Tiniplase mRNA (messenger Ribonucleic Acid) medicine and application thereof

Abstract

The invention discloses a teneplase mRNA (messenger Ribonucleic Acid) medicine and application thereof. The active ingredient of the medicine provided by the invention is mRNA (messenger ribonucleic acid) as shown in SEQ ID NO.5. The invention also protects a recombinant plasmid of the teneplase as shown in SEQ ID NO. 4. Specific DNA molecules are obtained through a series of design and sequence optimization, specific recombinant plasmids are synthesized through gene synthesis, the specific recombinant plasmids are subjected to in-vitro transcription, cell supernatant protein is obtained through transfection of mRNA of different amounts at different time points after transfection, and the cell supernatant protein is used for preparing the recombinant protein. According to the present invention, it is detected through ELISA that the tinipase mRNA can be effectively expressed in different cells of different species, such that the pioneering significance is provided for the development of the tinipase mRNA drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a tenecteplase mRNA drug and application thereof. BACKGROUND

[0002] Tenecteplase is a multi-site variant of tissue plasminogen activator (tPA). Tenecteplase is a thrombolytic drug and is the third generation of fibrinolytic agents. It is mainly used for thrombolytic therapy in clinical practice. At present, the administration mode of tenecteplase is mainly to inject tenecteplase protein,

[0003] Messenger RNA (mRNA) is transcribed from a DNA template strand, and its base sequence is the same as the gene coding strand and is complementary to the template strand. In eukaryotes, mRNA carrying genetic information obtained by gene transcription is composed of protein-coding exons and non-coding introns arranged at intervals. The mRNA directly transcribed from the gene needs to be correctly modified and spliced into mature mRNA before being transported to the cytoplasm for translation to produce proteins.

[0004] Compared with proteins, mRNA: 1) can be encapsulated and delivered by liposomes, which reduces its immunogenicity and can also extend its half-life; 2) in vitro transcription makes its yield higher and the production time shorter; 3) can be quickly modified to regulate half-life and immunogenicity characteristics, which is conducive to personalized treatment and emergency response to emerging pathogens. In view of the advantages of mRNA, the development of mRNA drugs has a very good development prospect. In addition, mRNA vaccine drugs for various viral (Ebola virus, Zika virus, influenza virus) infections and cancers are also attempted to be developed and induce safe and acceptable effective protective immune responses. However, the design of mRNA sequence is a complex process involving multiple factors, including mRNA stability, translation efficiency, and immunogenicity, etc., which limits its wide development and application. SUMMARY

[0005] The purpose of the present application is to provide a tenecteplase mRNA drug which has high expression in different cell strains and can achieve therapeutic effect with a small amount of mRNA, and is safer.

[0006] The purpose of the present application is achieved by the following technical solutions.

[0007] The present invention provides an mRNA molecule, characterized in that: the nucleotide sequence of the mRNA contains the sequence shown in SEQ ID NO. 6, or a derived sequence of SEQ ID NO. 6 by substitution, addition and / or deletion of one or more amino acids and having the same function as SEQ ID NO. 6, and the uracil in the mRNA molecule is all 1-N-Me-PseudoUTP.

[0008] In some embodiments, the mRNA molecule of the present invention has untranslated regions added to both the 5' and 3' ends of the sequence shown in SEQ ID NO. 6; preferably, the 5' untranslated region sequence is shown in SEQ ID NO. 7, and the 3' untranslated region sequence is shown in SEQ ID NO. 8.

[0009] In some embodiments, the mRNA molecule of the present invention has an mRNA cap structure added to the 5' end of the sequence shown in SEQ ID NO.6. The mRNA cap structure can be a commonly used mRNA cap structure in the art.

[0010] In some embodiments, the mRNA molecule of the present invention has a polyadenosine and / or nucleotide-like tail added to the 3' end of the sequence shown in SEQ ID NO.6. The polyadenosine or nucleotide-like tail can be conventional in the art, and more preferably, the degree of polymerization of the polyadenosine is 100 to 300.

[0011] In some embodiments, the nucleotide sequence of the mRNA of the present invention is the sequence of SEQ ID NO.5, or a derived sequence of SEQ ID NO.5 with one or more amino acids replaced, added and / or deleted and having the same function as SEQ ID NO.5, and the uracil in the mRNA molecule is 1-N-Me-PseudoUTP.

[0012] This invention also protects the gene encoding the mRNA molecule described herein.

[0013] In one specific instance, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0014] The present invention also provides recombinant plasmids containing the coding gene described herein. These recombinant plasmids can be formed by integrating the coding gene described herein into commonly used plasmids, such as the pUC57-kan plasmid, using conventional methods in the art. In a more specific example, the recombinant plasmid is shown in SEQ ID NO. 4.

[0015] The present invention also provides a pharmaceutical composition comprising the mRNA molecule, the DNA molecule or the recombinant plasmid described herein, and a pharmaceutically acceptable vector.

[0016] The present invention also provides the use of the mRNA molecule, the DNA molecule, the recombinant plasmid, or the pharmaceutical composition thereof in the preparation of tenecteplase mRNA drugs.

[0017] The present invention also provides the use of the mRNA molecule, the DNA molecule, the recombinant plasmid, or the pharmaceutical composition thereof in the preparation of thrombolytic drugs.

[0018] The present invention also provides a method for preparing the mRNA described in the present invention, wherein a recombinant plasmid containing the DNA molecule described in the present invention is taken, digested with restriction endonuclease Bsa I and the linearized plasmid is recovered, and then tenecteplase mRNA is obtained by in vitro transcription.

[0019] In one specific example, the method for preparing the tenecteplase mRNA includes the following steps in sequence:

[0020] (1) Take the recombinant plasmid containing the DNA molecule described in this invention, digest it with restriction endonuclease Bsa I, and recover the linearized plasmid;

[0021] (2) Transcription yields mRNA;

[0022] (3) After completing step (2), add DNase I to digest the DNA template in the transcription system;

[0023] (4) After completing step (3), the mRNA is purified using the reagent LiCl provided in the transcription kit to obtain the purified mRNA, which is tenecteplase mRNA.

[0024] In some embodiments, the reaction system (20 μl) for step (2) consists of: 1 μg of the linearized plasmid prepared in step (1), 2 μl of ATP, 2 μl of GTP, 2 μl of CTP, 2 μl of 1-N-Me-PseudoUTP, 4 μl of 5× reaction buffer, 1.5 μl of T7 transcriptase mixture, and the remainder being nuclease-free water. The T7 transcriptase mixture is a mixture of T7 transcriptase and RNsae inhibitor. The concentrations of ATP, GTP, CTP, and 1-N-Me-PseudoUTP in the reaction system are all 100 mM.

[0025] In some embodiments, the reaction conditions for step (2) are: 37°C, 2h.

[0026] In some embodiments, step (3) involves adding 2 μl of DNase I to the RNA solution after transcription and incubating at 37°C for 30 min.

[0027] The present invention also protects a tenecteplase drug comprising the mRNA, the DNA molecule, or the recombinant plasmid.

[0028] The inventors of this invention obtained a specific DNA molecule through a series of sequence designs and optimizations, and further constructed a specific recombinant plasmid. By introducing the specific recombinant plasmid into host cells and culturing them (in vitro transcription), tenecteplase protein secreted into the supernatant can be obtained. This invention has the following advantages over existing technologies:

[0029] (1) The tenecteplase mRNA described in this invention is highly expressed in different cell lines, especially in human cells, which can achieve therapeutic effects with a small amount of mRNA and has higher safety.

[0030] (2) The untranslated regions added to the N-terminus and C-terminus of the mRNA described in this invention can enhance the stability of the mRNA obtained by transcription, while also enabling higher expression of the mRNA in cells and reducing the immunogenicity of the mRNA. Attached Figure Description

[0031] Figure 1 The images show the electrophoresis diagrams of the linearized plasmid and the recombinant plasmid before enzyme digestion in Example 2.

[0032] Figure 2 This is an ELISA result of the cell culture supernatant of HEK293T cells transfected with mRNA 24 hours after Example 3.

[0033] Figure 3 The image shows the ELISA results of the cell culture supernatant of CHOK1Q cells and AC16 cells transfected with mRNA 72 hours after Example 4 and Example 5. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0036] Example 1: Construction of recombinant plasmids

[0037] The tenecteplase gene sequence was obtained through a query in NCBI GeneBank, as shown in SEQ ID NO.1 of the sequence listing. After optimization, the DNA molecule shown in SEQ ID NO.2 of the sequence listing was obtained, encoding the tenecteplase protein.

[0038] A T7 promoter sequence and a 5' untranslated region (5'UTR) were added to the N-terminus of the tenecteplase gene, and a 3' untranslated region (3'UTR) and a poly(A) tail with 128 A's were added to the C-terminus to obtain the DNA molecule shown in SEQ ID NO.3 of the sequence listing. In SEQ ID NO.3, nucleotides 1-21 form the T7 promoter, nucleotides 22-72 form the 5'UTR, nucleotides 73-1766 encode the tenecteplase protein, nucleotides 1767-1900 form the 3'UTR, and nucleotides 1901-2028 form the poly(A) tail.

[0039] The DNA molecule shown in SEQ ID NO.3 of the sequence listing was inserted into the pUC57-kan plasmid (purchased from GenScript Biotech Co., Ltd.) via homologous recombination to obtain the recombinant plasmid pUC57-kan-tPA mRNA, the sequence of which is shown in SEQ ID NO.4.

[0040] In an mRNA transcription system (where the UTPs required for RNA formation are replaced with 1-N-Me-PseudoUTPs), the DNA molecule shown in SEQ ID NO.3 is transcribed to the mRNA shown in SEQ ID NO.5, where U in SEQ ID NO.5 represents 1-N-Me-PseudoUTP. The mRNA shown in SEQ ID NO.5 is named tenecteplase mRNA. Translation of tenecteplase mRNA yields tenecteplase.

[0041] Example 2: Preparation and transfection of tenecteplase mRNA

[0042] 1. Recombinant plasmid pUC57-kan-tPA mRNA was digested with restriction endonuclease BsaI, and the linearized plasmid was recovered. Electrophoresis images of the linearized plasmid and the recombinant plasmid before digestion are shown below. Figure 1 M is the nucleic acid marker, A is the linearized plasmid, and B is the plasmid before enzyme digestion.

[0043] 2. Transcription to obtain mRNA

[0044] Reaction system (20 μl): 1 μg linearized plasmid prepared in step 1, 2 μl ATP, 2 μl GTP, 2 μl CTP, 2 μl 1-N-Me-PseudoUTP, 4 μl 5× reaction buffer, 0.8 μl Cap analog, 1.5 μl T7 transcriptase mixture, balance Nuclease-free water. The concentrations of ATP, CTP, GTP, and 1-N-Me-PseudoUTP in the reaction system were all 100 mM. 1-N-Me-PseudoUTP: Shanghai Zhaowei Biotechnology Co., Ltd., product catalog number R5-027. ATP, CTP, GTP, and T7 transcriptase mixture: Shanghai Zhaowei Biotechnology Co., Ltd., product catalog number ON-040. Cap analog: Shanghai Zhaowei Biotechnology Co., Ltd., product catalog number ON-134. Reaction conditions: Incubation at 37℃ for 2 hours.

[0045] 3. After completing step 2, add 2 μl of DNase I according to the transcription kit instructions and incubate at 37°C for 0.5 h.

[0046] 4. After completing step 3, according to the transcription kit instructions, add 30 μl LiCl and 30 μl Nuclease-free water, incubate at -20℃ for 30 min, centrifuge at 4℃ for 15 min, discard the supernatant, add 500 μl 70% ethanol, centrifuge at 4℃ for 10 min, discard the supernatant, and the precipitate is the tenecteplase mRNA shown in SEQ ID NO.5 (where U in SEQ ID NO.5 represents 1-N-Me-PseudoUTP).

[0047] 5. After completing step 4, dissolve the mRNA obtained in step 4 with an appropriate volume of RNase-free H2O according to the instructions of the transcription kit, and determine the RNA concentration using NanoDrop.

[0048] 6. According to RNAiMAX transfection reagent recommends the following mRNA transfection dosage, using Opti-MEM and... RNAiMAX transfection reagent was used to transfect different amounts of tenecteplase mRNA into cells that had been seeded in 96-well plates and cultured for 24 hours, using Opti-MEM and RNAiMAX transfection reagent mixture was used as a blank control. Opti-MEM: Gibco, catalog number 31985-062. RNAiMAX: Invitrogen, 13778-030.

[0049] 7. After a certain period of time following mRNA transfection, collect the cell supernatant and store it temporarily at -20℃.

[0050] Example 3: Expression of tenecteplase mRNA in human embryonic kidney cells (HEK293)

[0051] 1. Take a 96-well plate, seed HEK293 cells (20,000 cells / well), and culture in DMEM medium containing 10% serum and 1% penicillin antibiotics for 24 hours. DMEM medium: Sangon Biotech (Shanghai) Co., Ltd., product catalog number E600003-0500.

[0052] 2. After completing step 1,

[0053] Following steps 6 and 7 of Example 2, 8 pmol or 20 pmol mRNA was transfected into the wells of the experimental cells, respectively. The cell supernatant was collected 24 hours after mRNA transfection and stored temporarily at -20°C.

[0054] Example 4: Expression of tenecteplase mRNA in Chinese hamster ovary cells (CHO-K1)

[0055] 1. Inoculate 20,000 CHO-K1Q cells per well in a 96-well plate and culture for 24 hours in IMDM medium containing 10% serum and 1% penicillin antibiotics. IMDM medium: Gibco, catalog number 31980030.

[0056] 2. After completing step 1,

[0057] Following steps 6 and 7 of Example 2, 8 pmol or 20 pmol mRNA was transfected into the experimental cell wells, respectively. Cell supernatants were collected 24, 48, and 72 hours after mRNA transfection and stored temporarily at -20°C.

[0058] Example 5: Expression of tenecteplase mRNA in human cardiomyocytes (AC16)

[0059] 1. Take a 96-well plate, seed AC16 cells (20,000 cells / well), and culture in DMEM medium containing 10% serum and 1% penicillin antibiotics for 24 hours. DMEM medium: Sangon Biotech (Shanghai) Co., Ltd., product catalog number E600003-0500.

[0060] 2. After completing step 1,

[0061] Following steps 6 and 7 of Example 2, 8 pmol or 20 pmol mRNA was transfected into the experimental cell wells, respectively. Cell supernatants were collected 24, 48, and 72 hours after mRNA transfection and stored temporarily at -20°C.

[0062] Example 6: Detection of tenecteplase mRNA expression level in cells using ELISA technology.

[0063] 1. Based on the sample grouping quantity obtained in Examples 3, 4 and 5, determine the number of wells required for detection. Use 400 μl / well Wash Buffer to wash the 96-well plate coated with tenecteplase antibody carried in the kit. Each wash should keep the Wash Buffer in the well for 10-15 seconds, and wash twice.

[0064] 2. Dilute the tenecteplase protein standard in the kit with sample diluent in a concentration gradient to 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 pg / ml.

[0065] 3. Dilute the supernatant collected in Examples 3, 4, and 5 by 10, 50, 100, or 200 times respectively with sample diluent.

[0066] 4. Take 100 μl of each diluted protein standard solution and 100 μl of the diluted supernatant, and add them to the 96-well plate coated with tenecteplase antibody provided in the kit.

[0067] 5. Add 50 μl of HRP-Conjugate diluted with Assay Buffer at a ratio of 1:100 to the standard wells and the supernatant sample wells, respectively.

[0068] 6. Cover the plate with an adhesive film and incubate it on a shaker at room temperature for 2 hours.

[0069] 7. Peel off the adhesive membrane, discard the supernatant in the plate, and wash the wells 6 times with 400 μl / well Wash Buffer as in step 1.

[0070] 8. Add 100 μl of TMB Substrate Solution to each well.

[0071] 9. Cover the plate with an adhesive film and incubate at room temperature in the dark for 10 minutes.

[0072] 10. Quickly add 100 μl of Stop Solution to each well.

[0073] 11. Quickly measure the absorbance of the solution in the plate at 620 nm using an ELISA reader.

[0074] 12. Calculate the tenecteplase expression concentration in each sample based on the standard curve obtained from the absorbance values ​​of the standard solutions. Tenecteplase ELISA kit: Invitrogen, catalog numbers BMS258 and BMS258-2TEN.

[0075] 13. Detection Results: The tenecteplase mRNA molecule described in this invention was significantly expressed in the HEK293 cell line, such as...Figure 2 As shown in Table 1, the optimal expression conditions for tenecteplase protein in the HET293 cell line, calculated based on the standard curve, were approximately 16.5 ng / ml tenecteplase obtained after transfection with 20 pmol mNRA for 24 h. High levels of tenecteplase protein secretion were observed in both the CHO-K1 and AC16 cell lines. Figure 3 As shown in Table 2, the optimal expression conditions for tenecteplase protein in the CHOK1 cell line, calculated according to the standard curve, were approximately 173.3 ng / ml tenecteplase after transfection with 20 pmol mNRA for 72 h. In contrast, the optimal expression conditions for tenecteplase protein in the AC16 cell line were approximately 138.2 ng / ml tenecteplase after transfection with 20 pmol mNRA for 72 h.

[0076] Table 1. Protein expression concentration of tenepramase mRNA in HEK293 cell line 24 hours after transfection in Example 3.

[0077] mRNA transfection amount Tenecteplase protein expression concentration (μg / L) Blank control 3.53 8 pmol 14.1 20 pmol 16.5

[0078] Table 2. Protein expression concentration of tenepramase mRNA in CHOK1 cell line 72 hours after transfection in Example 4.

[0079]

[0080] Table 3. Protein expression concentration of tenepramase mRNA in AC16 cell line 72 hours after transfection in Example 5.

[0081]

[0082]

[0083] The above results indicate that the tenecteplase mRNA described in this invention is highly expressed in different cell lines, especially in human cells, which allows for therapeutic effects with a small amount of mRNA and has higher safety.

[0084] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application includes any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An mRNA molecule, characterized in that: The nucleotide sequence of the mRNA includes the sequence shown in SEQ ID NO.6, or a derived sequence of SEQ ID NO.6 with one or more amino acids replaced, added and / or deleted and having the same function as SEQ ID NO.6, and the uracil in the mRNA molecule is 1-N-Me-PseudoUTP.

2. The mRNA molecule according to claim 1, characterized in that: The mRNA molecule has untranslated regions added at both the 5' and 3' ends of the sequence shown in SEQ ID NO. 6; preferably, the 5' untranslated region sequence is shown in SEQ ID NO. 7, and the 3' untranslated region sequence is shown in SEQ ID NO.

8.

3. The mRNA molecule according to claim 1, characterized in that: The mRNA molecule has an mRNA cap structure added to the 5' end of the sequence shown in SEQ ID NO.6; preferably, the mRNA molecule has a polyadenosine and / or nucleotide-like tail added to the 3' end of the sequence shown in SEQ ID NO.6; more preferably, the degree of polymerization of the polyadenosine is 100 to 300.

4. The mRNA molecule according to claim 1, characterized in that: The nucleotide sequence of the mRNA is the sequence of SEQ ID NO.5, or a derived sequence of SEQ ID NO.5 with one or more amino acids replaced, added and / or deleted and having the same function as SEQ ID NO.5, and the uracil in the mRNA molecule is 1-N-Me-PseudoUTP.

5. A DNA molecule encoding the mRNA molecule of claim 1; preferably, the nucleotide sequence of the DNA molecule is as shown in SEQ ID NO.2 or SEQ ID NO.

3.

6. A recombinant plasmid containing the DNA molecule of claim 5; preferably, the recombinant plasmid sequence is as described in SEQ ID NO.

4.

7. A pharmaceutical composition comprising an mRNA molecule according to any one of claims 1 to 4, a DNA molecule according to claim 5 or a recombinant plasmid according to claim 6, and a pharmaceutically acceptable vector.

8. The use of the mRNA of any one of claims 1 to 4, the DNA molecule of claim 5, the recombinant plasmid of claim 6, or the composition of claim 7 in the preparation of tenecteplase mRNA drugs.

9. The use of the mRNA of any one of claims 1 to 4, the DNA molecule of claim 5, the recombinant plasmid of claim 6, or the composition of claim 7 in the preparation of a thrombolytic drug.

10. A method for preparing mRNA according to any one of claims 1-4, characterized in that, Take a recombinant plasmid containing the gene encoding the mRNA molecule as described in any one of claims 1-4, digest it with the restriction endonuclease Bsa I and recover the linearized plasmid, and then obtain tenecteplase mRNA by in vitro transcription.

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