure; (3) minimization of tandemly repeated codons or base runs that may impair gene construction or expression; (4) customization of transcription and translation control regions; and (5) reduction or elimination of problematic secondary structures within polynucleotides.

[0033] The “collagen” in the present disclosure may be recombinant collagen or wild-type collagen.

[0034] The "recombinant collagen" disclosed in the present invention is collagen that has been artificially mutated and modified. The mutation and modification (including but not limited to amino acid residue substitution, amino acid residue insertion and / or addition, amino acid residue deletion and covalent modification of the amino acid sequence, etc.) is the amino acid sequence of collagen that meets the requirements obtained by mutation and modification of wild-type collagen.

[0035] The term "wild-type collagen" is to be understood as the naturally occurring collagen amino acid sequence.

[0036] In some embodiments, the collagen disclosed herein is wild-type collagen. Furthermore, the collagen is selected from collagen COL1A2 or COL1A1.

[0037] In some embodiments, the collagen is collagen COL1A2, and the amino acid sequence of the collagen COL1A2 is shown in Seq ID NO.7.

[0038] In some embodiments, the collagen is collagen COL1A2, and the nucleotide sequence of the mRNA ORF encoding the collagen COL1A2 is shown in Seq ID NO.8.

[0039] In some embodiments, the collagen is collagen COL1A1, and the amino acid sequence of the collagen COL1A1 is shown in Seq ID NO.9.

[0040] In some embodiments, the collagen is collagen COL1A1, and the nucleotide sequence of the mRNA ORF encoding the collagen COL1A1 is shown in Seq ID NO.10.

[0041] In some embodiments, the collagen disclosed herein is recombinant collagen. Furthermore, the collagen is selected from collagen COL1A2 or COL1A1.

[0042] In some embodiments, the collagen is collagen COL1A2, and the amino acid sequence of the collagen COL1A2 is selected from the wild-type collagen COL1A2 truncation shown in Seq ID NO.7.

[0043] In some embodiments, the collagen is collagen COL1A2, and the mRNA ORF nucleotide sequence encoding the collagen COL1A2 is selected from any section of nucleotides shown in Seq ID NO.8.

[0044] In some embodiments, the collagen is collagen COL1A1, and the amino acid sequence of the collagen COL1A1 is selected from the wild-type collagen COL1A1 truncated form shown in Seq ID NO.9.

[0045] In some embodiments, the collagen is collagen COL1A1, and the nucleotide sequence of the mRNA ORF encoding the collagen COL1A1 is selected from any section of nucleotides shown in Seq ID NO.10.

[0046] Example 1 Preparation of mRNA 1. Synthesize RNA sequences using linearized plasmid DNA as template by in vitro transcription (IVT): Add the following into the reactor: 1.0 μg template DNA, 2.0 μL 10× transcription buffer, 0.2 μL NTP (25 mM each), 20 U RNase inhibitor, 3000 U T7 RNA polymerase, 20.0 μL dH2O; incubate at 37°C for 3 to 5 hours. The transcription buffer consists of 400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, and 10 mM spermidine.

[0047] 2. Enzymatic end-capping: The RNA obtained by in vitro transcription in the above step was purified, and 60 μg of the obtained RNA was mixed with 72 μL of dH2O, incubated at 65°C for 5 minutes to denature the RNA, and the RNA denatured mixture was transferred to ice.

[0048] The RNA denaturation mixture, 10.0 μL 10× blocking buffer, 5.0 μL 20 mM GTP, 2.5 μL 20 mM S-adenosylmethionine, 100 U RNase inhibitor, 400 U 2'-O-methyltransferase, 40 U vaccinia blocking enzyme, and 28 μL dH2O were mixed and incubated at 37°C for 30 min.

[0049] The blocking buffer composition is 0.5M Tris-HCl (pH 8.0), 60mM KCl and 12.5mM MgCl2.

[0050] 3. Polyadenylation tailing reaction: If the template DNA does not have a poly-T tail or in order to obtain a longer poly-A tail, a poly-A tailing reaction can be performed after enzymatic end-capping. Mix 100 μL RNA obtained after enzymatic end-capping, 20U RNase inhibitor, 12.0 μL 10× tailing buffer, 6.0 μL 20mM ATP, 20U poly-A polymerase and 123 μL dH2O, and incubate at 37°C for 30 minutes.

[0051] The tailing buffer consisted of 0.5M Tris-HCl (pH 8.0), 2.5M NaCl and 100mM MgCl2.

[0052] Example 2 Preparation of nucleic acid compositions This embodiment provides a nucleic acid composition, comprising mRNA encoding collagen, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' end to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0053] The lipid nanoparticles include Dlin-MC3-DMA 50%, DSPC 10%, cholesterol 38.5% and PEG-DMG 1.5% by molar percentage. The preparation method is as follows: (a) RNA was dissolved in pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0054] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0055] (c) The aqueous phase of step (a) and the organic phase of step (b) are mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, the mixed solution is immediately diluted 100 times with the mixed solution, and the ethanol component in the solution is removed by tangential flow filtration (TFF), and then concentrated until the concentration of mRNA in the system is not less than 100 μg / mL, to obtain a nucleic acid composition.

[0056] Example 3 Effects on cell migration of HaCaT cells Human immortalized epidermal cell line HaCaT cells were purchased from Henan Industrial Microbial Strain Engineering Technology Research Center; DMEM culture medium was purchased from Excell Bio.

[0057] (1) Take a 6-well plate with HaCaT cells attached to the wall, use a pipette tip to make a vertical scratch in each well, and gently rinse the scratched area with PBS; (2) Add 2% FBS DMEM medium and samples to each well, and no sample is added to the blank group; (3) Place the 6-well plate in a 37°C incubator and continue culturing. Take photos every 24 hours until the scratch on any well of the 6-well plate has healed.

[0058] Example 4

[0059] Wound healing experiment in mice The animal model used in this experiment was 6-8 week old KM mice (purchased from Bestone), with 6 mice in each group.

[0060] (1) After the mice were depilated, they were anesthetized using a small animal anesthesia machine (brand: Rayward, model: CL500IP) and each mouse was injected with 6.5% chloral hydrate; (2) Use scissors to create a wound model on the back of the mouse. The wound is to cut off the mouse skin and create a circular wound with a diameter of 8 mm2; (3) The mice were divided into a blank control group and an experimental group. The blank control group received no treatment; the experimental group mice were injected with the sample solution at the center of the circular wound. (4) Take photos every day to record the wound healing status.

[0061] Example 5

[0062] HaCaT cell cck-8 cell viability detection Human immortalized epidermal cell line HaCaT cells were purchased from Henan Industrial Microbial Strain Engineering Technology Research Center; CCK8 detection kit was purchased from Biotime.

[0063] (1) Take 2 mL EP tubes and add 2 mL of HaCaT cell suspension to each tube. The cell concentration in the suspension is 2 × 104. Add the respective sample solutions to different EP tubes. (2) Take a sterile 96-well plate, add 100 μL of the cell suspension prepared in step (1) to each well in the middle, add 100 μL of PBS around the well to seal the plate, and place it in a 37°C incubator for 24 hours.

[0064] (3) Discard the culture medium; add PBS buffer to wash, and then discard the liquid; (4) Add 100 μL of CCK-8 working solution and sample solution to the 96-well plate; (5) After 2 hours, the samples were detected at 450 nm using a continuous wavelength multifunctional microplate reader.

[0065] Example 6

[0066] The mRNA encoding collagen was prepared according to Example 1; the nucleic acid composition encoding collagen was prepared according to the method of Example 2. The empty LNP containing no nucleic acid was prepared according to the method of Example 2.

[0067] The amino acid sequence of the collagen is shown in Seq ID NO.7, and the nucleotide sequence of the collagen is shown in Seq ID NO.8.

[0068] This experimental example detects the expression of collagen at the cell level in vitro. The specific method is as follows: (1) Transfection Add 250 μl of opti-MEM to each tube, 10 μL of lip 2000 to tube A, and 5 μg of COL1A2 and the same volume of opti-MEM and LIP 2000 to tube B, respectively. Let stand for 5 minutes, mix, and let stand for another 30 minutes. Replace the culture medium in the 6-well plate with new DMEM complete culture medium. Add the prepared liquid to the well and shake well. Add 5 μg of COL1A2 and the same volume of LNP directly to the cell supernatant, shake well, and incubate in a cell culture incubator.

[0069] (2) Detection of intracellular collagen protein expression using Western Blot Remove the cell supernatant, wash the cells with PBS, add 1 mL PBS to the well, collect the cells in a centrifuge tube, centrifuge and remove the supernatant. Add cell lysis solution (containing PMSF) to the centrifuge tube, mix and vortex evenly. Use the BCA kit to measure the protein concentration of the cells. Then use precast gel for electrophoresis, add recombinant Anti-Collagen antibody and GoatAnti-Rabbit IgG H&L (HRP), and perform chemiluminescence on the PVDF membrane for color development.

[0070] The results are as follows Figure 1 As shown, 1 and 6 are PBS buffer blank control groups; 2 and 7 are empty LNP groups; 3 and 8 are mRNA groups encoding collagen prepared according to Example 1; 4 and 9 are nucleic acid composition groups encoding collagen prepared according to Example 2; and 5 is a marker.

[0071] from Figure 1 It can be seen that in the in vitro HaCaT cells, the collagen expression levels of the mRNA group encoding collagen prepared according to Example 1 and the nucleic acid composition group encoding collagen prepared according to Example 2 were significantly higher than those of the other experimental groups.

[0072] Example 7 The mRNA encoding collagen was prepared according to Example 1; the nucleic acid composition encoding collagen was prepared according to the method of Example 2, wherein the amino acid sequence of the collagen is shown in Seq ID NO.7, and the nucleotide sequence of the collagen is shown in Seq ID NO.8.

[0073] Empty LNPs containing no nucleic acid were prepared according to the method of Example 2.

[0074] The experiment on the influence on the cell migration of HaCaT cells was carried out according to the method of Example 3, and the cells were divided into groups 1-5; wherein the experiment on the influence on the cell migration of HaCaT cells was carried out according to the method of Example 3, and the cells were divided into groups 1-5; wherein group 1 added 6.74 μL 1x PBS buffer to the corresponding wells of the 6-well plate, group 2 added 3.57 μL Lip 2000 PBS solution to the corresponding wells of the 6-well plate, group 3 added 3.57 μL of a nucleic acid concentration of 1.4 μg / μL mRNA-lip 2000 mixed solution encoding collagen was prepared in this embodiment to the corresponding wells of the 6-well plate, group 4 added 6.74 μL of an empty LNP PBS solution to the corresponding wells of the 6-well plate, and group 5 added 6.74 μL of a nucleic acid concentration of 371 μg / mL PBS solution encoding a nucleic acid composition encoding collagen was prepared in this embodiment to the corresponding wells of the 6-well plate.

[0075] The experimental results are as follows Figure 2 shown.

[0076] The results are as follows Figure 2 1 is a PBS buffer blank control group; 2 is an empty Lip 2000 group; 3 is an mRNA group encoding collagen prepared according to Example 1; 4 is an empty LNP group; and 5 is a nucleic acid composition group encoding collagen prepared according to Example 2.

[0077] from Figure 2 It can be seen that the scratch healing speed of the nucleic acid composition group encoding collagen prepared according to Example 2 is the fastest, and the scratch healing speed of the mRNA group encoding collagen prepared according to Example 1 is the second fastest.

[0078] Example 8

[0079] The mRNA encoding collagen was prepared according to Example 1; the nucleic acid composition encoding collagen was prepared according to the method of Example 2, wherein the amino acid sequence of the collagen is shown in Seq ID NO.7, and the nucleotide sequence of the collagen is shown in Seq ID NO.8.

[0080] Empty LNPs containing no nucleic acid were prepared according to the method of Example 2.

[0081] The wound healing effect experiment on mice was carried out according to the method of Example 4, and the mice were divided into 1-4 groups; Group 1 was injected with 50 μL 1x PBS buffer, Group 2 was injected with 50 μL 1x PBS buffer containing LNP, Group 3 was injected with 50 μL 1x PBS buffer containing 2.5 μg of mRNA encoding collagen prepared according to Example 1, and Group 4 was injected with 50 μL 1x PBS buffer containing 2.5 μg of the nucleic acid composition encoding collagen prepared according to the method of Example 2.

[0082] The results of the experiment on the effect of wound healing on mice are shown in Figure 3 1 is a PBS buffer blank control group; 2 is an empty LNP group; 3 is a collagen encoding mRNA group prepared according to Example 1; and 4 is a collagen encoding nucleic acid composition group prepared according to Example 2.

[0083] It can be seen from the figure that the wounds of mice in the group of nucleic acid compositions encoding collagen prepared according to Example 2 healed fastest.

[0084] Example 9

[0085] A nucleic acid composition encoding collagen was prepared according to the method of Example 2, wherein the amino acid sequence of the collagen was shown in Seq ID NO.7, and the nucleotide sequence of the collagen was shown in Seq ID NO.8.

[0086] Empty LNPs containing no nucleic acid were prepared according to the method of Example 2.

[0087] The HaCaT cell cck-8 cell viability detection experiment was carried out according to the method of Example 5, and the cells were divided into a Control group, a LNP group, and a COL1A2 1.25 μg group.

[0088] The Control group was loaded with 100 μL 1x PBS buffer, the LNP group was loaded with 100 μL 100 μL / mL LNP PBS solution, and the COL1A2 1.25 μg group was loaded with 1.25 μg of a nucleic acid composition encoding collagen.

[0089] The experimental results are shown in Figure 4 As shown in the figure, it can be seen that the addition amount of COL1A2 1.250 μg group has little effect on the viability of HaCaT cell cck-8 cells.

Claims

1. A nucleic acid composition encoding collagen, characterized in that The nucleic acid composition comprises mRNA encoding collagen.

2. Use of the collagen encoding nucleic acid composition as claimed in claim 1 in the preparation of a drug for promoting wound healing.

3. Use of the nucleic acid composition encoding collagen as claimed in claim 1 in the preparation of a drug for promoting keratinocyte migration.

4. An isolated nucleic acid encoding collagen, characterized in that The isolated nucleic acid is at least one of a linear DNA molecule, a circular DNA molecule, mRNA or a circular RNA.

5. The nucleic acid composition according to claim 1 to 3, characterized in that: The collagen is selected from at least one of collagen COL1A2 and collagen COL1A1.

6. The nucleic acid composition according to claim 5, wherein The collagen is collagen COL1A2, and the amino acid sequence of the collagen COL1A2 is shown in Seq ID NO.7; Preferably, the nucleic acid composition comprises mRNA encoding collagen COL1A2, and the nucleotide sequence of collagen COL1A2 is shown in Seq ID NO.

8.

7. The nucleic acid composition according to claim 5, wherein The collagen is collagen COL1A1, and the amino acid sequence of the collagen COL1A1 is shown in Seq ID NO.

9.

8. Preferably, the nucleic acid composition comprises mRNA encoding collagen COL1A1, and the nucleotide sequence of collagen COL1A2 is shown in Seq ID NO.

10.

9. The nucleic acid composition of claim 1 or the isolated nucleic acid of claim 4, wherein: The mRNA includes a 5'UTR, an ORF region and a 3'UTR; Preferably, the nucleotide sequence of the 5'UTR is shown in SEQ ID NOs. 1 to 3; and / or, the nucleotide sequence of the 3'UTR is shown in SEQ ID NOs. 4 to 6; Preferably, the mRNA further contains a 3' end poly (A), the length of the poly (A) is 50-200, preferably 80-200; Preferably, one or more uridines in the mRNA molecule are replaced with modified nucleosides; Preferably, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U) (m5U); Preferably, the modified nucleoside is N1-methyl-pseudouridine (m1ψ); The isolated nucleic acid is DNA, which is obtained by reverse transcription of the aforementioned mRNA, or the DNA is a sequence that can transcribe the aforementioned mRNA.

10. A biological material, comprising any one of an expression cassette, a vector, an engineered bacterium or a cell line, wherein the biological material contains or expresses the aforementioned mRNA or the aforementioned isolated DNA.

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