WO2024051748A1 - 用于治疗骨关节炎的mRNA及其制备方法与应用 - Google Patents

用于治疗骨关节炎的mRNA及其制备方法与应用 Download PDF

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WO2024051748A1
WO2024051748A1 PCT/CN2023/117283 CN2023117283W WO2024051748A1 WO 2024051748 A1 WO2024051748 A1 WO 2024051748A1 CN 2023117283 W CN2023117283 W CN 2023117283W WO 2024051748 A1 WO2024051748 A1 WO 2024051748A1
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mrna
seq
present
preparation
sequence
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French (fr)
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胡勇
张昊
韩金雨
艾亮霞
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深圳瑞吉生物科技有限公司
武汉瑞佶生物科技有限公司
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Publication of WO2024051748A1 publication Critical patent/WO2024051748A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the invention belongs to the technical field of osteoarthritis treatment, and particularly relates to mRNA for treating osteoarthritis and its preparation method and application.
  • Osteoarthritis is a degenerative disease that commonly occurs in the elderly population and is one of the 10 most common causes of disability in the elderly. According to statistics, from 2008 to 2011, the revenue loss caused by OA cost US$80 billion per year to compensate. According to incomplete statistics, 500 million people around the world are currently affected by osteoarthritis. Traditional drug treatments all focus on symptoms to relieve pain. They cannot fundamentally treat OA, nor can they delay the development of OA. Some injection drugs also have serious liver and kidney risks.
  • CN113244413A and CN113425855A disclose technical solutions for stimulating the expression of cartilage growth factors in articular cavity chondrocytes through mRNA technology encoding, thereby promoting cartilage regeneration and treating osteoarthritis, and disclose mRNA drugs that are effective in treating osteoarthritis.
  • the above-mentioned mRNA drug has cartilage repair function.
  • the technical effect of this technical solution has great room for improvement.
  • the disclosed mRNA drugs still have problems such as insufficient intracellular specific expression levels, and further optimization and iteration are needed.
  • One object of the present invention is to provide an mRNA for treating osteoarthritis.
  • Another object of the present invention is to provide a method for preparing mRNA for treating osteoarthritis.
  • Another object of the present invention is to provide an application of mRNA for treating osteoarthritis.
  • the invention provides an mRNA encoding an amino acid sequence such as SEQ ID No. 12.
  • the mRNA is synthesized in vitro and can be used as an active ingredient to treat osteoarthritis.
  • the coding region sequence of the mRNA is as shown in any sequence of SEQ ID No. 1-SEQ ID No. 11.
  • the 5' end of the mRNA is connected to the cap structure and the 5'UTR; the 3' end of the mRNA is connected to the 3'UTR and the poly-A tail.
  • the poly-A tail includes multiple A's with a length of 90-130, and the multiple A's are continuous or a linker with 1-12 nt inserted in the middle and not all A's;
  • sequence of the poly A tail is AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
  • the cap structures used are m7G(5')ppp(5')(2'OMeA)pG, m7G(5')ppp(5')(2'OMeA), G(5 ')ppp(5')G, m7G(5')ppp(5')G, 3'-O-Me-m7G(5')ppp(5')G or m7(3'OMeG)(5') ppp(5')(2'OMeA)pG.
  • the 5'UTR sequence is AGGGAGATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCGCCACC (SEQ ID No. 16).
  • the 3'UTR sequence is GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID No. 17).
  • the present invention provides a DNA template that can be transcribed to obtain the mRNA.
  • the present invention provides the preparation method of mRNA, which includes:
  • the recombinant plasmid is transferred into host cells to obtain recombinant cells, and the mRNA is obtained by in vitro transcription and synthesis.
  • the method of the present invention may also include routine operations in the field, such as propagation of recombinant cells, extraction of plasmids, etc.
  • the conditions for in vitro synthesis of the mRNA are 36-38°C, 8-12 hours.
  • the present invention also provides the use of the mRNA in the preparation of medicaments for treating articular cartilage damage and/or osteoarthritis.
  • the present invention also provides a pharmaceutical preparation for treating articular cartilage damage and/or osteoarthritis, which contains the active ingredient mRNA, and the mRNA includes one or more than two of the aforementioned mRNAs.
  • the active component mRNA includes an mRNA with a coding region sequence as shown in SEQ ID No. 1 and/or an mRNA with a coding region sequence as shown in SEQ ID No. 5.
  • the pharmaceutical preparation is an injection preparation
  • the concentration of the active ingredient mRNA in the pharmaceutical preparation is 200-10000 ⁇ g/ml;
  • the dosage of the pharmaceutical preparation is 10-20 ⁇ g per joint, preferably 14-16 ⁇ g, and most preferably 15 ⁇ g.
  • the present invention optimizes the sequences through biological information means, and finally selects 11 optimized mRNAs through data screening, that is, the mRNAs with coding region sequences as shown in SEQ ID No. 1 to SEQ ID No. 11 in the present invention. And to this 11 The mRNA is tested to screen out a new generation of mRNA with better overall performance for treating osteoarthritis.
  • the present invention evaluates the efficacy of the mRNA preparation on the rat osteoarthritis model induced by medial meniscal transection (MMT) through joint histopathological analysis.
  • the weight of the animals increased steadily within 4 weeks after administration, and there was no significant difference in weight between the groups; in actual observation, compared with the control group, the pain symptoms of osteoarthritis in the experimental animals were significantly relieved.
  • One week after administration the swelling of the rat's knee joints was significantly relieved.
  • the results of joint histopathological analysis and evaluation showed that compared with the model group, the main tissue properties, surface regularity, and structural integrity of the mRNA preparation group tended to decrease 4 weeks after administration.
  • the present invention After different optimization and design of the mRNA encoding FGF18, the present invention finally obtains a new generation of mRNA therapeutic drugs with higher expression level, longer half-life, lower immunogenicity and better therapeutic effect.
  • the protein growth factor mRNA provided by the present invention has a higher specific expression level and a longer half-life in cells that can stimulate cartilage regeneration; after intramuscular injection of the mRNA preparation provided by the present invention, the concentration of protein growth factor in mouse serum The level of TNF ⁇ is significantly lower, indicating that the mRNA therapeutic drug provided by the present invention has lower immunogenicity and better safety; in terms of evaluation of therapeutic effect, the mRNA of the present invention also has better performance.
  • Figure 1 shows the difference chart of key indicators for sequence calculation optimization.
  • Figure 2 is a schematic structural diagram of mRNA encoding a protein growth factor that stimulates cartilage regeneration.
  • Figure 3 is the plasmid map of pRhe plasmid.
  • Figure 4 shows the expression of cartilage growth factor mRNA in cells detected by WesternBlot.
  • Figure 5 shows the TNF ⁇ level of mice injected with the osteoarthritis pharmaceutical preparation in the form of mRNA provided by the present invention.
  • Figure 6 shows the results of joint sections and safranin-fast green staining of rats in each experimental group to observe the cartilage repair.
  • Figure 7 shows the OARSI scores of joint sections of rats in each experimental group, showing the repairing effect of mRNA pharmaceutical preparations on osteoarthritis.
  • Figure 8 shows the gait analysis data of rats in each experimental group.
  • Figure 9 shows the digital DR measurement data of rats in each experimental group.
  • Figure 10 shows the relationship between the concentration of the corresponding protein in the blood and time after each experimental group was injected with the mRNA preparation or protein standard.
  • Figure 11 shows the detection of double-stranded RNA residues in the RNA preparation process using dot blot.
  • the present invention provides an osteoarthritis pharmaceutical preparation in the form of an mRNA dosage form, including the active ingredient mRNA.
  • the coding region sequence of the mRNA is as shown in SEQ ID No. 1 to SEQ ID No. 11.
  • T is used to represent uracil (U) of RNA.
  • T can also represent pseudouridine ( ⁇ ).
  • T can also represent N1-methylpseudouridine (m1 ⁇ ). );details as follows:
  • the 5’ end of the mRNA is preferably connected to the cap structure and the 5’ UTR; the 3’ end of the mRNA is preferably connected to the 3’ UTR and the modified poly-A tail.
  • the schematic structural diagram of the mRNA is shown in Figure 2.
  • the osteoarthritis pharmaceutical preparation is preferably a liquid preparation, and more preferably an injection preparation.
  • the concentration of the active ingredient mRNA in the osteoarthritis pharmaceutical preparation is preferably 200-10000 ⁇ g/ml, more preferably 200-5000 ⁇ g/ml, and more preferably 250-2500 ⁇ g/ml.
  • the solvent for the active ingredient mRNA is preferably physiological saline.
  • the present invention has no special limitations on the physiological saline and its preparation, and conventional physiological saline in this field can be used.
  • the present invention has no special limitations on the preparation method of the injection preparation, as long as it meets the requirements of conventional injection preparations in this field.
  • the invention also provides a method for preparing the osteoarthritis pharmaceutical preparation, which includes the steps of: 1) synthesizing the DNA fragment corresponding to the active ingredient mRNA in the osteoarthritis pharmaceutical preparation, and cloning the DNA fragment to express Obtain the recombinant plasmid from the plasmid; 2) Transfer the recombinant plasmid into host cells to obtain recombinant cells, extract the plasmid from the amplified recombinant cells, and use the extracted plasmid as a template to perform PCR amplification to obtain a DNA template for in vitro expression of mRNA; 3) Construct an in vitro RNA synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active component mRNA.
  • the DNA fragment transcribing the mRNA is synthesized, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid.
  • the present invention has no special limitations on the method of synthesizing the DNA fragment corresponding to the mRNA. Conventional DNA synthesis methods in the field can be used. During the specific implementation of the present invention, it is preferable to entrust a biotechnology company to synthesize. In the present invention, the specific sequence of the DNA fragment is determined based on the principle of complementary base pairing.
  • the expression plasmid is preferably a pRhe plasmid.
  • the plasmid map of the pRhe plasmid is as shown in Figure 3.
  • the DNA fragment is preferably cloned into an expression plasmid through enzyme digestion and ligation; in the present invention, the DNA fragment is preferably double digested with BamHI and NheI enzymes to obtain an enzyme-digested NDA fragment;
  • the expression plasmid is preferably double digested with BamHI and NheI enzymes to obtain the enzyme-digested plasmid; and then the enzyme-digested DNA fragment and the enzyme-digested plasmid are ligated to obtain the recombinant plasmid.
  • the present invention has no special limitations on the specific operations of the double enzyme cleavage and ligation, and conventional double enzyme cleavage and ligation operations in this field can be used.
  • the present invention transfers the recombinant plasmid into host cells to obtain recombinant cells, extracts the plasmid from the amplified recombinant cells, and uses the extracted plasmid as a template to perform PCR amplification to obtain in vitro expression of mRNA. DNA template.
  • the host cell is preferably an Escherichia coli competent cell; the present invention has no special limitation on the transfer method, and conventional transfer methods in this field can be used. After obtaining recombinant cells in the present invention, it is preferable to screen positive recombinant cells and sequence colonies.
  • the screening of positive recombinant cells is preferably performed on amp-resistant solid culture medium.
  • single colonies on the amp-resistant solid culture medium are selected for colony PCR, and colonies whose colony PCR results contain the target band are selected for sequencing.
  • the primers for colony PCR include primer F and primer R; the sequence of primer F is as follows: GAAATATAAGAGCCGCCACC (SEQ ID No. 13); the sequence of primer R is as follows: CCTACTCAGGCTTTATTCAAAGAC (SEQ ID No. 14);
  • the present invention has no special limitations on the specific steps of colony PCR, and conventional colony PCR steps in this field can be used.
  • the plasmids of the recombinant cells with correct sequencing are extracted; the present invention has no special limitations on the extraction method of the plasmids, and it is preferably carried out using a plasmid extraction kit.
  • the extracted plasmid is used as a template for PCR amplification to obtain a DNA template for in vitro expression of mRNA.
  • the PCR amplification system is calculated in 50 ⁇ l, preferably as follows:
  • the initial concentration of primer F and primer R is preferably 10 ⁇ mol/L; the concentration of the DNA template is preferably 1 ng/ ⁇ l.
  • the sequence of the primer F is as follows: GAAATATAAGAGCCGCCACC (SEQ ID No. 13); the sequence of the primer R is as follows: CCTACTCAGGCTTTATTCAAAGAC (SEQ ID No. 14).
  • the PCR amplification program is preferably as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; final extension at 72°C, 10 minutes.
  • the amplification product is preferably tested by agarose gel electrophoresis to determine whether the reaction is successful; the parameters of the agarose gel electrophoresis test are preferably as follows: 1.5% agar Sugar, 5V/min, 40 minutes. In the present invention, the presence of a band of the desired size in agarose gel electrophoresis is considered to be a successful reaction.
  • the amplification product is preferably concentrated and purified.
  • the concentration is preferably carried out using Millipore 30Kd ultrafiltration tubes; the purification is preferably carried out by FPLC; after the purification, the present invention preferably uses NanoDrop to detect the concentration of the purified template, and 260/280, A ratio of 260/230.
  • the preferred range of 260/280 is 1.8-2.1, and the range of 260/230 is greater than 2.0.
  • the present invention constructs an in vitro RNA synthesis system including the DNA template to perform in vitro synthesis of mRNA to obtain the active ingredient mRNA.
  • the RNA in vitro synthesis system includes the following components in 1600 ⁇ l:
  • the conditions for in vitro synthesis of RNA are preferably 36-38°C for 8-12 hours, and more preferably 37°C for 10 hours.
  • the RNA in vitro synthesis is preferably carried out in a constant temperature reactor; the RNA in vitro synthesis system is preferably placed in a 2ml RNase-free Tube tube, and multiple tubes can be reacted simultaneously at one time; in the RNA in vitro synthesis system Reagents are added in the order above.
  • the present invention preferably further includes the steps of removing the DNA template, recovering the mRNA, and purifying the mRNA.
  • the removal of the DNA template is preferably achieved by DNase I digestion; the digestion preferably includes mixing the solution after the in vitro synthesis reaction of DNase I and RNA; the solution after the in vitro synthesis reaction between DNase I and RNA is carried out.
  • the volume ratio of the solution is preferably 3:40; the mixing is preferably achieved by inverting the RNase-free Tube upside down, and the number of inversions is preferably 8 to 12 times, more preferably 10 times; the present invention is After the above mixing, it is preferred to centrifuge and collect the solution to the bottom of the RNase-free Tube.
  • the centrifugal speed is preferably 800 to 1200 rpm, more preferably 1000 rpm; the centrifugal time is preferably 8 to 12 seconds, more preferably 10 seconds.
  • the temperature of the digestion is preferably 37°C; the time of the digestion is preferably 1 hour. In the present invention, after the digestion is completed, DNA fragment residue detection is preferably performed.
  • the recovery of mRNA is preferably achieved by precipitating the ammonium acetate solution; for specific implementation methods, please refer to the examples; after recovering the mRNA in the present invention, the quality of the mRNA is detected; the quality detection includes the concentration of the mRNA , the ratio of 260/280 and 260/230 of mRNA, the value of A260/A280 of pure mRNA is 2.0 ⁇ 2.1, and the range of A260/A230 is 1.8 ⁇ 2.2.
  • the purified mRNA is achieved by FPLC purification. After purifying the mRNA according to the present invention, the purified mRNA is preferably aliquoted.
  • the invention provides the use of the osteoarthritis pharmaceutical preparation in preparing medicine for treating articular cartilage damage.
  • the dosage of the pharmaceutical preparation is preferably 10 to 20 ⁇ g per joint, more preferably 14 to 16 ⁇ g, and most preferably 15 ⁇ g.
  • the method of using the drug is joint intraarticular injection.
  • the present invention has no special limitations on the specific method of joint intraarticular injection. Conventional joint intraarticular injection operations in this field can be used.
  • the medicine for treating articular cartilage damage uses the osteoarthritis pharmaceutical preparation as an active ingredient or may also include other active ingredients; the dosage form of the medicine is preferably an injection preparation.
  • This embodiment provides a process for computational optimization of mRNA sequences.
  • sequence design and optimization are carried out on several important factors affecting mRNA stability and translation efficiency.
  • MFE Minimum Free Energy
  • CAI Codon Adaptation Index
  • the RH1231A sequence is significantly better than the randomly generated sequence in GC content, MFE and CAI invention.
  • the GC content increased by about 23.3% on average
  • the MFE increased by about 88.9% on average
  • the CAI increased by about 38.4% on average.
  • the present invention screened and optimized 11 sequences from 200,000 random sequences, namely SEQ ID No. 1-SEQ in this article ID No.11.
  • This embodiment provides a method for preparing the mRNA.
  • the 5' end of the mRNA is connected to the cap structure and the 5'UTR; the 3' end is connected to the 3'UTR and the modified poly-A tail.
  • the schematic structural diagram of mRNA in this embodiment is shown in Figure 2.
  • the coding region sequence of the prepared mRNA is shown in SEQ ID No.1-SEQ ID No.11; the cap structure is m7G(5')ppp(5')(2'OMeA)pG; the sequence of 5'UTR is AGGGAGATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCGCCACC (SEQ ID No.16);
  • the 3'UTR sequence is GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG (SEQ ID No.17);
  • the modified poly A tail sequence is AAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAA AAAAAAAAAAAAAAA
  • the synthetic sequence is 5'UTR, a section of SEQ ID No.1-SEQ ID No.11, 3'UTR, and a modified polyA tail DNA fragment (abbreviated as Seq1- in the examples and drawings). Seq11); 2) Double-digest the target fragment and pRhe plasmid with BamHI and NheI respectively; 3) Use T4 ligase for in vitro ligation; 4) Transform E. coli into competent cells and culture on amp solid medium for 12 hours; 5) Colony PCR, select the colonies containing the target band for sequencing; 6) Expand the colonies that have been sequenced correctly, and extract the plasmid.
  • the colony PCR steps in step 5) are as follows:
  • the initial concentration of primer F and primer R is preferably 10 ⁇ mol/L; the concentration of the DNA template is preferably 1 ng/ ⁇ l.
  • the sequence of the primer F is as follows: GAAATATAAGAGCCGCCACC (SEQ ID No. 13); the sequence of the primer R is as follows: CCTACTCAGGCTTTATTCAAAGAC (SEQ ID No. 14).
  • the PCR amplification program is preferably as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; final extension at 72°C, 10 minutes.
  • step 6 For the plasmid extraction steps described in step 6), refer to the instructions of omega D6915 Endo-free Plasmid Midi Kit.
  • the obtained plasmid is linearized to the DNA template according to the following reaction system:
  • Qualifying standards 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • Millipore 30Kd ultrafiltration tube concentrates the DNA template purified by FPLC, and elutes and dissolves it with RNase-free water. Use NanoDrop to detect the concentration of the template after ultrafiltration and the ratios of 260/280 and 260/230. Finally dilute to 150ng/ ⁇ l with RNase-free water.
  • RNA in vitro synthesis of mRNA is performed. Proceed according to the following synthesis system (reaction reagents are added from top to bottom): reaction volume, 1600 ⁇ l (placed in a 2ml RNase-free Tube tube, it is the reaction volume of a single tube, and multiple tubes can be reacted at one time).
  • reaction volume 1600 ⁇ l (placed in a 2ml RNase-free Tube tube, it is the reaction volume of a single tube, and multiple tubes can be reacted at one time).
  • the RNA in vitro synthesis system includes the following components based on 1600 ⁇ l:
  • RNA samples were 37°C and 10 hours.
  • DNA residue detection method quantitative real-time PCR detection method is used. The specific steps are as follows:
  • test solution Take an appropriate amount of sample, dilute it 10 times with enzyme-free water, mix well.
  • the PCR program settings are as follows:
  • Calculation formula Use the Ct value of the standard to plot the logarithm of its concentration, linear regression analysis, substitute the sample Ct value into the equation, calculate the "detection value of the logarithm of the concentration after dilution 10 times", and calculate the "DNA residue”.
  • DNA residual concentration (copies/ ⁇ l) detection value ⁇ dilution factor
  • the Ct difference between three parallel wells should be less than 1.0; except for samples with Ct values greater than 35.
  • Linear correlation coefficient R 2 >0.99.
  • the non-template control NTC should not be detectable or have a Ct value greater than 2 Ct values greater than the lowest concentration of the standard curve.
  • Standard regulations should not be higher than 10ng/dose.
  • the coding region sequence of the mRNA prepared in this example is as shown in SEQ ID NO.1-SEQ ID NO.11, and the 5' end is connected to the cap structure (m7G(5')ppp(5')(2'OMeA)pG) and 5' UTR (SEQ ID No. 16); the 3' end is connected to 3' UTR (SEQ ID No. 17) and modified poly A tail (SEQ ID No. 15), abbreviated in the embodiments and figures is Seq1-Seq11.
  • This embodiment provides a method for detecting the expression level of mRNA encoding cartilage growth factor.
  • Cell preparation Prepare cells for detection 1-3 days in advance. 293T cells purchased from the Cell Bank of the Chinese Academy of Sciences were passaged in cell culture bottles to ensure that the cells were in the logarithmic growth phase when used.
  • Cell dilution Take 1 ml of cell suspension, dilute it to 5 ⁇ 10 5 cells/ml with DMEM medium containing 10% FBS, and pipet to mix.
  • Cell seeding Add 2 ml of cell suspension into a 6-well plate. It is necessary to prepare 2 wells of parallel cells for each mRNA sample, 1 well of cells for the control sample (GFP-mRNA), and 1 well for the blank control. Place the 6-well plate in a (37 ⁇ 1)°C, (5 ⁇ 0.5)% CO2 incubator for overnight culture.
  • a) Prepare transfection system Take 200 ⁇ l opti-MEM, add 10 ⁇ g mRNA sample (Seq1-Seq11) or negative control GFP-mRNA, gently pipet and mix with a pipette tip, then add 60 ⁇ l PEI (concentration 1mg/ml), immediately Place on a vortex shaker and shake 10 times, 1 second each time, mix thoroughly, and let stand for 10 minutes.
  • Opti-MEM Take 200 ⁇ l opti-MEM, add 10 ⁇ g mRNA sample (Seq1-Seq11) or negative control GFP-mRNA, gently pipet and mix with a pipette tip, then add 60 ⁇ l PEI (concentration 1mg/ml), immediately Place on a vortex shaker and shake 10 times, 1 second each time, mix thoroughly, and let stand for 10 minutes.
  • PEI concentration 1mg/ml
  • the medium was changed 6 hours after transfection, the old medium was aspirated, and each well was replaced with 2 ml of fresh medium (90% DMEM+10% FBS).
  • BCA kit (Thermo Scientific, P/N 23225), operating steps Refer to the kit instructions for the steps, as follows: A. Dilute the BSA standard with ultrapure water, prepare a standard series of 2000, 1000, 500, 250, 125, 62.5, 31.25 ⁇ g/ml, and add another ultrapure water as a blank For each sample, prepare 100 ⁇ L. B. Take 10 ⁇ l of supernatant, add 90 ⁇ l of ultrapure water and mix well. C. Prepare 1X working reagent: Take 50 volumes of reagent A, add 1 volume of reagent B, and mix. D.
  • Preparation of protein samples to be tested After protein quantification, take 50 ⁇ g protein, adjust the volume to 20 ⁇ L with lysis buffer, add 5 ⁇ L 5 ⁇ Loading buffer (purchased from Sangon Bioengineering (Shanghai) Co., Ltd., P/N C508320), and mix Homogenize a total of 25 ⁇ L, incubate at 95°C for 5 minutes, and then place at room temperature;
  • 5 ⁇ Loading buffer purchased from Sangon Bioengineering (Shanghai) Co., Ltd., P/N C508320
  • 6% separating gel corresponds to 4% stacking gel
  • 12% separating gel corresponds to 5% stacking gel
  • the stacking gel is prepared according to the formula in Table 2.
  • the results are shown in Figure 4.
  • the test sample should show obvious color bands
  • GAPDH is the internal reference protein
  • 1-11 Represent Seq1-Seq11 respectively, among which the protein expression levels of Seq1, Seq2, Seq3, Seq4, and Seq5 are relatively high. After screening, it was finally decided to select Seq1-Seq5 as subsequent test samples for animal experiments.
  • This example provides a method for detecting the immunogenicity of mRNA encoding cartilage growth factor.
  • the evaluation standard is the level of TNF ⁇ in the serum after intramuscular injection of the mRNA into mice.
  • balb/c mice purchased from Beijing Vitong Lever
  • the mice shown in Seq1-Seq11 were The mRNA was injected intramuscularly into balb/c mice, and the injection dose of each mouse was 100 ⁇ g. After 24 hours, blood was taken from the orbit of the mice and the serum was separated. Enzyme-linked immunosorbent assay (ELISA) was performed using mouse TNFa kit (RayBio). The experimental results are shown in Figure 5.
  • the TNF ⁇ expression level was lower than the treatment and control group injected with FGF18 protein (CN113425855A superior group, i.e., Seq 2-1 group). It can be seen that the Seq1 in the mRNA dosage form provided by the present invention , Seq5 has lower immunogenicity than the protein control group and control group, and has better safety. Seq1 and Seq5 will be subject to subsequent animal experiments.
  • This example studies the repair of articular cartilage damage in rats by mRNA encoding cartilage growth factor.
  • the OARSI scoring criteria are as follows:
  • This embodiment provides a method for verifying the effect of mRNA encoding cartilage growth factors on modeled rat joints using a gait behavior system.
  • Lewis rats aged 6 to 8 weeks were raised in ventilated cages under SPF conditions with a 12-hour light and 12-hour dark cycle, and the medial collateral ligament + medial half moon were surgically cut.
  • the rat osteoarthritis model was established on a plate. The specific operations are as follows: After anesthetizing the rats in the model group with isoflurane inhalation (1% to 4% for induction anesthesia, 0.25% to 2% for maintenance anesthesia), prepare the joint surgical sites of the rats. After disinfecting the skin with iodine, make an incision through the inside of the knee joint of the right hind limb to expose the medial collateral ligament and medial meniscus of the knee joint cavity.
  • the day of surgery was counted as D0, and administration began on the 7th postoperative day (D7).
  • Each administration group was given RNA solution of corresponding concentration at 50uL/only through the knee joint of the right hind limb each time.
  • the administration groups included protein control group, CN113425855A
  • the medium and excellent group Seq 2-1 group, the Seq1 group and the Seq5 group of the present invention; the normal control group and the model control group were injected with an equal volume of 0.9% sodium chloride injection for 4 consecutive times, with an interval between each administration. 1 week. Gait data were collected every week to analyze and compare the changes in the rat's hindfoot support force.
  • This example provides histopathological examination to verify the effect of mRNA encoding cartilage growth factor on modeled rat joints.
  • Lewis rats aged 6 to 8 weeks were raised in ventilated cages under SPF conditions with a 12-hour light and 12-hour dark cycle, and the medial collateral ligament + medial half moon were surgically cut.
  • a rat osteoarthritis model was established on a plate. The specific operations are as follows: rats in the model group were anesthetized by isoflurane inhalation (1% to 4%). After anesthesia (maintenance anesthesia with 0.25% to 2%), the skin of the rat joint surgery site was prepared and disinfected with iodine.
  • the day of surgery was counted as D0, and administration began on the 7th postoperative day (D7).
  • Each administration group was given RNA solution of corresponding concentration at 50uL/only through the knee joint of the right hind limb each time.
  • the administration groups included protein control group, CN113425855A
  • the medium-effective group Seq 2-1 and the Seq1 group and Seq5 group of the present invention; the normal control group and the model control group were respectively injected with an equal volume of 0.9% sodium chloride injection for 4 consecutive times, with an interval between each administration. 1 week.
  • the rats in the administration group were anesthetized by isoflurane inhalation and euthanized by bleeding from the abdominal aorta.
  • Digital DR data were collected to observe the morphological changes of the cartilage tissue and synovial tissue of the rats in each group.
  • This example detects the expression level and half-life of the mRNA of the present invention.
  • the evaluation standard is to detect the FGF18 protein concentration in the synovial fluid after injecting an equal amount of the mRNA drug into the articular cavity of rats in rats.
  • the cartilage growth factor mRNA preparation provided by the present invention was injected into the articular cavity of rats, the protein expression level was higher than that of the injected FGF18 protein for a long time. It can be seen that the mRNA dosage form provided by the present invention has a higher expression amount and a longer expression time, and the protein content in the blood is higher. Longer half-life.
  • This example provides an experiment using dot blot to detect double-stranded RNA residues in the RNA preparation process.
  • the test solution is dropped onto a solid carrier (such as a nylon membrane).
  • the solid carrier has a positive charge and can adsorb negatively charged RNA.
  • Double-stranded RNA on a solid-phase carrier is used as an antigen, and the corresponding double-stranded RNA antibody (called For the first antibody) to initiate an immune reaction, a specific second antibody is used to recognize the first antibody.
  • the second antibody contains horseradish peroxidase (HRP), which can catalyze the luminol reagent in the presence of hydrogen peroxide to make it Luminescence, this process becomes a color reaction, and the signal of the substrate can be captured by the imaging system to form a spot blot.
  • the gray value of the spot represents the content.
  • test samples Take an appropriate amount of the test sample (sample in CN113425855A, sample in the present invention) and dilute it with enzyme-free water to contain 500ng/ ⁇ L mRNA to obtain the test solution; 2) In a ultra-clean workbench, use Use clean scissors to cut out a nylon membrane of appropriate size; 3) Take 4 ⁇ L of the diluted test sample and drop it on the nylon membrane respectively, and do 2 repetitions; 4) After all the samples have been dropped, leave a blank in the lower right corner of the nylon membrane Cut out chamfers to identify the front and back sides; air-dry the nylon membrane for 30 minutes; 5) Wear clean enzyme-free gloves, roll the membrane into a 50mL centrifuge tube, make sure the front side faces the inside of the tube and not the tube wall, add 10ml PBST

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Abstract

用于治疗骨关节炎的mRNA及其制备方法与应用。首先提供一种mRNA，其可编码序列如SEQ ID No.12的氨基酸。还提供所述mRNA的制备方法。还提供一种用于治疗关节软骨损伤和/或骨关节炎的药物制剂，其包含所述mRNA。还提供所述mRNA和mRNA剂型的骨关节炎药物制剂的应用。所提供的mRNA在细胞内的特异性表达水平更高；进行肌肉注射后，小鼠血清中TNFα水平明显更低；治疗效果更好。

Description

用于治疗骨关节炎的mRNA及其制备方法与应用 技术领域
本发明属于骨关节炎治疗技术领域，尤其涉及用于治疗骨关节炎的mRNA及其制备方法与应用。
背景技术
骨关节炎(OA)是一种常发于老年人口的退行性疾病，是老年人最常见的10种残疾原因之一。据统计，2008年至2011年，OA导致的收入损失需要花费800亿美元每年来补偿。据不完全统计，目前全世界已有5亿人受累于骨关节炎。传统的药物治疗手段，均从症状着手以缓解疼痛，无法从根本上治疗OA，也无法延缓OA的发展，部分注射类药物还存在较严重肝肾风险。
CN113244413A和CN113425855A公开了通过mRNA技术编码刺激软骨生长因子在关节腔软骨细胞中表达，从而促进软骨再生、起到治疗骨关节炎效果的技术方案，并公开了有效治疗骨关节炎的mRNA药物，所述mRNA药物具有软骨修复功能。但随着研究的进展和数据的不断收集，我们发现该技术方案的技术效果有较大的提升空间，其中公开的mRNA药物尚存在细胞内特异性表达水平不够高等问题，需进一步优化迭代。
发明内容
本发明的一个目的在于提供一种用于治疗骨关节炎的mRNA。
本发明的另一目的在于提供一种用于治疗骨关节炎的mRNA的制备方法。
本发明的另一目的在于提供一种用于治疗骨关节炎的mRNA的应用。
为了实现上述发明目的，本发明提供了以下技术方案：
一方面，本发明提供一种mRNA，可编码序列如SEQ ID No.12的氨基酸。所述mRNA是体外合成的，所述mRNA能够作为活性成分用于治疗骨关节炎。
根据本发明的具体实施方案，其中，所述mRNA的编码区序列如SEQ ID No.1-SEQ ID No.11任一序列所示。
根据本发明的具体实施方案，其中，所述mRNA的5’端连接帽子结构和5’UTR；所述mRNA的3’端连接3’UTR和多聚A尾。
根据本发明的具体实施方案，其中，所述多聚A尾包括长度为90-130的多个A，多个A为连续的或中间插入1-12nt且不全部为A的连接子；
优选地，所述多聚A尾的序列为AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID No.15)。
另一方面，本发明中，所采用的帽子结构为m7G(5')ppp(5')(2'OMeA)pG、m7G(5')ppp(5')(2'OMeA)、G(5')ppp(5')G、m7G(5')ppp(5')G、3′-O-Me-m7G(5')ppp(5')G或m7(3'OMeG)(5')ppp(5')(2'OMeA)pG。
根据本发明的具体实施方案，其中，所述5’UTR序列为AGGGAGATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCGCCACC(SEQ ID No.16)。
根据本发明的具体实施方案，其中，所述3’UTR序列为GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG(SEQ ID No.17)。
另一方面，本发明提供一种DNA模板，其可以转录得到所述的mRNA。
另一方面，本发明提供所述的mRNA的制备方法，其包括：
将能转录所述mRNA的DNA片段克隆至表达质粒获得重组质粒；
将所述重组质粒转入宿主细胞获得重组细胞，体外转录合成获得所述mRNA。
可以理解，本发明的方法还可以包括所述领域的常规操作，如重组细胞的扩繁操作、提取质粒的操作等。
根据本发明的具体实施方案，其中，所述mRNA体外合成的条件为36-38℃，8-12小时。
另一方面，本发明还提供所述的mRNA在制备用于治疗关节软骨损伤和/或骨关节炎的药物中的应用。
另一方面，本发明还提供一种用于治疗关节软骨损伤和/或骨关节炎的药物制剂，其包含活性成分mRNA，所述mRNA包括前面所述的mRNA中的一种或两种以上。
根据本发明的具体实施方案，其中，活性成分mRNA包括编码区序列如SEQ ID No.1所示的mRNA和/或编码区序列如SEQ ID No.5所示的mRNA。
根据本发明的具体实施方案，其中，所述药物制剂为注射制剂；
优选地，所述活性成分mRNA在药物制剂中的浓度为200～10000μg/ml；
优选地，所述药物制剂的使用剂量，以每一关节计，为10～20μg，优选为14～16μg，最优选为15μg。
本发明通过生物信息手段对序列进行优化，经过数据筛选最终选定11条优化后的mRNA，即本发明中编码区序列如SEQ ID No.1～SEQ ID No.11所示的mRNA。并对这11 条mRNA进行测试，筛选得到综合表现更好的新一代治疗骨关节炎的mRNA。
本发明通过关节组织病理学分析评价mRNA制剂对内侧半月板横断(MMT)诱导的大鼠骨关节炎模型的药效。在给药后4周内动物体重增长平稳，各组之间体重无显著性差异；实际观察中相比于对照组，实验动物的骨关节炎疼痛症状有明显缓解。给药后一周大鼠膝关节肿胀有明显缓解。关节组织病理学分析评价结果显示，与模型组相比较，mRNA制剂组在给药后4周后对主要组织性质、表面规则性、结构完整性均有降低的趋势。
本发明在对编码FGF18的mRNA进行不同的优化与设计之后，最终得到了表达水平更高、半衰期更长、免疫原性更低、治疗效果更好的新一代mRNA治疗药物。与现有技术相比，本发明提供的具有刺激软骨再生的蛋白生长因子mRNA在细胞内的特异性表达水平更高、半衰期更长；本发明提供的mRNA制剂进行肌肉注射后，小鼠血清中TNFα水平明显更低，说明本发明提供的mRNA治疗药物的免疫原性更低，安全性更好；在治疗效果的评价方面，本发明mRNA亦具有更好的表现。
附图说明
图1为序列计算优化关键指标差异图。
图2为编码刺激软骨再生的蛋白生长因子mRNA的结构示意图。
图3为pRhe质粒的质粒图谱。
图4为WesternBlot检测软骨生长因子mRNA在细胞内的表达情况。
图5为小鼠注射本发明提供的mRNA剂型的骨关节炎药物制剂的TNFα水平。
图6为各实验组大鼠关节切片和番红固绿染色结果，观察软骨修复情况。
图7为各实验组大鼠关节切片OARSI评分，显示mRNA药物制剂对骨关节炎的修复作用。
图8为各实验组大鼠步态分析数据。
图9为各实验组大鼠数字DR测量数据。
图10为各实验组注射mRNA制剂或蛋白标品之后血液中相应蛋白的浓度与时间的关系。
图11为斑点印迹法(dot blot)检测RNA制备工艺中双链RNA残留情况。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术 方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
本发明提供了一种mRNA剂型的骨关节炎药物制剂，包括活性成分mRNA，在本发明中，所述mRNA的编码区序列如SEQ ID No.1～SEQ ID No.11所示。本发明中使用“T”代表RNA的尿嘧啶(U)，本发明中“T”也可以代表假尿苷(ψ)，本发明中“T”也可以代表N1-甲基假尿苷(m1ψ)；具体如下：
SEQ ID No.1：
SEQ ID No.2：
SEQ ID No.3：

SEQ ID No.4：
SEQ ID No.5：
SEQ ID No.6：

SEQ ID No.7：
SEQ ID No.8：
SEQ ID No.9：

SEQ ID No.10：
SEQ ID No.11：
SEQ ID No.12：
在本发明中，所述mRNA的5’端优选的连接帽子结构和5’UTR；所述mRNA的3’端优选的连接3’UTR和修饰的多聚A尾。在本发明中，所述mRNA的结构示意图如图2所示。
在本发明中，所述骨关节炎药物制剂优选为液体制剂，更优选为注射制剂。在本发明中，所述活性成分mRNA在骨关节炎药物制剂中的浓度优选为200～10000μg/ml，更优选为200-5000μg/ml，更优选为250～2500μg/ml。所述活性成分mRNA的溶剂优选为生理盐水。本发明对所述生理盐水及其制备没有特殊限定，采用本领域常规的生理盐水即可。本发明对所述注射制剂的制备方法没有特殊限定，满足本领域常规注射制剂的要求即可。
本发明还提供了所述的骨关节炎药物制剂的制备方法，包括步骤：1)合成所述的骨关节炎药物制剂中的活性成分mRNA对应的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒；2)将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板；3)构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。
在本发明中，合成转录所述的mRNA的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒。本发明对合成所述的mRNA对应的DNA片段的方法没有特殊限定，采用本领域常规的DNA合成方法即可，在本发明具体实施过程中，优选的委托生物技术公司合成。在本发明中，所述DNA片段的具体序列根据碱基互补配对原则确定。在本发明中，所述表达质粒优选为pRhe质粒，在本发明中，所述pRhe质粒的质粒图谱如图3所示。在本发明中，优选的通过酶切连接的方法将所述DNA片段克隆至表达质粒中；在本发明中，所述DNA片段优选的通过BamHI和NheI酶进行双酶切获得酶切NDA片段；所述表达质粒优选的通过BamHI和NheI酶进行双酶切后获得酶切质粒；然后将所述酶切DNA片段和酶切质粒连接获得重组质粒。本发明对所述双酶切和连接的具体操作没有特殊限定，采用本领域常规的双酶切和连接的操作即可。
本发明在获得所述重组质粒后，将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。在本发明中，所述宿主细胞优选为大肠杆菌感受态细胞；本发明对所述转入的方法没有特殊限定，采用本领域常规的转入方法即可。本发明在获得重组细胞后，优选的进行阳性重组细胞的筛选和菌落测序。在本发明中，所述阳性重组细胞的筛选优选的在amp抗性的固体培养基上进行。在本发明中，挑选所述amp抗性的固体培养基上的单菌落进行菌落PCR，选取菌落PCR结果含目的条带的菌落进行测序。在本 发明中，所述菌落PCR的引物包括引物F和引物R；所述引物F的序列如下：GAAATATAAGAGCCGCCACC(SEQ ID No.13)；所述引物R的序列如下：CCTACTCAGGCTTTATTCAAAGAC(SEQ ID No.14)；本发明对所述菌落PCR的具体步骤没有特殊限定，采用本领域常规的菌落PCR步骤即可。
在本发明中，提取测序正确的重组细胞的质粒；本发明对所述质粒的提取方法没有特殊限定，优选的采用质粒提取试剂盒进行。在本发明中，以提取获得的质粒为模板进行PCR扩增获得体外表达mRNA的DNA模板。在本发明中，所述PCR扩增的体系以50μl计，优选的如下：
在本发明中，所述引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μl。在本发明中，所述引物F的序列如下：GAAATATAAGAGCCGCCACC(SEQ ID No.13)；所述引物R的序列如下：CCTACTCAGGCTTTATTCAAAGAC(SEQ ID No.14)。在本发明中，所述PCR的扩增程序优选的如下：预变性98℃3分钟；变性98℃10秒，退火60℃5秒，延伸72℃2分钟，34个循环；最后延伸72℃，10分钟。
在本发明中，所述PCR扩增反应结束后，优选的对扩增产物进行琼脂糖凝胶电泳检测以确定反应是否成功；所述琼脂糖凝胶电泳检测的参数优选的如下：1.5％琼脂糖，5V/min，40分钟。在本发明中，琼脂糖凝胶电泳出现目的大小的条带认为是反应成功。
本发明在所述PCR扩增反应结束后，优选的将所述扩增产物进行浓缩和纯化。在本发明中，所述浓缩优选的采用Millipore 30Kd超滤管进行；所述纯化优选的采用FPLC进行；本发明在所述纯化后优选的采用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。优选260/280范围为1.8-2.1，260/230范围为大于2.0。
本发明在获得所述DNA模板后，构建包括所述DNA模板的RNA体外合成体系进行mRNA的体外合成获得所述活性成分mRNA。在本发明中，所述RNA体外合成体系以1600μl计，包括以下组分：

在本发明中，所述RNA体外合成的条件优选的为36～38℃，8～12小时，更优选为37℃，10小时。在本发明中，所述RNA体外合成优选的在恒温反应器中进行；所述RNA体外合成体系优选的置于2ml RNase-free Tube管中，一次同时反应多管；所述RNA体外合成体系中的反应试剂按照上述顺序添加。
本发明在所述RNA体外合成结束后，优选的还包括去除DNA模板、回收mRNA和纯化mRNA的步骤。在本发明中，所述去除DNA模板优选的通过DNase I消化实现；所述消化优选的包括将DNase I与RNA体外合成反应后的溶液混合后进行；所述DNase I与RNA体外合成反应后的溶液的体积比优选为3:40；所述混合优选的通过上下颠倒所述RNase-free Tube管实现，所述上下颠倒的次数优选为8～12次，更优选为10次；本发明在所述混合后，优选的进行离心将溶液收集至RNase-free Tube管底部。在本发明中，所述离心的转速优选为800～1200rpm，更优选为1000rpm；所述离心的时间优选为8～12秒，更优选为10秒。所述消化的温度优选为37℃；所述消化的时间优选为1小时。本发明在所述消化结束后，优选的进行DNA片段残留检测。在本发明中，所述回收mRNA优选的通过将所述醋酸铵溶液沉淀实现；具体实施方法参见实施例记载；本发明在回收mRNA后，进行mRNA的质量检测；所述质量检测包括mRNA的浓度、mRNA的260/280、260/230的比值，纯mRNA的A260/A280的值为2.0～2.1，A260/A230范围为1.8～2.2。在本发明中，所述纯化mRNA通过FPLC纯化实现。本发明所述纯化mRNA后，优选的对纯化后的mRNA进行分装。
本发明提供了所述的骨关节炎药物制剂在制备治疗关节软骨损伤的药物中的应用。在本发明中，所述药物制剂的使用剂量，以每一关节计，优选为10～20μg，更优选为14～16μg，最优选为15μg。在本发明中，所述药物的使用方法为关节腔注射，本发明对所述关节腔注射的具体方法没有特殊限定，采用本领域常规的关节腔注射操作即可。本发明中，所述治疗关节软骨损伤的药物以所述骨关节炎药物制剂为活性成分或，还可以包括其他活性成分；所述药物的剂型优选为注射制剂。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
本实施例提供mRNA序列计算优化的过程。
本发明中对影响mRNA稳定性及翻译效率的几个重要影响因素进行了序列设计优化。其中，提高GC含量，降低最小自由能(Minimum Free Energy，MFE，单位为kcal/mol)能提升mRNA的稳定性，避免稀有密码子的使用，提高密码子适应指数(Codon Adaptation Index，CAI)能增加mRNA翻译效率。如图1所示，RH1231A序列在GC含量、MFE和CAI发明均显著优于随机生成的序列。其中，GC含量平均提升约23.3％，MFE平均提升约88.9％，CAI平均提升约38.4％，本发明从20万条随机序列中筛选优化得到11条序列，即本文中SEQ ID No.1-SEQ ID No.11。
实施例2
本实施例提供所述mRNA的制备方法。
本实施例中mRNA的5’端连接帽子结构和5’UTR；3’端连接3’UTR和修饰的多聚A尾。本实施例mRNA的结构示意图如图2所示。其中，制备的mRNA的编码区序列如SEQ ID No.1-SEQ ID No.11所示；帽子结构为m7G(5')ppp(5')(2'OMeA)pG；5’UTR的序列为AGGGAGATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCGCCACC(SEQ ID No.16)；3’UTR序列为GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG(SEQ ID No.17)；修饰的多聚A尾的序列为AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID No.15)。
制备mRNA的具体步骤如下。
重组质粒制备：
1)合成序列依次为5’UTR、SEQ ID No.1-SEQ ID No.11中的一段、3’UTR、修饰的多聚A尾的DNA片段(各实施例及附图中简写为Seq1-Seq11)；2)将目的片段和pRhe质粒分别进行BamHI和NheI双酶切；3)使用T4连接酶进行体外连接；4)转化大肠杆菌感受态，在amp固体培养基上培养12小时；5)菌落PCR，选取含目的条带的菌落进行测序；6)测序正确的菌落扩大培养，质粒提取。
步骤5)中菌落PCR步骤如下：
(1)单菌落挑取：把LB培养基倒入排枪槽中，然后用排枪加400μl LB培养基到48孔深孔板，用镊子拿已灭菌的小白枪头挑取平板上的单菌落并放到48孔深孔板中，同时在48孔深孔板和相应的表单上做好记录。挑好的48孔深孔板用封口膜盖好并做好相 应标记(日期、板号等)，用针头在封口膜打孔，把它放在37℃摇床上摇1小时。
(2)菌落PCR反应：配制好如下PCR反应体系，把配好的反应液加到96孔板中，用排枪加2μl菌液到其中，按照PCR程序进行扩增：
在本发明中，所述引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μl。在本发明中，所述引物F的序列如下：GAAATATAAGAGCCGCCACC(SEQ ID No.13)；所述引物R的序列如下：CCTACTCAGGCTTTATTCAAAGAC(SEQ ID No.14)。在本发明中，所述PCR的扩增程序优选的如下：预变性98℃3分钟；变性98℃10秒，退火60℃5秒，延伸72℃2分钟，34个循环；最后延伸72℃，10分钟。
(3)琼脂糖凝胶电泳：先配制1％琼脂糖凝胶(称取1g琼脂糖加入100ml的TAE溶液中)，在完成PCR反应的96孔板中加入0.5μl溴酚蓝，震荡混匀，然后点样，电泳好的琼脂糖凝胶拍照、保存。
(4)判断阳性克隆并测序：根据琼脂糖凝胶电泳条带图来判断阳性克隆，把阳性克隆的菌液进行扩大培养，进行测序验证，选取具备完全正确序列的克隆进行下一步操作。
步骤6)中所述的质粒提取的步骤参考omega D6915 Endo-free Plasmid Midi Kit说明书。
得到的质粒按如下反应体系进行DNA模板的线性化：
标准反应体系：
反应结束后，将反应液合并于1.5ml Tube管中。取10μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖，5V/min，40分钟)。根据电泳目的条带的大小对反应成功与否进行确认。合格标准：电泳检测出现单一的条带，且大小正确。
测定结果：条带大小单一，大小符合要求。
DNA模板超滤
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。
DNA模板FPLC纯化
将上述超滤得到的DNA，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇＝25/24/1)，充分震荡后，12000g离心15分钟。去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(pH 5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静至30分钟。4℃，12000g离心10分钟，弃上清。用70％乙醇洗涤沉淀，12000g离心5分钟，取上清，于超净台晾干5分钟。用适当的RNase-free水溶解纯化后的DNA模板。用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40分钟)。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：浓度为500ng/μl，260/280＝1.93，260/230＝1.75。
FPLC纯化后模板超滤
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。
测定结果：浓度为150ng/μl，260/280＝1.96，260/230＝1.87。
mRNA的体外合成
在恒温反应器中，进行mRNA的体外合成。按照如下合成体系进行(反应试剂按照从上至下添加)：反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)。所述RNA体外合成体系以1600μl计，包括以下组分：
所述RNA体外合成的条件为37℃，10小时。
DNase I消化去除DNA模板
向mRNA体外合成后的每个Tube管中各加入120μl DNase I。上下颠倒10次混匀，1000rpm离心10秒。重新置于恒温反应器中，37℃，1小时。反应结束后，将反应液合并到RNase-free 50ml Tube管中，检测DNA片段的残留。
DNA残留检测的方法：采用定量实时PCR检测方法，具体操作步骤如下：
(1)供试品溶液配制：取样品适量，用无酶水稀释10倍，混匀，混得。
(2)标准品溶液配制：用无酶水将标定过的质粒标准品进行稀释至1E+08copies/μl，然后再进行梯度稀释。具体操作如下：
ERC的制备：取20μl供试品溶液，加入20μl ST4，混匀，即得。
MIX反应液配制：以SuperFast Probe Mixtuure：W2306F：W2521R：W2430P：H2O＝10:0.6:0.6:0.4:7.4的比例进行配制，混匀，即得。
PCR管加样：每孔加入19μl qPCR MIX、再依次加入1μl标准曲线(ST1/ST2/ST3/ST4/ST5/ST6)、ERC、NTC、供试品溶液，混匀。每个样各做三个平行。
PCR程序设定如下：
计算公式：以标准品的Ct值对其浓度的对数值做图，线性回归分析，将样品Ct值代入方程，计算“稀释10倍后的浓度对数的检测值”，计算“DNA残留”。
DNA残留浓度(copies/μl)＝检测值×稀释倍数
结果判定：三个平行孔间的Ct差值应小于1.0；Ct值大于35的样本除外。线性相关系数R2>0.99。无模板对照NTC应不得检出或大于标准曲线最低浓度2个Ct值。
标准规定：应不高于10ng/剂量。
mRNA沉淀回收
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。上下颠倒10次混匀。置于-20℃2小时，沉淀。17000g，4℃离心，30分钟。去掉上清，用70％乙醇洗涤沉淀。17000g，4℃离心，10分钟。去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。静置10分钟后，用枪头轻吹混匀。用NanoDrop检测回收后的mRNA浓度为5.06μg/μl，A260/A280为1.81、A260/A230为1.95。取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。检测结果为，条带符合大小，片段完整。
LiCl沉淀纯化mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。加入原mRNA1.5倍体积-20预冷的LiCl溶液，混匀。然后于-20℃静至2小时。16000g离心20分钟。弃上清，用70％乙醇洗涤沉淀，16000g离心15分钟。取上清，于超净台晾干5分钟。用适当的RNase-free水溶解纯化后的mRNA。纯化后的mRNA浓度为2μg/μl，A260/A280为1.95、A260/A230为1.84。
mRNA分装
将上一步骤中纯化后的mRNA分装至西林瓶。
本实施例制备的mRNA，其编码区序列如SEQ ID NO.1-SEQ ID NO.11所示，5’端连接帽子结构(m7G(5')ppp(5')(2'OMeA)pG)和5’UTR(SEQ ID No.16)；3’端连接3’UTR(SEQ ID No.17)和修饰的多聚A尾(SEQ ID No.15)，在各实施例和附图中简写为Seq1-Seq11。
实施例3
本实施例提供编码软骨生长因子的mRNA表达水平检测的方法。
实验方法：体外效力-Western Blot检测
293T细胞的接种
1)细胞准备：提前1-3天准备检测用细胞。取购自中科院细胞库的293T细胞，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。
2)细胞消化计数：取生长状态良好的293T细胞，去掉培养基，以10ml PBS清洗细胞后，加入体积百分含量0.25％胰酶(T75瓶加1ml 0.25％胰酶，T175瓶加3ml 0.25％胰酶)消化5分钟，然后加入含10％FBS的DMEM培养基(T75瓶用9ml培养基，T175瓶用17ml培养基)中和胰酶，吹打细胞并转至50ml离心管，反复吹打混匀，然后取0.3～0.5ml的细胞悬液，计数。
3)细胞稀释：取1ml细胞悬液，用含10％FBS的DMEM培养基稀释到5×105个/ml，吹打混匀。
4)细胞接种：取2ml细胞悬液加到6孔板内。每个mRNA样品需要准备2孔平行细胞，对照组样品(GFP-mRNA)需要准备1孔细胞，空白对照1孔。将6孔板放入(37±1)℃、(5±0.5)％CO2培养箱培养过夜。
细胞转染
接种完细胞后约24小时，观察6孔板内的细胞状态，汇合度在90％左右。在生物安全柜内，配制所需体积的90％DMEM+10％FBS培养基。转染前30分钟弃掉孔板的培养基，每孔加入1ml新鲜培养基(90％DMEM+10％FBS)。
a)配制转染体系：取200μl opti-MEM，加入10μg mRNA样品(Seq1-Seq11)或阴性对照GFP-mRNA，用枪头轻轻吹打混匀，再加入60μl PEI(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1秒，充分混匀，静置10分钟。
b)将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。
c)换液
转染后6小时换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。
d)收获
转染后30小时收获。吸掉旧的培养基，用1ml PBS清洗一遍。吸掉PBS，继续用1ml PBS将细胞吹打下来，收集于1.5ml离心管中，300g离心5分钟。将离心后的上清尽量吸去干净，沉淀的细胞用于Western blot检测。
蛋白提取
1.配制细胞裂解液：0.1％Triton X-100(sigma，P/N T9284)，150mM NaCl(sigma，P/N S5886)，50mM HEPES pH8(sigma，P/N V900477)，EDTA-free protease inhibitor cocktail(sigma，P/N 11873580001)；
2.在之前收集的细胞沉淀中加入100μL 4℃预冷的细胞裂解液，重悬细胞沉淀，4℃冰浴30分钟；
3.将细胞样品置于冰盒上进行超声破碎(小美超声仪器(昆山)有限公司，XM-20MINI)1分钟：功率10W，循环模式：1秒on/1秒off；
4.超声后置于离心机中，4℃，20000rcf，离心10分钟。收集上清液。
5.BCA法测上清液蛋白浓度，BCA试剂盒(Thermo Scientific,P/N 23225)，操作步 骤参见试剂盒说明书，具体如下：A.用超纯水稀释BSA标准品，配制2000、1000、500、250、125、62.5、31.25μg/ml的标准品系列，另外加一个超纯水作为空白样品，各准备100μL。B.取10μl上清液，加入90μl超纯水混匀。C.配制1X工作试剂：取50份体积试剂A，加入1份体积试剂B，混匀。D.将样品加到96孔板，每孔25μL，做3个平行。E.每孔加入200μL工作试剂，震荡30S混匀。F.将96孔板放置37℃孵育30分钟。G.孵育结束后，等待2分钟冷却到室温。用酶标仪对562nm读板。H.吸光值平均扣除空白对照。标准品扣除空白吸光值对浓度作图，代入样品吸光值计算。
SDS-PAGE
1.待测蛋白样品制备：蛋白定量后，取50μg蛋白，用裂解液定容至20μL，加入5μL 5×Loading buffer(购自生工生物工程(上海)股份有限公司，P/N C508320)，混匀后共25μL，使用95℃孵育5分钟，结束后室温放置；
按照表1的配方配制6％SDS-PAGE分离胶和12％SDS-PAGE分离胶(1mm厚度)
表1、6％SDS-PAGE分离胶和12％SDS-PAGE分离胶配方
6％分离胶对应4％浓缩胶，12％分离胶对应5％浓缩胶，按照表2的配方配制浓缩胶。
表2、4％SDS-PAGE浓缩胶和5％SDS-PAGE浓缩胶配方
2.配制电泳缓冲液：取100ml 10×蛋白电泳缓冲液(生工，P/N C520001)，用超纯水稀释至1000ml；
3.将凝胶夹紧到电泳槽中(Bio-rad，P/N 1658004)，灌入电泳缓冲液；
4.将25μL待测蛋白样品和5μL蛋白分子量Marker(thermo，P/N 26619)分别加到样品孔中；
5.使用80V电泳30分钟；
6.然后用140V电泳，对于6％分离胶，电泳至70kDa分子量Marker跑到底部但未跑出去为止；对于12％分离胶，电泳至25kDa分子量Marker跑到底部但未跑出去为止。
Western blot
使用半干转膜仪(Pyxis，型号SPJ-1000A)进行转膜，转膜前准备配套耗材(Pyxis，P/N SPJ-T20S)，内含Top buffer，Down Buffer，Balance buffer和滤纸，另需自行裁剪与滤纸大小相同的PVDF膜(millipore，P/N IPVH00010)；将滤纸分别放入两个方形培养皿，标记好Top和Down；将20ml Top、Down buffer倒入对应的方形培养皿中，浸泡滤纸5分钟至完全浸润；将10ml Balance倒入方形培养皿，放入PVDF膜，孵育5分钟；用撬板把凝胶玻璃板撬开，切去浓缩胶，小心取出凝胶，防止撕裂；凝胶泡入超纯水的方形培养皿；取出转膜仪的转印槽，去除盖子，将Down滤纸摆好在最底层；将PVDF膜小心地覆盖Down滤纸，确认无气泡；将凝胶覆盖在PVDF膜上，确认无气泡；将Top滤纸覆盖在凝胶上，用滚轮滚动排气泡；盖好转印槽盖子，放入转膜仪；对于6％分离胶，使用11分钟进行转膜，对于12％分离胶，使用14分钟进行转膜；转膜完成后，用镊子小心取出膜，在此后，膜应避免干燥；用超纯水洗5分钟；配制TBS：取100ml 10×TBS(生工，P/N C520002)，用水稀释至1000ml；配制TBST：取1000ml TBS加入1ml Tween-20(sigma，P/N P1379)；配制5％脱脂牛奶：取2.5g脱脂奶粉(伊利脱脂奶粉)溶解于50ml TBST；去除超纯水，用TBS洗5分钟；去除TBS，用TBST洗5分钟；用5％脱脂牛奶在室温封闭1小时；6％凝胶转的膜，用于检测S蛋白，12％凝胶转的膜，用于检测GAPDH蛋白；目的蛋白一抗孵育：取3μl FGF18一抗稀释到3ml 5％脱脂牛奶中(稀释比1:1000)，置于4℃摇床过夜孵育；GAPDH蛋白一抗孵育：取0.25μL GAPDH antibody(proteintech，P/N 60004-1-Ig)稀释到5ml 5％脱脂牛奶中(稀释比1:20000)，置于4℃过夜孵育；一抗孵育结束后，去除一抗溶液，用TBST洗三次，每次5分钟；二抗孵育：取1μL HRP-conjugated Affinipure Goat Anti-Mouse IgG(H+L)(proteintech，P/N SA00001-1)稀释到5ml 5％脱脂牛奶中(稀释比1:5000)，常温孵育1小时；二抗孵育结束后，去除二抗溶液，用TBST洗三次，每次5分钟；最后将膜浸泡在TBS中；准备好显影液(thermo，P/N 34580)，把成像仪(analytikjena，型号UVP Chemstudio touch)的相机和软件打开，进行预冷；预冷完成后，取500μL显影A液和500μL显影B液混合，把膜放在托盘中，向表面倒入500μL显影混合液，反应1分钟；在成像仪中选择ECL程序进行成像，曝光时间根据情况而定；保存图片。
结果判定：结果如图4所示，供试品应呈现明显色带，GAPDH为内参蛋白，1-11 分别代表Seq1-Seq11，其中Seq1、Seq2、Seq3、Seq4、Seq5的蛋白表达量相对较高，经过筛选，最终决定选择Seq1-Seq5作为后续的试验样品进行动物实验。
实施例4
本实施例提供检测编码软骨生长因子的mRNA的免疫原性的方法。
根据本发明的编码软骨生长因子的mRNA，检测其免疫原性，评价标准为将所述mRNA对小鼠进行肌肉注射后血清中TNFα水平。
将6-8周龄的balb/c小鼠(购自北京维通利华)在SPF条件下，并且保持12小时光亮和12小时黑暗循环下的通气笼中饲养，将Seq1-Seq11所示的mRNA对balb/c小鼠进行肌肉注射，每只小鼠的注射剂量为100μg，24小时后对小鼠进行眼眶取血，分离血清。用小鼠TNFa试剂盒(RayBio)进行酶联免疫吸附分析(ELISA)。实验结果如图5所示。
小鼠注射本发明提供的软骨生长因子mRNA制剂后，TNFα表达水平低于注射FGF18蛋白的处理及对照组(CN113425855A优效组，即Seq 2-1组)，可见本发明提供的mRNA剂型中Seq1、Seq5免疫原性相比于蛋白对照组及对照组更低，安全性更好，将Seq1和Seq5进行后续的动物实验。
实施例5
本实施例研究编码软骨生长因子的mRNA修复大鼠关节软骨损伤的状况。
将6～8周龄的SD大鼠(购自北京维通利华)在SPF条件下，并且保持12小时光亮和12小时黑暗循环下的通气笼中饲养，膝关节注射单点乙酸(MIA)诱导关节软骨损伤(详细步骤参见参考文献Takahashi I,Matsuzaki T,Kuroki H,et al.Induction of osteoarthritis by injecting monosodium iodoacetate into the patellofemoral joint of an experimental rat model[J].PLoS One,2018,13(4):e0196625.)，待造模成功后(通过大鼠膝关节组织切片番红固绿染色和HE染色，观察到明显的软骨组织损伤；认为是造模成功)，将TNFα表达水平低的Seq1、Seq5所示的mRNA以及对照组(CN113425855A优效组，即Seq 2-1组)mRNA对大鼠进行关节腔注射，每只大鼠关节的注射剂量为50μl(15μg)，同时设置蛋白对照组，每只大鼠关节注射10μg相应蛋白药物(FGF18蛋白)，注射频率为每周一次，持续4周，随后对大鼠关节处进行切片和染色，观察软骨修复状况。
OARSI评分标准如下：
0正常
1表面轻微纤维化，但没有软骨损失
2关节软骨表面出现轻微裂痕；
3关节软骨表面裂痕磨损<25％；
4关节软骨表面裂痕磨损25％～50％；
5关节软骨表面裂痕磨损50％～75％；
6关节软骨表面裂痕磨损>75％。
结果如图6和图7所示，相比于蛋白药物和对照组(CN113425855A优效组，即Seq 2-1组)对骨关节软骨治疗效果，本发明的mRNA药物对骨关节炎有更加明显的治疗效果，其中Seq1、Seq5均有良好的软骨保护效果，综合比较Seq1的保护效果相对更加优秀。
实施例6
本实施例提供采用步态行为系统验证编码软骨生长因子的mRNA对造模大鼠关节的影响的方法。
将6～8周龄的Lewis大鼠(购自北京维通利华)在SPF条件下，并且保持12小时光亮和12小时黑暗循环下的通气笼中饲养，采用手术切断内侧副韧带+内侧半月板建立大鼠骨关节炎模型，具体操作如下：将模型组大鼠采用异氟烷吸入(1％～4％诱导麻醉，0.25％～2％维持麻醉)麻醉后，对大鼠关节手术部位备皮，采用碘酒消毒后，经右后肢膝关节内侧行切口，暴露膝关节腔内侧副韧带和内侧半月板，并行内侧副韧带切断和内侧半月板横断术，用0.9％氯化钠注射液溶液冲洗干净后逐层缝合关节腔。剩余12只大鼠经膝关节内侧行切口，显露内侧副韧带，但不进行切除，逐层缝合切口，作为假手术组。术后给予肌肉注射青霉素20万U，每天2次，连续3天。
手术当天计为D0，术后第7天(D7)开始给药，各给药组每次按50uL/只经右后肢膝关节注射给予相应浓度的RNA溶液，给药组包括蛋白对照组、CN113425855A中优效组Seq 2-1组、本发明Seq1组和Seq5组；正常对照组和模型对照组分别注射给予等体积的0.9％氯化钠注射液，连续给药4次，每次给药间隔1周。每周采集步态数据，分析比较大鼠后足支撑力的变化情况。
结果如图8所示，mRNA药物Seq1组和Seq5组对骨关节炎有明显的治疗效果，并且优于蛋白阳性对照组和CN113425855A筛选的RNA序列。
实施例7
本实施例提供组织病理学检查验证编码软骨生长因子的mRNA对造模大鼠关节的影响。
将6～8周龄的Lewis大鼠(购自北京维通利华)在SPF条件下，并且保持12小时光亮和12小时黑暗循环下的通气笼中饲养，采用手术切断内侧副韧带+内侧半月板建立大鼠骨关节炎模型，具体操作如下：将模型组大鼠采用异氟烷吸入(1％～4％诱导麻醉， 0.25％～2％维持麻醉)麻醉后，对大鼠关节手术部位备皮，采用碘酒消毒后，经右后肢膝关节内侧行切口，暴露膝关节腔内侧副韧带和内侧半月板，并行内侧副韧带切断和内侧半月板横断术，用0.9％氯化钠注射液溶液冲洗干净后逐层缝合关节腔。剩余12只大鼠经膝关节内侧行切口，显露内侧副韧带，但不进行切除，逐层缝合切口，作为假手术组。术后给予肌肉注射青霉素20万U，每天2次，连续3天。
手术当天计为D0，术后第7天(D7)开始给药，各给药组每次按50uL/只经右后肢膝关节注射给予相应浓度的RNA溶液，给药组包括蛋白对照组、CN113425855A中优效组Seq 2-1和本发明的Seq1组和Seq5组；正常对照组和模型对照组分别注射给予等体积的0.9％氯化钠注射液，连续给药4次，每次给药间隔1周。给药组大鼠自末次给药后第7天，经异氟烷吸入麻醉后，腹主动脉放血安乐死，采集数字DR数据，分观察各组大鼠软骨组织和滑膜组织形态学变化。
结果如图9所示，Seq1组和Seq5组的mRNA药物对骨关节炎有明显的治疗效果，并且优于蛋白阳性对照组和前期筛选的RNA序列，其中Seq1组步态分析结果最好，证明其治疗效果更加有效。
实施例8
本实施例检测本发明mRNA表达水平和半衰期。
注射本发明的编码软骨生长因子的mRNA，检测其表达产物的表达水平和半衰期，评价标准为将等量mRNA药物关节腔注射大鼠后检测关节液中的FGF18蛋白浓度。
将12周龄的SD大鼠(购自北京维通利华)在SPF条件下，并且保持12小时光亮和12小时黑暗循环下的通气笼中饲养，将Seq1和Seq5所示的mRNA、对照组(CN113425855A优效组，即Seq 2-1组)mRNA以及FGF18蛋白对大鼠进行膝关节腔注射，每只大鼠的注射剂量为250μg，蛋白用量为500μg，24小时后打开大鼠关节腔，收集关节液。通过酶联免疫吸附分析(ELISA)检测FGF18蛋白含量。实验结果如图10所示。
大鼠关节腔注射本发明提供的软骨生长因子mRNA制剂后，蛋白表达水平长时间高于注射FGF18蛋白，可见本发明提供的mRNA剂型表达量更高，表达时间也更长，血液中蛋白含量的半衰期更长。
实施例9
本实施例提供采用斑点印迹法(dot blot)检测RNA制备工艺中双链RNA残留的实验。
将供试品溶液滴加到固相载体(例如尼龙膜)上，固相载体带有正电荷，可以吸附带有负电荷的RNA。以固相载体上的双链RNA作为抗原，与对应的双链RNA抗体(称 为第一抗体)起免疫反应，使用特定的第二抗体去识别第一抗体，第二抗体带有辣根过氧化物酶(HRP)，能够在过氧化氢存在下催化鲁米诺试剂使其发光，这个过程成为显色反应，经过成像系统拍摄即可捕捉底物的信号，形成斑点印迹图，斑点的灰度值高低代表含量的高低，对比印迹图中的供试品与双链RNA标准品的灰度值高低，可以定性地检测供试品的双链RNA残留含量。具体检验步骤：1)取供试品(CN113425855A中样品、本发明中样品)适量，用无酶水稀释至含500ng/μL mRNA，即得供试品溶液；2)在超净工作台中，用洁净的剪刀剪出合适大小的尼龙膜；3)取4μL稀释后的供试品，分别滴在尼龙膜上，做2个重复；4)所有样品都滴加完毕后，在尼龙膜右下角空白处剪出倒角，用于识别正反面；将该尼龙膜风干30分钟；5)佩戴干净无酶的手套，将膜卷入50mL离心管，并确保正面朝向管内而非管壁，加入10ml PBST，缓慢上下颠倒润洗10次，倒掉PBST；6)加入10mL 5％脱脂牛奶，置于滚轴混匀仪，设置20rpm，室温孵育1小时；7)倒去脱脂牛奶，重新加入3mL脱脂牛奶和3μL J2抗体，混匀，置于滚轴混匀仪，设置20rpm，4℃孵育过夜；8)倒去离心管中的抗体溶液，加入20mL PBST，置于滚轴混匀仪，洗涤5分钟倒去PBST，重复三次；9)重新加入5mL脱脂牛奶和1μL二抗，混匀，置于滚轴混匀仪，设置20rpm，室温孵育1小时；10)去除离心管中的抗体溶液，加入20mL PBST，置于滚轴混匀仪，洗涤5分钟，重复三次；11)将显影剂AB液按1:1混合，共1mL，将尼龙膜铺平在成像仪样品盘中央，排除气泡，将AB混合液快速均匀地滴加在尼龙膜表面；12)使用智能成像仪进行化学发光的曝光保存；13)用核酸染料按1:5000稀释于TAE buffer，将膜泡入染料1分钟，取出，用智能成像仪分别在化学发光模式和核酸成像模式下分析；保存图片。
结果见图11，本发明中使用的更新的工艺，其中RNA双链残留含量更低，产品质量更优异。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (13)

  1. 一种mRNA，其可编码序列如SEQ ID No.12的氨基酸。
  2. 根据权利要求1所述的mRNA，其中，所述mRNA的编码区序列如SEQ ID No.1-SEQ ID No.11任一序列所示。
  3. 根据权利要求2所述的mRNA，其中，所述mRNA的5’端连接帽子结构和5’UTR；所述mRNA的3’端连接3’UTR和多聚A尾。
  4. 根据权利要求2所述的mRNA，其中，所述多聚A尾包括长度为90-130的多个A，多个A为连续的或中间插入1-12nt且不全部为A的连接子；
    优选地，所述多聚A尾的序列为AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID No.15)。
  5. 根据权利要求3或4所述的mRNA，其中，所述帽子结构为m7G(5')ppp(5')(2'OMeA)pG、m7G(5')ppp(5')(2'OMeA)、G(5')ppp(5')G、m7G(5')ppp(5')G、3′-O-Me-m7G(5')ppp(5')G或m7(3'OMeG)(5')ppp(5')(2'OMeA)pG。
  6. 根据权利要求3-5中任意一项所述的mRNA，其中，所述5’UTR序列为AGGGAGATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCGCCACC(SEQ ID No.16)。
  7. 根据权利要求3-6中任意一项所述的mRNA，其中，所述3’UTR序列为GCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAG(SEQ ID No.17)。
  8. 一种DNA模板，其可以转录得到权利要求1-7任一项所述的mRNA。
  9. 权利要求1-7任一项所述的mRNA的制备方法，其包括：
    将能转录权利要求1-7任一项所述mRNA的DNA片段克隆至表达质粒获得重组质粒；
    将所述重组质粒转入宿主细胞获得重组细胞，体外转录合成获得所述mRNA。
  10. 权利要求1-7任一项所述的mRNA在制备用于治疗关节软骨损伤和/或骨关节炎的药物中的应用。
  11. 一种用于治疗关节软骨损伤和/或骨关节炎的药物制剂，其包含活性成分mRNA，所述mRNA包括权利要求1-7任一项所述的mRNA中的一种或两种以上。
  12. 根据权利要求11所述的药物制剂，其中，活性成分mRNA包括编码区序列如SEQ ID No.1所示的mRNA和/或编码区序列如SEQ ID No.5所示的mRNA。
  13. 根据权利要求11或12所述的药物制剂，其为注射制剂；
    优选地，所述活性成分mRNA在药物制剂中的浓度为200～10000μg/ml；
    优选地，所述药物制剂的使用剂量，以每一关节计，为10～20μg，优选为14～16μg，最优选为15μg。
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