CN117187255B - mRNAs encoding FGF18 or rhFGF18 and use thereof in the treatment of osteoarthritis - Google Patents
mRNAs encoding FGF18 or rhFGF18 and use thereof in the treatment of osteoarthritis Download PDFInfo
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- CN117187255B CN117187255B CN202311465945.6A CN202311465945A CN117187255B CN 117187255 B CN117187255 B CN 117187255B CN 202311465945 A CN202311465945 A CN 202311465945A CN 117187255 B CN117187255 B CN 117187255B
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Abstract
The invention discloses a nucleic acid for encoding human fibroblast growth factor 18 and application thereof, wherein the sequence of the nucleic acid is shown as SEQ ID No.1 or SEQ ID No. 2. The invention improves the expression level of the human fibroblast growth factor 18 in a cell body by optimizing the nucleic acid sequence of the human fibroblast growth factor 18, and is beneficial to improving the treatment effect on OA.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a nucleic acid sequence for encoding human fibroblast growth factor 18 and application thereof in osteoarthritis treatment.
Background
Osteoarthritis (OA) is a common chronic, degenerative joint disease. Current OA clinical treatments are limited to symptomatic treatments such as weight reduction, drug analgesia (including anti-inflammatory and analgesic drugs), nutritional cartilage, and end-stage joint replacement, but none of these effectively alter the pathobiological processes of the disease. The number of new cases of OA in china exceeds ten millions each year since 2015, and the rate of disability loss healthy life Years (YLD) increases year by year since 1990. However, OA clinical treatment is limited to symptomatic treatment and end-stage joint replacement by weight reduction, drug analgesia (including anti-inflammatory and analgesic drugs), nutritional cartilage, etc., but none of these effectively alters the pathobiological processes of the disease. As the front edge of the innovative field of gene therapy, nucleic acid drugs mainly comprising siRNA and mRNA are listed in the important development field of China, and a new opportunity is provided for OA treatment.
The human fibroblast growth factor 18 (Fibroblast growth factor, FGF 18) gene encodes a protein sequence of 207 amino acids with a molecular mass of about 23kDa. 1998. In the years, japanese scientist Ohbayashi isolated FGF18 from mouse embryos for the first time. FGF18 plays an important role in skeletal growth and development, and is also involved in cortical neuronal activity, survival, differentiation and proliferation of the pituitary glands and in regulating hair growth and skin repair. Several studies in vitro and in vivo have shown that FGF18 also plays an important role in morphogenesis, angiogenesis, tumor growth and other cellular development processes. Recent studies have found that FGF18 participates in the whole life activity process by regulating the expression of FGFR, twist1 and BMP (bone morphogenic protein), FGF18 can induce proliferation of chondrocytes and promote synthesis of extracellular matrix. Currently, 3 phase II clinical trials have been completed with recombinant human fibroblast growth factor 18 (rhFGF 18), also known as Sprifermin, developed by the combination of merck and northern european biosciences. Therefore, FGF18 has potential clinical application value for treating bone diseases such as cartilage disorder, achondroplasia, bone repair and the like.
mRNA is messenger RNA, messenger ribonucleic acid. mRNA consists essentially of a 5' cap (cap for short), a 5' untranslated region (untranslated region, UTR), an open reading frame (open reading frame, ORF), a 3' untranslated region, and a Poly A tail [ Poly (A) ] structure. mRNA uses gene in cell as template, and after transcription and generation according to base complementary pairing principle, it contains base sequence correspondent to some functional fragments in DNA molecule, so that it can be used as direct template for protein biosynthesis. mRNA only accounts for 2% -5% of total RNA, but has a wide variety of metabolism, very active metabolism and extremely short half-life, and can be decomposed even within minutes after synthesis. The advantages of mRNA are expressed in: (1) The central rule shows that mRNA is synthesized through in vitro transcription, the process is simpler, and mRNA can theoretically express any protein, so that the mRNA has the potential of treating various diseases; (2) DNA serves as a genetic core and is transcribed into mRNA only into the nucleus. mRNA does not need to enter the nucleus, protein translation can be started in cytoplasm, and the efficiency is greatly higher than that of DNA; (3) mRNA can not be inserted into genome like DNA and virus vector to affect genetic information, mRNA encoding protein can be expressed instantaneously and then degraded quickly, there is no risk of gene integration, and safety is high; (4) Compared with protein and virus, mRNA can translate required protein in cells quickly, and has quick response. And the production is simple, the cost is low, and the industrial production is easy.
The application of mRNA therapeutic technology is initially limited by its high immunogenicity, low stability, production and preparation limitations, and the like. However, in recent years, with the development of in vitro transcribed mRNA (in vitro transcribed mRNA, IVT mRNA) purification and modification technology and the continuous optimization of various novel delivery systems, the stability and translation efficiency are greatly improved, and the immunogenicity is gradually controlled. mRNA treatment, as a novel therapeutic approach, is showing great potential for development and can effectively replace traditional therapies based on DNA and recombinant proteins. At present, mRNA protein supplementation therapy has entered a clinical stage, and in vivo expression of the supplementation protein is realized by delivering mRNA, so that the mRNA protein supplementation therapy is used for treating different diseases such as type II diabetes, heart failure, methylmalonic acidemia, cystic fibrosis and the like. Notably, studies have been made to use chemically modified cytokine mRNA (Chemically modified mRNA, modRNA) for OA treatment.
The native FGF18mRNA sequence is as follows:
ATGTATTCAGCGCCCTCCGCCTGCACTTGCCTGTGTTTACACTTCCTGCTGCTGTGCTTCCAGGTACAGGTGCTGGTTGCCGAGGAGAACGTGGACTTCCGCATCCACGTGGAGAACCAGACGCGGGCTCGGGACGATGTGAGCCGTAAGCAGCTGCGGCTGTACCAGCTCTACAGCCGGACCAGTGGGAAACACATCCAGGTCCTGGGCCGCAGGATCAGTGCCCGCGGCGAGGATGGGGACAAGTATGCCCAGCTCCTAGTGGAGACAGACACCTTCGGTAGTCAAGTCCGGATCAAGGGCAAGGAGACGGAATTCTACCTGTGCATGAACCGCAAAGGCAAGCTCGTGGGGAAGCCCGATGGCACCAGCAAGGAGTGTGTGTTCATCGAGAAGGTTCTGGAGAACAACTACACGGCCCTGATGTCGGCTAAGTACTCCGGCTGGTACGTGGGCTTCACCAAGAAGGGGCGGCCGCGGAAGGGCCCCAAGACCCGGGAGAACCAGCAGGACGTGCATTTCATGAAGCGCTACCCCAAGGGGCAGCCGGAGCTTCAGAAGCCCTTCAAGTACACGACGGTGACCAAGAGGTCCCGTCGGATCCGGCCCACACACCCTGCCTGA
the rhFGF18 mRNA sequence was as follows:
ATGGAAGAGAACGTTGATTTTAGGATTCACGTGGAGAACCAGACCAGGGCCAGGGACGACGTGAGCAGGAAGCAGCTGAGGCTGTACCAGCTGTACAGCAGGACCAGCGGCAAGCACATCCAGGTGCTGGGCAGGAGGATCAGCGCCAGGGGCGAGGACGGCGACAAGTACGCCCAGCTGCTGGTGGAGACCGACACCTTCGGCAGCCAGGTGAGGATCAAGGGCAAGGAGACCGAGTTCTACCTGTGCATGAACAGGAAGGGCAAGCTGGTGGGCAAGCCCGACGGCACCAGCAAGGAGTGCGTGTTCATCGAGAAGGTGCTGGAGAACAACTACACCGCCCTGATGAGCGCCAAGTACAGCGGCTGGTACGTGGGCTTCACCAAGAAGGGCAGGCCCAGGAAGGGCCCCAAGACCAGGGAGAACCAGCAGGACGTGCACTTCATGAAGAGGTACCCCAAGGGCCAGCCCGAGCTGCAGAAGCCCTTCAAGTACACCACCGTGACCAAGTGA
however, the natural FGF18mRNA and rhFGF18 mRNA have low sequence stability, short half-life and easy degradation. Therefore, how to improve the stability of the nucleic acid sequence of mRNA so that it can translate FGF18 protein remains a technical problem to be solved by the person skilled in the art.
Disclosure of Invention
For the above reasons, the present invention aims to provide nucleic acids encoding FGF18 and rhFGF18 and the use thereof in the treatment of osteoarthritis by increasing the expression levels of FGF18 and rhFGF18 proteins. In the early-stage mouse osteoarthritis model and the treatment thereof, obvious curative effect is seen, and the method has great clinical application prospect. Specifically, in order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:
one aspect of the invention relates to nucleic acids encoding FGF18 and rhFGF18, the sequences of which are shown in SEQ ID No.1 or SEQ ID No. 2. The invention also relates to a human fibroblast growth factor 18, the nucleotide sequence of which is shown as SEQ ID No.1 or SEQ ID No.2
"nucleic acid" refers to a polymer of nucleotides linked by 3',5' -phosphodiester linkages. The nucleic acid is a single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule and a hybrid molecule thereof. Examples of nucleic acid molecules include, but are not limited to, messenger RNAs (mrnas), micrornas (mirnas), small interfering RNAs (sirnas), self-amplifying RNAs (sarnas), and antisense oligonucleotides (ASOs). Nucleotides are a class of compounds consisting of three substances, purine or pyrimidine bases, ribose or deoxyribose, and phosphate. "polypeptide" or "protein" refers to a polymer of amino acids joined by peptide bonds. The amino acids include 20 kinds of natural amino acids and other unnatural amino acids. The 20 natural amino acids are glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine. The phenomenon in which the same amino acid has two or more codons is called degeneracy of the codons. Synonymous codons are typically different at base 3. The presence of synonymous codons allows the presence of multiple different nucleic acid coding sequences for a single protein or polypeptide sequence, which differ significantly in stability and protein expression efficiency in cells. The sequence design for searching the better effect is one of the key research contents of nucleic acid drug development, and the invention improves the expression level of the nucleic acid sequences for coding FGF18 and rhFGF18 in a cell body by optimizing the nucleic acid sequences, thereby being beneficial to improving the therapeutic effect on OA.
Another aspect of the invention also relates to a plasmid comprising a nucleic acid as shown in SEQ ID No.1 or SEQ ID No. 2.
In a further aspect the invention relates to a pharmaceutical composition comprising a nucleic acid or plasmid as described above. "pharmaceutical composition" refers to a composition comprising an active ingredient, which may further comprise pharmaceutically acceptable excipients and other optional therapeutic ingredients. The pharmaceutical compositions of the present invention include those suitable for oral, rectal, topical and parenteral (including subcutaneous, intramuscular, intravenous) administration. The pharmaceutical compositions of the present invention may be conveniently presented in unit dosage form well known in the art and prepared by any of the methods of manufacture well known in the pharmaceutical arts.
In a preferred embodiment of the invention, the pharmaceutical composition further comprises a phosphorylation inhibitor; preferably, the phosphorylation inhibitor is delatinib (Derazantinib).
Another aspect of the invention relates to the use of the above plasmid or nucleic acid for the preparation of a medicament for the treatment of Osteoarthritis (OA).
In another aspect, the invention also relates to a method for preparing FGF18 and rhFGF18 mRNA, comprising the steps of transfecting said plasmids into 293T cells for expression and extracting FGF18 and rhFGF18 mRNA.
Advantageous effects
The nucleic acid sequence of the present invention optimizes ORF region codon and increases 5 'UTR, 3' UTR and polyA structure, and this can raise the stability of mRNA in vitro transcription and translation and has protein expression level superior to that of natural sequence. In vitro cell experiments, the inventor of the application found that mRNA transcribed from the FGF18 and rhFGF18 coding nucleic acid sequences can be stably expressed in eukaryotic cells, and cell culture solutions (Conditioned Media, CM) after transfection of FGF18 and rhFGF18 mRNA are collected, so that the expression of FGF18 and rhFGF18 proteins can be detected. Meanwhile, the expression quantity of FGF18 and rhFGF18 is detected to be increased by extracting transfected eukaryotic cell proteins, so that FGF18 and rhFGF18 can be effectively expressed in 293T cells, FGF18 receptor FGFR3 can be phosphorylated, MAPK signal pathway is activated, ERK1/2 is phosphorylated, and biological functions are exerted. Thus, the human FGF18 and rhFGF18 encoding nucleic acid sequences are predicted to be useful in the clinical induction of chondrocyte proliferation and stimulation of extracellular matrix synthesis, as well as in the treatment of osteoarthritis and cartilage defects.
Drawings
Fig. 1: FGF18 DNA template agarose gel electrophoresis;
fig. 2: bands of FGF18 protein expression in 293T cells;
fig. 3: FGF18mRNA transfection 293T cell downstream pathway activation assay results;
fig. 4: SM102 encapsulation efficiency, particle size and PDI characterization results;
fig. 5: results of biochemical indicators of animal experiment blood, wherein fig. 5A is glutamic pyruvic transaminase and alkaline phosphatase, fig. 5B is glutamic pyruvic transaminase and lactic dehydrogenase, fig. 5C is creatine kinase and creatinine, fig. 5D is urea and uric acid, fig. 5E is white blood cell and monocyte, fig. 5F is lymphocyte and neutrophil, fig. 5G is red blood cell and hemoglobin, and fig. 5H is platelet;
fig. 6: HE staining results of pathological sections of important internal organs of animal experiments;
fig. 7: hot plate experiment results;
fig. 8: reconstructing a micro CT image of the knee joint;
fig. 9: the analysis result of the knee joint micro CT subchondral bone parameters;
fig. 10: the red O solid green dyeing result of the knee joint;
fig. 11: knee joint HE staining results;
FIG. 12; OARSI score.
Detailed Description
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1
1. Plasmid preparation:
ORF sequences SEQ ID NO.1 (NM-003862.3) and SEQ ID NO.2 encoding the same human FGF18 native protein are synthesized, the synthesized sequences are amplified by primer CGGCGCCGCCACCATGTACTCTGCTCCTTCCGCC and primer GCCCAAGGGGCAAGAAGCTAGGCCACCGAGGCTCCAGCCTATTATCAGGCGGGGTGGGTG, and the template plasmid is digested with restriction enzymes NcoI and BpmI, and the ORF sequences are inserted into the template SEQ ID NO.6 using seamless cloning. A DNA plasmid containing the ORF sequence and its flanking upstream and downstream 5 'UTR, 3' UTR regulatory sequences encoding the mRNA product FGF18 is amplified and extracted using conventional methods. The 5 'UTR, 3' UTR and polyA sequences in the upstream and downstream regulatory sequences are SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.
SEQ ID NO.1：
FGF18：
ATGTACTCTGCTCCTTCCGCCTGCACATGCCTGTGCCTGCACTTCCTGCTGCTGTGCTTCCAGGTGCAGGTGCTGGTGGCCGAGGAGAACGTGGACTTCAGGATCCACGTGGAGAACCAGACCAGGGCCAGGGACGACGTGAGCAGGAAGCAGCTGAGGCTGTACCAGCTGTACAGCAGGACCAGCGGCAAGCACATCCAGGTGCTGGGCAGGAGGATCAGCGCCAGGGGCGAGGACGGCGACAAGTACGCCCAGCTGCTGGTGGAGACCGACACCTTCGGCAGCCAGGTGAGGATCAAGGGCAAGGAGACCGAGTTCTACCTGTGCATGAACAGGAAGGGCAAGCTGGTGGGCAAGCCCGACGGCACCAGCAAGGAGTGCGTGTTCATCGAGAAGGTGCTGGAGAACAACTACACCGCCCTGATGAGCGCCAAGTACAGCGGCTGGTACGTGGGCTTCACCAAGAAGGGCAGGCCCAGGAAGGGCCCCAAGACCAGGGAGAACCAGCAGGACGTGCACTTCATGAAGAGGTACCCCAAGGGCCAGCCCGAGCTGCAGAAGCCCTTCAAGTACACCACCGTGACCAAGAGGAGCAGGAGGATCAGGCCCACCCACCCCGCCTGA
SEQ ID NO.2：
rhFGF18：
ATGGAAGAGAACGTTGATTTTAGGATTCACGTGGAGAACCAGACCAGGGCCAGGGACGACGTGAGCAGGAAGCAGCTGAGGCTGTACCAGCTGTACAGCAGGACCAGCGGCAAGCACATCCAGGTGCTGGGCAGGAGGATCAGCGCCAGGGGCGAGGACGGCGACAAGTACGCCCAGCTGCTGGTGGAGACCGACACCTTCGGCAGCCAGGTGAGGATCAAGGGCAAGGAGACCGAGTTCTACCTGTGCATGAACAGGAAGGGCAAGCTGGTGGGCAAGCCCGACGGCACCAGCAAGGAGTGCGTGTTCATCGAGAAGGTGCTGGAGAACAACTACACCGCCCTGATGAGCGCCAAGTACAGCGGCTGGTACGTGGGCTTCACCAAGAAGGGCAGGCCCAGGAAGGGCCCCAAGACCAGGGAGAACCAGCAGGACGTGCACTTCATGAAGAGGTACCCCAAGGGCCAGCCCGAGCTGCAGAAGCCCTTCAAGTACACCACCGTGACCAAGTGA
SEQ ID NO.3：
GGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACC
SEQ ID NO.4：
TCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCA
SEQ ID NO.5：
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO.6
CGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACATTATTCAGATTGGGCTTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGTAATACGACTCACTATAAGGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGACCCCGGCGCCGCCACCATGGTGCCCAAGAAGAAGAGGAAAGTCTCCAACCTGCTGACTGTGCACCAAAACCTGCCTGCCCTCCCTGTGGATGCCACCTCTGATGAAGTCAGGAAGAACCTGATGGACATGTTCAGGGACAGGCAGGCCTTCTCTGAACACACCTGGAAGATGCTCCTGTCTGTGTGCAGATCCTGGGCTGCCTGGTGCAAGCTGAACAACAGGAAATGGTTCCCTGCTGAACCTGAGGATGTGAGGGACTACCTCCTGTACCTGCAAGCCAGAGGCCTGGCTGTGAAGACCATCCAACAGCACCTGGGCCAGCTCAACATGCTGCACAGGAGATCTGGCCTGCCTCGCCCTTCTGACTCCAATGCTGTGTCCCTGGTGATGAGGAGAATCAGAAAGGAGAATGTGGATGCTGGGGAGAGAGCCAAGCAGGCCCTGGCCTTTGAACGCACTGACTTTGACCAAGTCAGATCCCTGATGGAGAACTCTGACAGATGCCAGGACATCAGGAACCTGGCCTTCCTGGGCATTGCCTACAACACCCTGCTGCGCATTGCCGAAATTGCCAGAATCAGAGTGAAGGACATCTCCCGCACCGATGGTGGGAGAATGCTGATCCACATTGGCAGGACCAAGACCCTGGTGTCCACAGCTGGTGTGGAGAAGGCCCTGTCCCTGGGGGTTACCAAGCTGGTGGAGAGATGGATCTCTGTGTCTGGTGTGGCTGATGACCCCAACAACTACCTGTTCTGCCGGGTCAGAAAGAATGGTGTGGCTGCCCCTTCTGCCACCTCCCAACTGTCCACCCGGGCCCTGGAAGGGATCTTTGAGGCCACCCACCGCCTGATCTATGGTGCCAAGGATGACTCTGGGCAGAGATACCTGGCCTGGTCTGGCCACTCTGCCAGAGTGGGTGCTGCCAGGGACATGGCCAGGGCTGGTGTGTCCATCCCTGAAATCATGCAGGCTGGTGGCTGGACCAATGTGAACATTGTGATGAACTACATCAGAAACCTGGACTCTGAGACTGGGGCCATGGTGAGGCTGCTCGAGGATGGGGACTACCCATATGATGTGCCCGACTATGCTTGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGATGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCG
mRNA preparation:
2.1 restriction enzyme digestion of plasmid DNA
Vortex mixing, centrifuge instantaneous separation for 10s, and reaction for 3h in 37 deg.C metal bath. If the amount of plasmid DNA added varies, the amounts of other additives are adjusted accordingly. After linearizing the DNA plasmid containing FGF18 coding sequence, enzyme cutting is carried out, and agarose gel electrophoresis detection shows that the band is bright, single and has no tail and good integrity.
2.2 purification of plasmid DNA products
Product purification using the vazyme kit, cat: L/N7F612L2
(1) After the end of the DNA electrophoresis, the gel containing the target DNA fragment is rapidly cut under an ultraviolet lamp, and it is recommended to suck out the gel surface liquid with a paper towel and cut up, and to remove the excess gel as much as possible. Weigh the gel (remove empty tube weight), 100mg gel equivalent to 100ul volumes as one gel volume.
(2) Equal volumes of Buffer GDP were added. And (3) carrying out water bath for 7-10 min at 50-55 ℃ and properly adjusting the time according to the size of the gel to ensure that the gel block is completely dissolved. The sol is accelerated by reversing and mixing for 2 times during the water bath.
(3) The droplets on the walls of the tube were collected by brief centrifugation. Placing FastPure DNA Mini Columns-G adsorption column on Collection Tubes
In a 2ml collection tube, transferring the sol solution of 700.ltoreq. 700 ul to an adsorption column, centrifuging at 12,000 rpm (13,800×9) for 30-60 sec if the sol volume is >700 ul, placing the adsorption column in a recovery header, transferring the rest sol solution to the adsorption column, and centrifuging at 12,000 rpm (13,800×g) for 30-60 sec.
(4) The filtrate was discarded and the column was placed in a collection tube. 300 ul Buffer GDP was added to the column. Standing for 1min. Centrifugation at 12,000 rpm (13,800 Xg) for 30-60 sec.
(5) The filtrate was discarded and the column was placed in a collection tube. 700 ul Butfer GW (absolute ethanol has been added) was added to the column. Centrifugation is carried out at 12,000 rom (13,800 Xg) for 30-60 sec.
(6) And (5) repeating the step 5.
(7) The filtrate was discarded and the column was placed in a recovery header. Centrifuge at 12,000 rpm (13,800 Xg) for 2 min.
(8) The column was placed in a 1.5ml sterilized centrifuge tube, 20-30 g ul Elution Buffer g was added to the center of the column, and left for 2 min. Centrifuge at 12,000 rpm (13,800 Xg) for 1min. The column was removed and the DNA was stored at-20 ℃.
2.3 in vitro transcription
In vitro transcription kit using vazyme, cat: DD4202-01. Cap analogs CAG primer (100 mM), cat No.: DD4118-PC-00.
(1) 10 XIVT Buffer was thawed at room temperature, 100mM NTP and CAG Trimer were thawed on ice, and T7 Enzyme Mix was placed in ice bin.
(2) Clean PCR tubes were taken and the following components were added sequentially to the tubes (20. Mu.l for example).
(3) Vortex mixing for 5 seconds, centrifuging for 10 seconds by a centrifuge, and placing in a constant temperature mixing instrument for incubation for 2-3 hours at 37 ℃.
(4) After the reaction was completed, 1. Mu.l DNaseI was added and incubated at 37℃for 15 minutes to remove the linearized template.
2.4 purification of mRNA by magnetic beads
(1) Taking out the RNA clear Beads from 2-8 ℃ in advance of 30min, balancing to room temperature, and reversing or vortex oscillating to fully and uniformly mix the magnetic Beads.
(2) 36 mu l RNA Clean Beads was added to the mixture after IVT.
(3) The mixture was blown with a pipette to mix thoroughly.
(4) The RNA was bound to the beads by incubation at room temperature for 5 min.
(5) The sample was placed on a magnetic rack for 5min and after the solution was clear, the supernatant was carefully removed.
(6) The samples were kept in the magnet holder all the time, and the beads were rinsed by adding 200. Mu.l of freshly prepared 80% ethanol and incubated at room temperature for 30s.
(7) The supernatant was removed and the previous step was repeated for a total of 2 rinses.
(8) The sample is kept in the magnetic rack all the time, and the magnetic beads are air-dried for 5-10 min after being uncapped.
(9) Taking out the sample from the magnetic rack, adding a proper volume of water without the nuclease, blowing for 10 times by using a pipette to fully mix, and standing for 5min at room temperature.
(10) The sample was placed on a magnetic rack for 5min, after which the solution was clarified, the supernatant was carefully transferred to a new centrifuge tube. mRNA concentration and OD detection Using NanoDrop 260/280 Is marked and stored in a refrigerator at-80 ℃.
mRNA in vitro transfection
3.1 Cell plating
293T cells were resuscitated and subcultured using DMEM medium containing 10% FBS and 1% P/S (hereinafter abbreviated as D10 medium). Selecting 293T cells with good growth state, digesting for 1min at 37deg.C with pancreatin, adding D10 culture medium, stopping digestion, centrifuging at 1000 rpm for 5min to obtain the final productThe cells were resuspended in D10 medium to adjust the cell density to 1X 10 6 Each of 2 mL/well was inoculated into 6-well cell culture plates, wherein the sample group (FGF 18 mRNA) and the blank group (idler) were each 3 replicates.
3.2 mRNA preparation
FGF18mRNA was diluted to 2 ug/. Mu.L with RNase-free water.
(1) mRNA dilution
mu.L of Opti-MEM was added to a centrifuge tube by taking 2ug of FGF18mRNA per well TM (Gibco) Medium, was thoroughly mixed.
(2) Lipofectamine RNAiMAX diluent
Taking 9 mu L Lipofectamine RNAiMAX transfection reagent from each hole, adding 150 mu L of Opti-MEM culture medium, and fully and uniformly mixing;
(3) Preparation of transfection complexes
And adding the mRNA diluent into the Lip 2000 diluent, fully and uniformly mixing, and standing at room temperature for 10 min.
3.3 mRNA transfection
To the transfection complex, 700. Mu.L of Opti-MEM medium was added, and the medium in the original cell culture plate was removed by pipetting, and 1mL of the prepared transfection complex mixture was added to each well.
3.4 Liquid exchange
After transfection of 4h, the cell status was observed and the medium in the cell culture plate was replaced with 2mL of complete medium.
3.5 Collecting a sample
3.5.1 293T cells were transfected with FGF18mRNA native and optimized sequences, respectively, CM collection after transfection:
FGF18mRNA transfected 293T cells with Lipofectamine RNAiMAX;
FGF18 starts to express after transfection for 6 hours, at the moment, the culture medium is replaced by a DMEM high-sugar culture medium for culture, the culture medium is collected after 6 hours, 12 hours, 24 hours and 48 hours after the culture medium is replaced, centrifugation is carried out at 3000 RPM for 5 minutes, and the supernatant is collected and stored at the temperature of minus 80 ℃;
FGF18 protein content detection in CM:
ELISA assays were performed on the collected supernatants, and FGF18 content in CM was measured as shown in Table 2.
Table 2: FGF18 protein content in CM
Extracting cell proteins for Western Blot experiments, and performing protein cleavage, protein denaturation, gel configuration, sample loading, electrophoresis, membrane transfer, rinsing, sealing, primary antibody incubation, secondary antibody incubation and chromogenic exposure to obtain the results shown in figure 2
From table 2, the results of fig. 2 show that FGF18mRNA is expressed in 293T cells and can be secreted into CM and demonstrate that the optimized sequences of the present invention have higher stability.
3.5.2 Collection of proteins after transfection of 293T cell FGF18mRNA
1) Sucking culture medium/culture solution of six-hole plate, and washing 3 times with PBS;
2) Adding 100-150uLRIPA lysate into each hole, and performing ice lysis for 15-30min, and shaking frequently to allow cells to be fully lysed;
3) Cells were scraped using a spatula and transferred to a 1.5mL/2mL EP tube;
4) Ultrasonic disruption of cells;
5) Pre-cooling 4 oC,12000rmp,10min by a centrifugal machine in advance, and taking a supernatant;
6) Adding Loading buffer, and heating at 95deg.C for 10min to denature protein.
7) BCA quantification, protein concentration was all matched to 2ug/ul.
4.293T cell downstream pathway activation assay
1) 293T cells were seeded in 3 6-well plates at a density of 1X 10 6 Culturing 293T cells at 37 ℃ under the condition of 5% CO2 after inoculating, and culturing for 24 hours by changing a culture medium into a DMEM high-sugar culture medium for later use;
2) The sample addition layout is detailed as follows: wherein the negative control group and the sample group (natural FGF18mRNA, natural FGF18 mRNA+phosphorylation inhibitor, sequence-optimized FGF18mRNA, sequence-optimized FGF18 mRNA+phosphorylation inhibitor, mRNA is 2 ug/hole, derazantinib concentration is 10 uM) are 3 repeats, and Derazantinib is the phosphorylation inhibitor;
cell resuspension: cell concentration was 1X 10 by re-suspension counting after pancreatin digestion after 24h of cell treatment 6 Per mL, 100uL of resuspended cells per tube for all groups of samples;
after cleavage of proteins, protein denaturation, gel configuration, loading, electrophoresis, transfer, rinsing, blocking, primary antibody incubation, secondary antibody incubation and chromogenic exposure, the results are shown in fig. 3:
the results of FIG. 3 demonstrate that following sequence optimized FGF18mRNA transfection into 293T cells, the downstream MAPK pathway is significantly more activated than in the control and natural FGF18mRNA groups, allowing ERK1/2 phosphorylation, and ERK1/2 phosphorylation is inhibited after the addition of the phosphorylation inhibitor Derazantinib. Meanwhile, after the FGF18mRNA subjected to sequence optimization is transfected for 293T, the intracellular expression quantity of the FGF18 protein is obviously increased, the FGF18 protein and the FGF18mRNA can activate a downstream ERK channel, the translated FGF18 protein can be identified by the same antibody, and a strip appears at the same position, so that the molecular weight and the function of the FGF18 protein are proved to be the same.
5. ICR mice joint cavity injection FGF18mRNA for osteoarthritis in vivo experiments
5.1 Preparation of FGF18 mRNA-LNP and rhFGF18 mRNA-LNP
The prepared cationic compounds SM102, DSPC, cholesterol, DMG-PEG2000 were formulated as ethanol phase with ethanol in a molar ratio of 50:10:38.5:1.5, and FGF18mRNA and rhFGF18 mRNA were formulated as aqueous phase with 50 mM citric acid buffer solution at ph=4. Mixing according to water phase and ethanol phase=3:1 (volume ratio) by microfluidic technology to obtain liposome nano particles, and then dialyzing and purifying in PBS to remove ethanol.
After the preparation is finished, the encapsulation efficiency and the particle size of the FGF18 mRNA-LNP and the rhFGF18 mRNA-LNP are detected, so that the encapsulation efficiency of the FGF18 mRNA-LNP and the rhFGF18 mRNA-LNP is high and can reach more than 80%, the particle size is stable, and the uniformity is good (shown in figure 4).
5.2 Establishing ICR mouse osteoarthritis model
Wild type 6-8 week old male ICR mice were selected from 6 groups including a sham operation group, a physiological saline group, an FGF18 mRNA-LNP group, an rhFGF18 mRNA-LNP group, a luciferase mRNA-LNP group, and an rhFGF18 protein group. 6 in each group, a hindlimb bilateral sham surgery (sham surgery group) and an ACLT surgery were performed, respectively.
ACLT surgery: the back skin was anesthetized, the skin, subcutaneous tissue and joint capsule were incised, the anterior cruciate ligament was exposed and severed, and after knee examination confirmed that the anterior drawer experiment was positive, joint capsule and skin were sutured layer by layer.
False operation: the back skin is anesthetized, the skin and the joint capsule are incised, the anterior cruciate ligament is not cut off, and the joint capsule and the skin are sutured layer by layer.
One week after the operation, the physiological saline group, the FGF18 mRNA-LNP group, the rhFGF18 mRNA-LNP group, the luciferase mRNA-LNP group and the rhFGF18 protein group were injected, and 20ul of physiological saline or FGF18 mRNA-LNP, rhFGF18 mRNA-LNP, luciferase mRNA-LNP and rhFGF18 protein were injected into each knee joint of each mouse once a week for 3 times. Each group was randomly selected for 3 of 4 weeks and 8 weeks for euthanasia and further follow-up.
5.3 Results of treatment of ICR mice with FGF18 mRNA-LNP and rhFGF18 mRNA-LNP
Mice in the physiological saline group, FGF18 mRNA-LNP group, rhFGF18 mRNA-LNP group, luciferase mRNA-LNP group, and rhFGF18 protein group were injected into the knee joint space at a dose of 20ul.
5.3.1 Toxicity assessment
The mice were euthanized at 4w and 8w, respectively, blood was collected from inner canthus after molding, blood was collected from the mice, blood biochemical indicators (fig. 5A to 5H) corresponding to organ functions (heart function, liver function, kidney function, etc.) of the mice were detected, and important organs (heart, liver, spleen, lung, kidney, brain, etc.) were collected for pathological section, HE staining analysis (fig. 6), and each data was compared with a control group to evaluate whether there was a potential side effect of LNP. The detection results show that the four groups of hematological and pathological section indexes of the FGF18 mRNA-LNP group, the rhFGF18 mRNA-LNP group, the luciferase mRNA-LNP group and the rhFGF18 protein group are not abnormal.
5.3.2 Behavioural assessment
Hot plate experiments were used to detect the pain level of the knee joint of the experimental mice at 4w and 8w after molding, respectively. The study was tested by placing each group of treated mice on a hotplate (55 ℃). Timing was started when four mice were exposed to the plate, and the response time for hind limbs to develop the following actions as response time for pain in mice, such as shaking, jumping, licking, etc. If the above reaction has not occurred for more than 60 seconds, the mice are immediately removed to prevent scalding the hind limbs. Each mouse was measured three times with 15 minutes intervals therebetween, and the average value was used as the response time of the mouse. The tester is not informed of the experimental group. The results of the tests are shown in FIG. 7, and the pain response time of FGF18 mRNA-LNP group, rhFGF18 mRNA-LNP group and rhFGF18 protein group is long, which indicates that the knee joint pain condition of mice is improved compared with the control group.
5.3.3 Imaging assessment
The micro-CT is adopted in the study to detect the formation of subchondral bone and osteophyte of knee joint of an experimental mouse. The knee joints of each group of experimental mice are taken out entirely for standby (without fixation treatment, can be stored at-80 ℃) and each group of specimens is scanned by micro-CT. micro-CT images were reconstructed three-dimensionally using Mimics Research software. Semi-quantitative analysis was performed using Inveon Research Workplace software, and the measured data included relative bone volume (trabecular bone volume per total volume, BV/TV) and bone trabecular pattern factor (trabecular bone pattern factor, tb. Pf). As shown in fig. 8, FGF18 mRNA-LNP group and rhFGF18 mRNA-LNP group showed reduced osteophyte formation, increased BV/TV, and decreased Tb. Pf (fig. 9) compared to the control group, suggesting an osteoarthritis treatment effect.
5.3.4 Histological evaluation
I safranine fast green dyeing
(1) Dewaxing: xylene substitute I15 min → xylene substitute II 15 min → absolute ethanol 5min → 95% ethanol 5min → 80% ethanol 5min → 70% ethanol 5min → PBS solution rinse 2 min.
(2) Dyeing: dip-dyeing in solid green dyeing liquid for 5 minutes, rinsing with PBS solution to remove floating color for 10 seconds, washing the slice with weak acid solution for 10-15 seconds, so as to remove residual solid green, rinsing with PBS solution for 10 seconds, dip-dyeing in safranin O dyeing liquid for 5 minutes, rinsing with PBS solution to remove floating color for 10 seconds.
(3) Dehydrating: 95% ethanol treatment for 30 seconds, absolute ethanol treatment for 30 seconds.
(4) And (3) transparency: rapidly transferred to xylene substitute solution for 1 minute.
(5) Sealing piece: taking out, airing in the air, then dripping a proper amount of neutral resin on the glass slide, and covering the glass slide with a cover glass sealing piece.
The staining results are shown in FIG. 10, with increased FGF18 mRNA-LNP group and rhFGF18 mRNA-LNP histone polysaccharide content and decreased OA phenotype.
II HE staining
(1) Dewaxing: xylene substitute I15 min → xylene substitute II 15 min → absolute ethanol 5min → 95% ethanol 5min → 80% ethanol 5min → 70% ethanol 5min → PBS solution rinse 2 min.
(2) The paraffin section is dyed with hematoxylin for 0.5 to 1min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, rinsed with tap water, then returned with 1% aqueous ammonia solution for 1min, rinsed with running water for several seconds, and dyed with eosin dye solution for several seconds, and rinsed with running water.
(3) Dehydrating: 95% ethanol treatment for 30 seconds, absolute ethanol treatment for 30 seconds.
(4) And (3) transparency: rapidly transferred to xylene substitute solution for 1 minute.
(5) Sealing piece: taking out, airing in the air, then dripping a proper amount of neutral resin on the glass slide, and covering the glass slide with a cover glass sealing piece.
The staining results are shown in FIG. 11, in which the FGF18 mRNA-LNP group and rhFGF18 mRNA-LNP group had a large number of chondrocytes and fewer hypertrophic chondrocytes.
OARSI scores (fig. 12) showed that the OA phenotypes of FGF18 mRNA-LNP group and rhFGF18 mRNA-LNP group were reduced.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (14)
1. An mRNA encoding human fibroblast growth factor 18, wherein the nucleotide sequence of said mRNA is shown in SEQ ID No. 1.
2. An mRNA encoding recombinant human fibroblast growth factor 18, wherein the nucleotide sequence of said mRNA is shown in SEQ ID No. 2.
3. A plasmid comprising the nucleotide sequence set forth in SEQ ID No. 1.
4. A plasmid comprising the nucleotide sequence shown in SEQ ID No. 2.
5. A human fibroblast growth factor 18 has a nucleotide sequence shown in SEQ ID No. 1.
6. A recombinant human fibroblast growth factor 18 has a nucleotide sequence shown in SEQ ID No. 2.
7. A pharmaceutical composition comprising the mRNA of claim 1 or 2 or the plasmid of claim 3 or 4.
8. The pharmaceutical composition of claim 7, further comprising a phosphorylation inhibitor.
9. The pharmaceutical composition of claim 8, wherein the phosphorylation inhibitor is delatinib.
10. Use of the plasmid of claim 3 or 4 for the preparation of a medicament for the treatment of osteoarthritis.
11. Use of an mRNA according to claim 1 or 2 for the preparation of a medicament for the treatment of osteoarthritis.
12. Use of a pharmaceutical composition according to claim 7 or 8 for the preparation of a medicament for the treatment of osteoarthritis.
13. A method of producing human fibroblast growth factor 18 comprising the step of transfecting the plasmid of claim 3 into 293T cells for expression and the step of extracting human fibroblast growth factor 18.
14. A method of producing recombinant human fibroblast growth factor 18 comprising the steps of transfecting the plasmid of claim 4 into 293T cells for expression and extracting recombinant human fibroblast growth factor 18.
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