CN114788876B - MRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes and preparation method and application thereof - Google Patents

MRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes and preparation method and application thereof Download PDF

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CN114788876B
CN114788876B CN202210176697.2A CN202210176697A CN114788876B CN 114788876 B CN114788876 B CN 114788876B CN 202210176697 A CN202210176697 A CN 202210176697A CN 114788876 B CN114788876 B CN 114788876B
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mrna
glp
lipid
preparation
receptor agonist
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CN114788876A (en
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郭立新
肖飞
潘琦
苏斐
王晓霞
张兰馨
邹丽辉
李贺鑫
吴萌
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Beijing Hospital
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Beijing Hospital
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Abstract

The invention relates to the technical field of biological medicine, in particular to an mRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes, a preparation method and application thereof. The fusion mRNA expressing the GLP-1 polypeptide receptor agonist and the Fc segment of the antibody is delivered into a body, and the GLP-1 polypeptide receptor agonist is autonomously expressed by human cells. The autonomous expressed GLP-1 polypeptide receptor agonist can be secreted outside cells to stimulate insulin secretion, thereby achieving the effect of treating diabetes. The pharmaceutical preparation provided by the invention has the advantages of rapid and simple method, high expression quantity, long time, high bioavailability, low immunogenicity, short drug onset time and long duration, can rapidly stimulate insulin production, and improves the compliance and comfort of patient administration.

Description

MRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes and preparation method and application thereof
Technical Field
The invention relates to the technical field of biological medicine, in particular to an mRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes, a preparation method and application thereof.
Background
Diabetes is one of three major chronic non-infectious diseases that threatens human life and develops when the pancreas does not produce enough insulin or the human body cannot effectively utilize the produced insulin. Diabetes can be further classified into type I diabetes and type II diabetes depending on whether insulin is relied on. In China, more than 90% of diabetics are type II diabetics. Type II diabetes, also known as non-insulin dependent diabetes mellitus, is caused by the inability of the human body to effectively utilize insulin, i.e., insulin resistance, or insulin compensatory hyposecretion.
GLP-1, which is collectively referred to as glucagon-like peptide-1, is a peptide-like hormone encoded by the human glucagon gene and secreted by intestinal L cells. GLP-1 can act on beta receptors on islet beta cells in a glucose-dependent manner, thereby promoting transcription of insulin genes and increasing insulin biology and synthesis and secretion. Meanwhile, GLP-1 can also stimulate proliferation and differentiation of beta cells, inhibit apoptosis of beta cells, further increase the quantity of islet beta cells, inhibit secretion of glucagon, inhibit appetite and ingestion, delay emptying of gastric contents, and be favorable for reducing postprandial blood sugar and stabilizing blood sugar level. The GLP-1 receptor agonist with biological activity in an organism is mainly GLP-1, but is easily hydrolyzed by dipeptidyl peptidase, and the half-life period is less than 5 minutes, so that the GLP-1 receptor agonist with long half-life period is one of the development directions of the existing diabetes medicines.
MRNA is also known as messenger RNA and is transcribed from one strand of DNA as a template. mRNA carries genetic information and can direct the synthesis of protein. As one of gene therapies, mRNA has a series of advantages over DNA: firstly, when mRNA enters cytoplasm, the translation process of protein can be started, and the efficiency is far higher than that of DNA; secondly, mRNA can not be inserted into a genome, only the encoded protein can be expressed transiently, and the safety is high; finally, mRNA can be formed by an in vitro transcription process, which is relatively inexpensive and can be rapidly applied to a variety of therapies. Meanwhile, in theory, mRNA can express any protein, so almost all diseases can be treated, and from the perspective of pharmaceutical industry, mRNA is a potential candidate drug, and can meet the related requirements of gene individuation treatment, tumor individuation treatment, vaccine and the like.
MRNA nanoliposome formulations have been successfully used in the preparation of partial vaccines, and there are reports disclosing modified polynucleotides for use in the treatment of otic diseases and disorders, comprising administering to a subject a pharmaceutical composition comprising a polynucleotide encoding at least one otic polypeptide, and wherein the polynucleotide is formulated in a pharmaceutically acceptable carrier or excipient. Although mRNA was constructed, it was not demonstrated that the preparation of mRNA nanolipids was effective in treating disease. Also disclosed is Sub>A polynucleotide encoding Sub>A Low Density Lipoprotein Receptor (LDLR), wherein the LDLR comprises at least one mutation in Sub>A domain selected from the group consisting of an EGF-Sub>A domain, an intracellular domain, and both an EGF domain and an intracellular domain, wherein the polynucleotide comprises (Sub>A) Sub>A first region of linked nucleosides, said first region encoding Sub>A polypeptide of interest for reducing cholesterol levels in Sub>A patient. Some studies disclose personalized vaccines for cancer comprising recombinant polypeptides comprising mutation-based neoepitopes produced by cancer-specific somatic mutation in tumor samples of cancer patients or nucleic acids encoding the polypeptides.
However, current mRNA preparations are still focused on anti-tumor studies, and there is little concern about mRNA preparations for the treatment of diabetes. Since mRNA formulations still present challenges such as unstable properties, chemical degradation of RNA in vitro mainly involves hydrolysis and oxidation, hydrolysis mainly occurring in the phosphodiester linkages of the mRNA molecular backbone. Naked mRNA is also highly susceptible to degradation and inactivation in vivo, and difficulties in delivering and efficiently expressing therapeutic amounts of protein in vivo, as well as possible side effects, are currently a serious challenge. Thus, there are positive clinical values to provide an mRNA formulation that can be used to treat other than viral vaccines, but there are challenges.
Disclosure of Invention
Accordingly, the present invention is directed to a pharmaceutical preparation of mRNA for treating diabetes, a preparation method and application thereof. The medicine for treating diabetes is an mRNA medicine, the invention utilizes a delivery carrier to deliver mRNA encoding polypeptide GLP-1 receptor agonist into a human body, and human cells are utilized to express GLP-1 receptor agonist polypeptide, then the polypeptide is further secreted outside the cells of the human body, GLP-1 receptors on the surfaces of islet beta cells are activated, insulin is secreted, and the purpose of treating diabetes is achieved.
The present invention provides a pharmaceutical formulation of an mRNA comprising a nucleic acid encoding a GLP-1 receptor agonist and a nucleic acid encoding an Fc region of an IgG antibody, and a delivery lipid.
In the mRNA pharmaceutical formulation of the present invention, the GLP-1 receptor agonist is selected from any one of the following:
i) At least one of the polypeptides having the amino acid sequences shown in any one of SEQ ID Nos. 1 to 13;
II) a polypeptide having at least 60% identity to the polypeptide of I);
III), a polypeptide having one or more amino acids substituted, deleted and/or inserted in the amino acid sequence of the polypeptide of I);
The nucleotide sequence of the coding mRNA is a nucleotide sequence optimized by human codons. In some embodiments, the nucleic acid sequence encoding a GLP-1 receptor agonist is selected from any one of the following i) to iii):
i) At least one of the nucleic acid sequences as set forth in any one of SEQ ID Nos. 14 to 26;
ii) a sequence having at least 60% identity to the sequence of i);
iii) A sequence of one or more nucleotides substituted, deleted and/or inserted in said sequence of i);
the Fc sequence of the antibody IgG is selected from Fc segments of heavy chains of antibodies IgG1, igG2, igG3 and IgG4, and comprises a hinge region, a CH2 region and a CH3 region.
In the mRNA pharmaceutical preparation of the invention, the Fc region of the IgG antibody is any one of A) to C):
A) An Fc region of an IgG1 antibody, an Fc region of an IgG2 antibody, an Fc region of an IgG3 antibody, or an Fc region of an IgG4 antibody;
B) A fragment having at least 60% identity to a);
c) A polypeptide having one or more amino acids substituted, deleted and/or inserted in the Fc region of a).
The nucleotide sequence of the coding mRNA is a nucleotide sequence optimized by human codons. In some embodiments, the nucleic acid sequence encoding an Fc region of an IgG antibody is selected from any one of a) to c):
a) At least one of the nucleic acid sequences as set forth in any one of SEQ ID Nos. 27 to 30;
b) A sequence having at least 60% identity to the sequence of a);
c) A sequence of one or more nucleotides substituted, deleted and/or inserted in said sequence of a).
The invention provides a pharmaceutical preparation, wherein mRNA (messenger ribonucleic acid) sequentially comprises the following components from a 5 'end to a 3' end: 5' -CAP, 5' -UTR, signalpeptide, nucleic acid encoding a GLP-1 receptor agonist, linker, nucleic acid encoding an Fc region of an IgG antibody, 3' -UTR and PolyA.
The linker in the pharmaceutical preparation is selected from any one of ①～③: .
① . The amino acid sequence of the linker is GGGGSGGGGSGGGGSA, GSAGSAAGSGEF, GGGGGGGG, EGKSSGSGSESKST or KESGSVSSEQLAQFRSLD;
② . A polypeptide having at least 70% identity to the amino acid sequence of ① linker;
③ . A polypeptide having one or more amino acids substituted, deleted and/or inserted in the sequence of amino acids ①.
In the present invention, a sequence having at least 60% identity refers to a sequence having 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to said sequence.
The sequence of one or more nucleotide substitutions, deletions and/or insertions in the sequence encodes a protein that has the same or similar function as the protein encoded by the prosequence.
In the pharmaceutical formulation provided by the present invention, the 5'CAP is selected from Cap0 (m 7 Gppp), cap1 (m 7 GpppmN), cap2 (m 7 GpppmNmN) or the anti-reverse Cap analogue ARCA (3' -O-Me-m7G (5 ') ppp (5') G).
In the pharmaceutical preparation provided by the invention, the length of the tail of the Poly A is 20-200 thymidines, preferably 100, 120, 150 and 160.
In some embodiments, the amino acid sequence of the signal peptide in the mRNA in the pharmaceutical preparation of the invention is shown in SEQ ID No. 31.
The delivery vehicle in the pharmaceutical formulation of the invention may be selected from the group consisting of non-viral delivery vehicles selected from the group consisting of ionizable liposomes, cationic polymers, lipid nanoparticles, and viral delivery vehicles. Delivery of the active ingredient mRNA of the present invention may also include use of protamine, albumin, or use of viral delivery vectors including lentiviral vectors, adenoviral vectors, retroviral vectors, adeno-associated viral vectors. In an embodiment of the invention, the delivery vehicle is a cationic liposome or a lipid nanoparticle.
In some embodiments, the delivery lipids provided herein consist of cationic lipids, PEG-lipid, ampholytic phospholipids, and cholesterol:
The cationic lipid is selected from: 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), (2, 3-dioleoyl-propyl) -trimethylamine, N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), 2, 3-dioleyloxy-N- [2 (spermine-carboxamide) ethyl ] -N, N-dimethyl-1-propanammonium trifluoroacetate (DOSPA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), dioctadecylaminoglycyl spermine (DOGS), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybut-4-yloxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), preferably 3- (N, N-dioleylamino) -1, 2-propanediol, N- (1- (2), 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride, 1, 2-dioleyloxy-N, N-dimethylaminopropane. Wherein the cationic lipid comprises 45% -55%, preferably 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53% or 54% of the delivered lipid, by molar ratio;
The PEG-lipid is selected from: one or more of PEG-phospholipid, PEG-dilauryloxypropyl, PEG-dialkoxypropyl, and PEG-diacylglycerol, preferably PEG-diacylglycerol, and PEG-phospholipid. Wherein PEG-lipid comprises 1% -2%, preferably 1.2%, 1.5% or 1.8% of said delivery lipid, in terms of molar ratio;
the ampholytic phospholipids are selected from: egg yolk phosphatidylcholine (EPC), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dimyristoyl-phosphatidylethanolamine (DMPE), ditolyl-phosphatidylethanolamine (DEPE). Preferably one or a combination of two or more of distearoyl-phosphatidylethanolamine, dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine. In molar ratio, wherein the amphipathic lipid comprises 9% to 11%, preferably 9.5%, 10% or 10.5% of the delivered lipid.
The cholesterol content of the delivery lipid is 33.5% -42%, preferably 35%, 36%, 37%, 38%, 39%, 40%, 41% by mole ratio.
In some embodiments, the cationic lipid, PEG-lipid, amphoteric phospholipid and cholesterol provided by the invention have a molar ratio of (45-55): (1-2): (9-11): (33.5-42).
The drug of the present invention may be administered parenterally or parenterally, and parenterally is selected from sublingual, intramuscular, intravenous, rectal, mucosal and respiratory administration, and in some embodiments, intramuscular or mucosal administration.
The solvent of the pharmaceutical preparation is selected from physiological saline, PBS solution with pH of 7.2-7.4, PBS solution containing 0.05-5% of chitosan or sodium alginate, or glucose solution with concentration of 5%. Preferably, the pharmaceutical dosage form is a liquid injection preparation, and the solvent is physiological saline.
The concentration of mRNA in the pharmaceutical preparation is 100-3000 mug/mL, and the preferred concentration of mRNA in the pharmaceutical preparation is 200-2000 mug/mL.
The invention also provides a preparation method of the pharmaceutical preparation, which comprises the following steps: mRNA is encapsulated into lipid delivery using either thin film hydration or organic solvent evaporation.
In the preparation method, the preparation of mRNA comprises the following steps:
S1: synthesizing cDNA fragments comprising fusion mRNA of a GLP-1 polypeptide receptor agonist and an antibody Fc, and constructing the cDNA fragments on the downstream of an RNA polymerase promoter to obtain recombinant plasmids;
S2: transfecting the recombinant plasmid into escherichia coli to obtain escherichia coli for expressing exogenous recombinant plasmid, and extracting the recombinant plasmid after amplifying the escherichia coli in large quantity, and taking the recombinant plasmid as a template to obtain a linearized mRNA fragment template comprising a T7, T3 or SP6 promoter;
S3: constructing an mRNA extracellular transcription system reaction to obtain the mRNA of the active ingredient;
Preferably, in the step S3, DNase I is added for reaction after the reaction system is completed, so as to remove the DNA template in the reaction system. After S3, obtaining the fusion mRNA of the coded GLP-1 polypeptide receptor agonist and the antibody FC, purifying the mRNA by adopting a magnetic bead method or an affinity column chromatography method, and subpackaging and preserving at-80 ℃ or normal temperature or 4 ℃ or-20 ℃.
Wherein the method for obtaining the linearized mRNA template comprises direct plasmid PCR with synthetic primers or cleavage of circular plasmids into linearized fragments by enzymatic cleavage.
The capping method of the mRNA extracellular transcription system of the present invention includes, but is not limited to, the following two methods: 1. ) Capping by in vitro co-transcription using a cap structural analogue; 2) Capping was performed after in vitro transcription using capping enzymes alone.
Wherein the RNA Polymerase used in the mRNA extracellular transcription system can be T7, SP6 or T3 RNA Polymerase.
Wherein ATP, GTP, CTP and UTP in the mRNA extracellular transcription system can be unmodified nucleotides or modified nucleotides including but not limited to m1A, m6A, m A m, m5C, m3C, m7G methylation modified nucleotides and pseudouracil modified, hypoxanthine modified non-methylation modified nucleotides.
The preferred in vitro mRNA synthesis system of the invention employs an in vitro transcription one-step capping method, taking 2000. Mu.L as an example, the reaction system is as follows:
TABLE 1 one-step capping in vitro transcription reaction System
Component (A) Volume of
ddH2O To 2000μL
ATP 10μL
GTP 10μL
UTP 10μL
CTP 10μL
CleanCap AG 8μL
10×Buffer 20μL
DNA template 2000ng
RNase Inhibitor 4μL
YIPP 3μL
RNA polymerase 30μL
DTT 10μL
Preferably, the in vitro reaction conditions of the mRNA are 37℃for 24h.
Preferably, after the preparation of mRNA in step 3) is obtained, the linearized DNA template is degraded by adding 10. Mu.L DNase I.
Preferably, the method for preparing a pharmaceutical preparation for treating diabetes further comprises encapsulating the mRNA in an ionizable or cationic liposome preparation by a thin film dispersion method.
The invention provides application of the mRNA, the polypeptide, the recombinant protein, the recombinant plasmid or the pharmaceutical preparation in preparing medicines for treating diabetes.
The invention also provides a medicine for treating diabetes, which comprises the pharmaceutical preparation.
The invention also provides a method of treating diabetes comprising administering a medicament of the invention.
The mRNA-based medicament for treating diabetes provided by the invention, namely, in-vitro synthesized fusion mRNA encoding a polypeptide GLP-1 receptor agonist and an antibody Fc end is delivered to a body, so that the GLP-1 receptor agonist can be effectively expressed, and the normal blood sugar level can be effectively reduced. Compared with the current GLP-1 polypeptide drug or Fc fusion polypeptide thereof, the mRNA preparation has higher bioavailability, rapid in-vivo drug response, more stable action, high expression quantity and long expression duration; on the other hand, the mRNA preparation has low immunogenicity, can quickly stimulate insulin production, has reliable biosafety, and greatly provides the comfort and compliance of the administration of patients.
Drawings
For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:
FIG. 1 shows a schematic diagram of the structure of the constructed mRNA;
FIG. 2 shows the effect of mRNA preparations encoding fusion of different Fc segments with GLP-1 receptor agonists on ADCC effects;
FIG. 3 shows the ability of mRNA encoding GLP-1, GLP-1-mIgG4 Fc, GLP-1-linker-mIgG4 Fc polypeptides to activate GLP-1 receptors, respectively;
FIG. 4 shows the IL-8 and TNF- α expression levels (pg/mL) in rat serum;
FIG. 5 shows the change in blood glucose concentration in rats after injection of mRNA liposome preparation;
FIG. 6 shows the content of polypeptide GLP-1 receptor agonist in rat blood of mRNA injected liposome preparation.
Detailed Description
The invention provides an mRNA pharmaceutical preparation for treating diabetes, a preparation method and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
TABLE 2 amino acid sequences and nucleic acid sequences involved in the invention
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 construction and amplification of recombinant plasmids
1. CDNA sequence of Fc fusion mRNA of polypeptide GLP-1 receptor agonist of SEQ ID No.10 and antibody IgG4 after partial site mutation is synthesized. The GLP-1 receptor agonist is connected with the polypeptide sequence of the antibody Fc through a GGGGSGGGGSGGGGSA flexible protein linker, and the front end of the GLP-1 is connected with a signal peptide sequence as shown in SEQ ID No. 31. The fusion polypeptide sequence coded by the target mRNA is named GLP-1-linker-mIgG4 Fc.
2. The DNA sequence encoding the fusion mRNA of the GLP-1 receptor agonist polypeptide and the Fc segment of the antibody was ligated to PRSF vector plasmids by SfiI cleavage ligation. First, the vector and the fragment are digested. The vector and fragment cleavage reaction system was (50. Mu.L) DNA vector/fragment: 5. Mu.L; 10x NEB Buffer: 5. Mu.L; ddH 2 O: 40. Mu.L; uniformly mixing the reaction systems, and reacting for 2 hours at 50 ℃; subsequently, ligation of the vector and fragment is performed. After the reaction product after enzyme digestion is recovered, 30 mu L of ddH 2 O is dissolved, then the concentration of the recovered product of the enzyme digestion and the recovery of the enzyme digestion of the carrier is measured by using NanoDrop, the concentration of the recovered product of the enzyme digestion and the recovery of the enzyme digestion of the carrier is 50 ng/. Mu.L, and the concentration of the recovered product of the enzyme digestion and the recovery of the enzyme digestion of the PCR fragment is 60 ng/. Mu.L. The carrier and fragment were added to the reaction system in a molar ratio of 1:3, and the reaction system in this example was configured as shown in Table 3 (20. Mu.L system) at 16℃overnight:
table 3: enzyme cutting connection reaction system
Component (A) Volume (mu L)
10x Buffer 2
Carrier body 1
Fragments 2
T4 ligase 1
ddH2O 44
3. The ligated plasmid was transformed into E.coli and cultured on solid medium overnight. Firstly, taking out the commercial sensory DH5 alpha from a refrigerator at the temperature of-80 ℃ and placing the refrigerator on ice for melting; then, 5. Mu.L of the post-ligation reaction solution was added to 100. Mu.L of the commercial competence, and gently shaken to uniformly distribute the post-ligation reaction solution in the competence. Placing the competence after adding the connection reaction liquid on ice for 30min, and then thermally exciting for 90s at 42 ℃ and immediately returning to ice for 2min; subsequently, 800. Mu.L of an LB medium without resistance was added thereto, and the mixture was placed on a shaking table and shaken at 37℃for 50 minutes; and finally, the competence after heavy shaking amplification is slightly turned for 30 seconds at 4000rpm, the supernatant is poured off, the sediment at the bottom of the EP tube is uniformly smeared on LB, and the LB is inverted in an incubator for overnight culture.
4. Colony PCR was performed and E.coli colonies with the correct band size were selected for sequencing. Firstly, transformants on LB culture plates are randomly selected at normal temperature, single colonies are picked up by sterilized toothpicks or gun heads, and the picked colonies are marked. Then, the toothpick or the tip stained with the bacterial cells was placed in a corresponding PCR reaction plate equipped with a PCR tube or 96 wells, and colony PCR reaction liquids were as shown in Table 4 below (reaction system 25. Mu.L). Finally, the PCR mixture mixed with the thalli is placed in a PCR instrument for amplification.
TABLE 4 colony PCR reaction System
Component (A) Volume (mu L)
Taq 47
Primer F 1
Primer R 1
Colony of bacteria
TABLE 5 colony PCR reaction conditions
5. Coli with correct sequencing was amplified and plasmids were extracted in large quantities. Firstly, adding 500 mu L of LB culture solution into a colony with the correct sequence after sequencing, and amplifying the colony by shaking for 1h in a shaking table at 37 ℃; subsequently, adding the amplified bacterial liquid into 100mL of LB culture medium containing kana resistance, and shaking overnight at 37 ℃; finally, endotoxin-free plasmids were extracted according to the instructions of TIANGEN kit, and the concentration of the extracted plasmids was found to be 809 ng/. Mu.L by NanoDrop measurement, and further stored in a-20℃refrigerator.
Example 2 in vitro transcription of target mRNA fragments (IVT)
1. Target fragment PCR: after obtaining the purified plasmid in large quantities, DNA sequences encoding the target mRNA, including the T7 sequence, are amplified using high fidelity Taq enzyme. The PCR amplification system is shown in Table 6 (50. Mu.L of the reaction system). The amplified reaction solution was added to 1% agarose gel, and the mixture was subjected to electrophoresis at a constant voltage of 100V for 20 minutes.
TABLE 6PCR reaction System
Component (A) Volume (mu L)
Taq 47
Primer F 1
Primer R 1
Plasmid template 1
TABLE 7 PCR reaction conditions for target fragments
2. Purification of target PCR fragment: firstly, cutting fragments with correct sizes observed under an ultraviolet lamp, adding 700 mu L of GSB solution, and dissolving in water bath at 60 ℃ for 10min; then, cooling the dissolved peptized solution containing the target fragments to room temperature, and pouring the cooled peptized solution into a peptized recovery column; then, centrifuging for 30s at normal temperature under the rotation speed of 12000rpm, rinsing twice by adopting a Wash Buffer, and airing the gel recovery column; subsequently, 30. Mu.L of ddH 2 O at 60℃was added thereto, and left standing at room temperature for 2 minutes; finally, the mixture was centrifuged at 12000rpm for 3min at room temperature, and the centrifugate was collected and the concentration of the PCR product was determined to be 58 ng/. Mu.L by NanoDrop.
3. The one-step capping technique is adopted to synthesize the needed target mRNA in vitro: first, the PCR product described above was added to 100. Mu.L of RNase-Free EP tube together with commercial Cap1 Cap analogue CLEANCAP AG of mRNA and T7 RNA polymerase, and the specific reaction system was shown in Table 8 (10. Mu.L); subsequently, the above reaction system was left to react at 37℃for 3 hours.
TABLE 8 one-step in vitro capping transcription System
Component (A) Volume of
ddH2O 1.8μL
ATP 0.5μL
GTP 0.5μL
UTP 0.5μL
CTP 0.5μL
CleanCap AG 0.4μL
10x Buffer 1μL
DNA template 3μL
RNase Inhibitor 0.1μL
YIPP 0.2μL
RNA polymerase 1μL
Poly(A)Polymerase 0.5μL
4. Purification of mRNA: the synthesized mRNA is purified by oligo (dT) -cellulose column chromatography, and the treatment process is as follows: first, the oligo (dT) -cellulose column was pretreated: (1) 0.5g to 1.0g oligo (dT) cellulose was suspended with 0.1mol/L NaOH. (2) The suspension was placed in a Pasteur pipette filled with DEPC water, autoclaved glass wool, and the oligo (dT) cellulose was rinsed with 3 times the bed volume of RNase-free sterile double distilled water at about 0.5 mL-1.0 mL bed. (3) The oligo (dT) cellulose was then washed with 3-5 fold bed wash buffer I until the pH of the effluent was less than 8.0. (4) Pouring the treated oligo (dT) cellulose from the Pasteur pipette, suspending with a suitable bed wash buffer I at a concentration of about 0.1g/mL, and storing at 4deg.C for use; next, the total RNA solution was transferred to a suitable RNase-Free centrifuge tube, and the concentration of total RNA was measured and diluted to 0.55mg/mL with RNase-Free double distilled water. (5) The RNA solution was heated in a 65℃water bath for 5min, and then rapidly inserted on ice for cooling. (6) Adding a certain volume of 5mol/L NaCl to adjust the salt concentration in the RNA solution to 0.5mol/L; Finally, mRNA in the reaction solution was purified: (7) The oligo (dT) -cellulose was resuspended in a pipette, an appropriate amount of oligo (dT) -cellulose was added to the RNA sample, the lid was closed, the oligo (dT) -cellulose was mixed with the in vitro transcription reaction solution by inverting it several times, and the mixture was incubated in a water bath at 37℃and gently swirled for 15min. The volume/mass ratio of oligo (dT) -cellulose to in vitro transcription reaction solution was 1:1, i.e., about 0.1mg total mRNA required 1mL of oligo (dT) -cellulose. (8) 1 syringe (5 ml) was used, the syringe was held on an RNase-free holder by tightening the syringe tip with a high temperature sterilized glass wool plug, oligo (dT) -cellulose/RNA suspension was added to the syringe, the plug was pushed to the bottom, and the effluent liquid was collected, i.e.the liquid containing unbound RNA was collected in an RNase-free centrifuge tube. (9) The wash buffer was slowly absorbed by syringe, gently shaken, mRNA-oligo (dT) -cellulose was well suspended, the plug was pushed, and the eluate was collected by RNase-Free centrifuge tube. The OD 260 of each tube was measured and the eluate was prepared for elution when the OD value in the eluate was 0. (10) The RNase-Free double distilled water was slowly sucked into the syringe by the syringe, the mRNA-oligo (dT) -cellulose was sufficiently resuspended, the plug was pushed, and the mixture was packed into RNase-Free centrifuge tubes at 1/3 to 1/2 bed volume. (11) The OD 260 of each tube was measured and the RNA-containing eluates were pooled. At 4 ℃,2500g, centrifuging for 2-3 min, and transferring the supernatant into a new RNase-Free centrifuge tube. (12) Adding 1/10 volume of 3mol/L NaAc (pH 5.2) into the supernatant, adding 2.5 times volume of ice-cold ethanol, mixing, and precipitating at-20deg.C for 30min or standing overnight. (13) The supernatant was carefully discarded by centrifugation at 12000rpm at 4℃for 15 min. The precipitate was rinsed with 70% ethanol, 12000g, centrifuged for 5min at 4℃and the supernatant carefully discarded. mRNA pellet was dissolved in an appropriate volume of RNase-Free ddH 2 O. The mRNA after purification and concentration was measured by NanoDrop at a concentration of 1. Mu.g/. Mu.L.
The target mRNA is prepared by purification, the 5' end of the target mRNA is sequentially connected with a signal peptide, a 5' -UTR and a 5' -CAP, the 3' end of the target mRNA is sequentially connected with a 3' -UTR and a poly A with the length of 150bp, and the sequence of the ORF region of the mRNA is shown in Table 2. The polypeptide sequence encoded by the target mRNA is GLP-1-linker-mIgG4 Fc.
EXAMPLE 3 preparation of Liposome preparation of fusion mRNA of polypeptide GLP-1 receptor agonist and antibody Fc segment
1. Respectively dissolving cationic lipid, PEG-lipid, amphoteric phospholipid and cholesterol in the lipid according to the prescription ratio of Table 8 in chloroform of a round-bottomed flask, and performing rotary evaporation at the water bath temperature of 50-70 ℃ to uniformly form a film on the bottom of the round-bottomed flask;
2. adding mRNA prepared in the example 2 and citric acid solution with pH of 5.5 into a round-bottom flask after film forming, and dispersing the film in the mRNA solution by shaking to obtain a multichamber liposome;
3. Carrying out probe ultrasonic treatment on the obtained multi-layer liposome solution for 5-20 min, wherein the ultrasonic working time is 1-5 s, the intermittent working power is 220W, and the single-layer liposome with relatively uniform particle size is obtained;
4. The monolayer liposome obtained above is filtered by a 100nm filter membrane and is extruded at 50-70 ℃ to obtain a liposome preparation loaded with coded polypeptide GLP-1 receptor agonist and antibody Fc fusion mRNA with uniform particle size;
5. Placing the liposome preparation loaded with the target mRNA in a microgel centrifugal column, centrifuging at the normal temperature at the speed of 2000rpm/min for 5min, concentrating and purifying the liposome preparation, and diluting with 0.9% physiological saline to ensure that the final concentration of the active mRNA in the preparation is 2 mg/mL-5 mg/mL. The liposome preparation of the prescription 1 to 4 is respectively prepared.
Table 9 lipid Material formulation
EXAMPLE 4 characterization of Liposome formulations of self-amplified mRNA of polypeptide GLP-1 receptor agonists
1. Measurement of particle size potential of the liposome prepared. Liposomes prepared as in example 3, prescription 1, were diluted hundreds of times with 1x PBS. The PBS is firstly used for measuring the background of the sample by a Mastersizer 3000, after the background is measured, the dispersed sample is added, and after the concentration of a dispersion system is stable, the measurement is started to obtain the potential and the particle size of the liposome loaded with the target mRNA. The particle size distribution of the finally measured cationic liposome is between 100nm and 120nm, and the Zeta potential value is about +10mV to +14mV;
2. And (5) determining the encapsulation rate of the prepared liposome on mRNA. The liposome preparation prepared in example 3 (prescriptions 1 to 4) was added with an equal amount of methanol-chloroform (volume ratio 1:2) and mixed uniformly, and demulsification was performed, and at the same time, the encapsulation efficiency of the liposome on the target mRNA was measured by using RiboGreen. The specific measurement steps are as follows: (1) And placing the liposome after demulsification in an ice tank. Then, setting excitation wavelength of 485nm and emission wavelength of 530nm of the fluorescence spectrophotometer; (2) Melting ReagentA staining solution in a refrigerator kit at-20 ℃ at room temperature, adding 2.5 mu L of ReagentA into 500 mu L of ReagentB diluent, uniformly mixing, wrapping with tinfoil, and placing in a darkroom for later use; (3) 5 1.5mL EP tubes were labeled 1/2/3/4/5 tubes each, and 100. Mu.L ReagentB was added each. Subsequently, 100. Mu.L of ReagentC were taken into a first tube, mixed well and diluted in a gradient in sequence, finally resulting in total RNA in 1/2/3/4/5 tubes at concentrations of 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL, 0. Mu.g/mL, respectively. And moving 100 mu L of the prepared Reagent A and Reagent B mixed solution into a cuvette, incubating for 5 minutes at room temperature to avoid illumination, and placing the cuvette in a fluorescence spectrophotometer for measurement and reading to obtain an RFU value. Respectively making standard curves of RNA standard substances with different gradient concentrations and corresponding RFUs; (4) And (3) re-taking 100 mu L of the prepared ReagentA and ReagentB dyeing working solution into a new cuvette, adding the demulsified liposome solution into the cuvette, and finally obtaining the RFU value of the demulsified liposome according to the prepared standard curve in the subsequent steps which are the same as those in the step (3). When 2mg/mL of mRNA was added, the encapsulation concentration of mRNA of the prepared liposome was 1.88mg/mL, and finally the encapsulation efficiency of GLP-1 receptor agonist-antibody Fc fusion mRNA of the liposome was calculated, and the results are shown in Table 10.
Table 10 encapsulation efficiency measurement results
Prescription 1 Prescription 2 Prescription 3 Prescription 4
Encapsulation efficiency (%) 94.1％ 92.9％ 95.2％ 88.8％
Example 5 identification of cellular protein expression levels of Liposome preparations based on fusion mRNA of polypeptide GLP-1 receptor agonist-antibody Fc segment
1. HEK-293T cells were seeded one day in advance in 6-well plates and incubated overnight with 10% fbs in high-sugar DMEM, attached the next day and grown to 70% confluency;
2. the liposome preparation (example 3, prescription 1) loaded with polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA prepared above is transfected into the adherent cells, and the adherent cells are kept stand in an incubator at 37 ℃ for 6 hours, and then fresh culture solution is replaced;
3. Taking the supernatant of the cell culture fluid, adopting an Elisa kit of an antibody Fc segment, operating according to the specification of the Elisa kit, measuring the content of target GLP-1 receptor agonist polypeptide secreted by cells, and finally measuring the content of the GLP-1 receptor agonist polypeptide in a 6-hole plate to be 300pg/mL.
Example 6 in vitro determination of ADCC Effect of Liposome formulations based on fusion mRNA of polypeptide GLP-1 receptor agonist-antibody Fc segment
1. Target mRNAs encoding the following fusion polypeptides were prepared separately as described in examples 1-2: the polypeptide GLP-1 receptor agonist of SEQ ID No.10 is linked to the Fc segment of IgG1, the Fc segment of IgG4 and the Fc segment of mutated anti-IgG 4 by a flexible protein linker (GGGGSGGGGSGGGGSA). The fusion polypeptides are respectively marked as GLP-1-linker-IgG1 Fc, GLP-1-linker-IgG4 Fc and GLP-1-linker-mIgG4 Fc, and the amino acid sequences are respectively shown in Table 2.
Wherein the target mRNA encoding the GLP-1-linker-mIgG4 Fc fusion polypeptide is shown in example 2, the sequence of the ORF region of the target mRNA encoding the GLP-1-linker-IgG1 Fc fusion polypeptide or the GLP-1-linker-IgG4 Fc fusion polypeptide is shown in Table 2, and the sequence of the signal peptide, 5' -UTR, 5' -Cap,3' -UTR and Poly A sequence of the target mRNA are the same as those of the corresponding portion of the target mRNA of example 2 except the sequence of the ORF region.
Then the 3 target mRNAs are referred to the formula of the prescription 1 of the embodiment 3 to prepare the nano liposome.
2. The liposome preparations of the polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA prepared as described above were each transfected into cells according to the method described in example 5, and cell supernatants containing GLP-1-linker-IgG1 Fc, GLP-1-linker-IgG4 Fc, GLP-1-linker-mIgG4 Fc fusion polypeptides were each collected and continuously cultured for one week, and affinity chromatography was performed using a ProteinA affinity chromatography column, and further hydrophobic chromatography was performed since the protein after affinity chromatography still contained degradation products and multimers. The fusion protein obtained by two-step purification shows that the protein purity is higher than 90% by HPLC analysis and SDS-PAGE analysis. The anti-GLP-1 antibody and the anti-IgG Fc antibody are respectively used as detection antibodies, and the fusion protein secreted by the cells is found to have better integrity after Western-blotting detection is carried out on the GLP-1-Fc fusion polypeptide obtained by purification. The concentration of the purified GLP-1-Fc fusion polypeptide is adjusted to 1mg/mL by PBS with pH7.4, and the GLP-1-linker-Fc fusion polypeptides obtained by the three plants are respectively diluted to 250 mug/mL, 125 mug/mL, 62.5 mug/mL, 31.3 mug/mL, 15 mug/mL, 10 mug/mL, 5 mug/mL, 2 mug/mL, 1 mug/mL and 0.5 mug/mL by PBS for standby;
3. constructing a lentivirus pWSLV-EF-1 alpha-GLP-1 receptor-T2A-CMV-luciferase vector, and co-transferring the vector and a packaging plasmid into 293T cells for virus packaging; collecting a supernatant culture solution after 72 hours, adding 1/5 volume of PEG 8000 into the supernatant culture solution, standing at 4 ℃ for 30 minutes at 4000rpm after standing overnight, re-suspending the centrifuged viral pellet by PBS, respectively dripping the viral pellet into HeLa cells, sorting GFP positive cells by a flow cell sorter after 48 hours, namely a HeLa cell line with high expression of GLP-1receptor and luciferases, and further amplifying and culturing;
4. The HeLa cell line, which highly expresses GLP-1receptor and luciferases, obtained in step 3 was co-cultured with human peripheral blood mononuclear cells isolated from human blood cells, and dilutions of the three sets of fusion polypeptides obtained in step 2) of example 6 were added thereto, respectively. In the co-culture system, a HeLa cell line highly expressing GLP-1receptor and luciferases is used as a target cell for ADCC, and human peripheral blood mononuclear cells separated from human blood cells are used as effector cells for ADCC. After GLP-1-Fc fusion polypeptides with different groups and different concentrations are respectively added into a co-culture system for a certain time, after cells are lysed after cell lysates are added, a substrate D-Luciferin is added into the cell lysates, and a microplate luminescence detector is used for detecting the OD value of target cells, namely HeLa cells which highly express GLP-1receptor and luciferases, the generated luciferases are lysed to react with the added D-Luciferin to release chemiluminescence intensity, and the luminescence intensity value of the luciferases reflects the ADCC effect of the GLP-1linker-Fc fusion polypeptides with different Fc segments.
The specific results are shown in FIG. 2, and the results show that the mRNA encoding the GLP-1-linker-IgG 1 Fc fusion polypeptide produces a stronger ACDD effect than the mRNA encoding the GLP-1-linker-IgG 4 Fc or GLP-1-linker-mIgG4 Fc fusion polypeptide. The low ADCC effect of the IgG4 Fc fragment fusion polypeptide after partial site-directed mutagenesis after expression of the target mRNA in vivo represents a lower affinity between Fc and Fc receptors located on the surface of effector cells, which means a reduced dose-dependent cytotoxicity, and mRNA encoding GLP-1-linker-IgG 4 Fc or GLP-1-linker-mIgG4 Fc fusion polypeptides will have lower side effects when administered in vivo.
EXAMPLE 7 Effect of GLP-1 receptor agonist and intermediate linker of IgG Fc segment on GLP-1 receptor agonist function
1. Target mRNAs encoding the following polypeptides were prepared and purified separately as described in example 6 (1): GLP-1 polypeptide, GLP-1-mIgG4 Fc polypeptide which is not connected by Linker and GLP-1-Linker-mIgG4 Fc polypeptide sequence which is connected by Linker sequence (GGGGSGGGGSGGGGSA) are marked as GLP-1, GLP-1-mIgG4 Fc and GLP-1-Linker-mIgG4 Fc respectively.
Wherein the target mRNA encoding the fusion polypeptide of GLP-1-linker-mIgG4 Fc is described in example 2, the sequence of the ORF region of the target mRNA encoding the GLP-1 polypeptide or the GLP-1-mIgG4 Fc polypeptide not linked by linker is described in Table 2, respectively, and the sequence of the target mRNA except the ORF region is different, and the rest of the signal peptide, 5' UTR, 5' cap,3' UTR and Poly A sequence are the same as those of the target mRNA of example 2. Then the 3 target mRNAs are referred to the formula of the prescription 1 of the embodiment 3 to prepare the nano liposome. The liposome preparations of mRNA prepared as described above were transfected into cells, respectively, according to the method of example 6, step (2).
2. A stable transgenic 293T cell line was prepared that highly expressed GLP-1receptor and was based on the CRE-BLAM reporting system of the lacZ high-expression cAMP response as described in example 6 (3);
3. The stable report 293T cell line from step (2) above was seeded in 24-well plates and cultured overnight at 27 ℃. After cell attachment, 10nM GLP-1, GLP-1-mIgG4 Fc, GLP-1-linker-mIgG4 Fc diluted with serum-free DMEM were added thereto, respectively. After 5h, 20. Mu.L of substrate lactamase was added thereto, and after 1h of substrate addition, the emitted fluorescence was measured to indicate the amount of aAMP activated, and at the same time to reflect the signal intensity generated by the action of the GLP-1 receptor agonist on the GLP-1 receptor.
The results are shown in FIG. 3. Experimental results show that when mRNA encoding a fusion polypeptide in which GLP-1 is directly linked to an IgG Fc segment is not passed through a suitable linker, the ability of the mRNA to activate GLP-1 receptor in vivo is instead decreased; when the mRNA encoding a fusion polypeptide of GLP-1 and IgG Fc segments joined by a suitable linker, the mRNA has a 4-fold capacity for GLP-1 activation of the GLP-1 receptor in vivo over the mRNA encoding GLP-1 alone.
Example 8 evaluation of immunogenicity in vivo of Liposome pharmaceutical formulations based on polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA
1. SD rats under SPF condition line were kept in ventilated cages under 12h light, 12h dark cycle conditions;
2. Different rats were injected intramuscularly with physiological saline injection of mRNA liposomes of the polypeptide GLP-1 receptor agonist (example 2, prescription 1), control group with the same dose of physiological saline, and commercially available semaglutin injection;
3. After 24h, rats of different groups were subjected to orbital bleeding, and serum was isolated and the concentrations of IL-8 and TNF- α in serum were determined separately using the Elisa kit.
The measurement result is shown in fig. 4, and it can be seen from the result that the immunogenicity of the prepared liposome pharmaceutical preparation of the fusion mRNA encoding the polypeptide GLP-1 receptor agonist-antibody Fc is not greatly different from that of commercially available semeglutide, and the immunogenicity of the liposome pharmaceutical preparation is not statistically different from that of commercially available semeglutide.
Example 9 evaluation of in vivo potency of Liposome pharmaceutical formulations based on mRNA encoding a polypeptide GLP-1 receptor agonist-antibody Fc fusion
1. Culturing male rats with SPF grade of 4W-6W and weight of 160 g-200 g in an aeration cage under 12h illumination and 12h dark circulation conditions;
2. Injecting streptozotocin into the abdominal cavity of the rat for 7 days, assisting with high-sugar diet, measuring the weight and the blood sugar concentration of the rat after 7 days, confirming that the rat has diabetes, and establishing a type II diabetes disease model of the rat;
3. intramuscular injection of the mRNA liposome pharmaceutical formulation of example 2, formulation 1, into model rats, resulted in a final mRNA concentration of 200 μg/each final injection of active ingredient per rat;
4. After injection of the preparation, rats were subjected to orbital bleeding daily, centrifuged at 3000rpm/min for 10min, and serum was isolated for use. In the measurement of the blood glucose protein in rat serum, first, o-xylylenediamine, 55.56mmol/L of standard glucose stock solution and 5.56mmol/L of standard glucose application solution are prepared; then, taking 3 test tubes of 10mL, respectively adding 0.1mL of distilled water, standard glucose application liquid and serum samples into the three test tubes after adding 5mL of o-toluidine reagent, placing the test tubes in an electrothermal incubator, boiling for 20min, taking out and cooling; finally, a 721 type spectrophotometer is adopted, a cuvette with an optical path of 1cm is used at the wavelength of 630nm, a blank tube is zeroed, and absorbance values A of the sample tube and a standard tube are respectively read. Finally, the blood glucose concentration (mmol/L) of the sample = (a sample/a standard) ×5.56mmol/L.
The results are shown in FIG. 5, and demonstrate that the blood glucose concentration of rats injected with a liposomal pharmaceutical formulation encoding the polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA gradually decreased and eventually returned to normal steady state levels.
Example 10 in vivo half-life and bioavailability of Liposome formulations based on mRNA encoding a polypeptide GLP-1 receptor agonist-antibody Fc fusion
1. SD rats under SPF condition line were kept in ventilated cages under 12h light, 12h dark cycle conditions;
2. different rats were injected intramuscularly at the same site with a physiological saline injection of liposomes (example 2, prescription 1) encoding the polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA at a concentration of 200 μg/mL, and the control group was injected with the same dose of commercially available semaglutin injection;
3. Rats were bled daily by orbital extraction, serum was isolated and GLP-1 content in serum was determined by Elisa kit according to Elisa kit instructions.
As a result, as shown in FIG. 6, the corresponding GLP-1 protein level in the serum of the rat injected with the protein-based drug rapidly decreased to less than 1/3 of the initial level in 14 days, and the injection of the preparation of mRNA encoding the polypeptide-based GLP-1 receptor agonist-antibody Fc fusion was able to maintain the expression of the GLP-1 receptor agonist at a higher level in 14 days without significant decrease. Therefore, the mRNA liposome preparation can continuously express GLP-1 protein for a long time, and plays a long-acting role.
From the above examples, the drug based on polypeptide GLP-1 receptor agonist-antibody Fc fusion mRNA provided by the invention has long duration of action, short onset time and low side effect, and can significantly reduce blood glucose level of type II diabetics.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> Beijing Hospital
<120> MRNA pharmaceutical preparation for treating diabetes mellitus, preparation method and application thereof
<130> MP21038377
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His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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tatgcggaag gcacctttat tagcgattat agcattgcga tggataaaat tcatcagcag 60
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acccag 126
<210> 17
<211> 90
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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tatgcggaag gcacctttat tagcgattat agcattgcga tggataaaat tcatcagcag 60
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<211> 132
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 19
catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60
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<211> 117
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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<210> 23
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<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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catggcgaag gcacctttac cagcgatgtg agcagctatc tggaagaaca ggcggcgaaa 60
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<211> 99
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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<210> 25
<211> 117
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
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catggcgaag gcagctttag cagcgaactg agcaccattc tggatgcgct ggcggcgcgc 60
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<210> 26
<211> 93
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 26
catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60
gaatttattg cgtggctggt gaaaggccgc ggc 93
<210> 27
<211> 696
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 27
gaaccgaaaa gctgcgataa aacccatacc tgcccgccgt gcccggcgcc ggaactgctg 60
ggcggcccga gcgtgtttct gtttccgccg aaaccgaaag ataccctgat gattagccgc 120
accccggaag tgacctgcgt ggtggtggat gtgagccatg aagatccgga agtgaaattt 180
aactggtatg tggatggcgt ggaagtgcat aacgcgaaaa ccaaaccgcg cgaagaacag 240
tataacagca cctatcgcgt ggtgagcgtg ctgaccgtgc tgcatcagga ttggctgaac 300
ggcaaagaat ataaatgcaa agtgagcaac aaagcgctgc cggcgccgat tgaaaaaacc 360
attagcaaag cgaaaggcca gccgcgcgaa ccgcaggtgt ataccctgcc gccgagccgc 420
gatgaactga ccaaaaacca ggtgagcctg acctgcctgg tgaaaggctt ttatccgagc 480
gatattgcgg tggaatggga aagcaacggc cagccggaaa acaactataa aaccaccccg 540
ccggtgctgg atagcgatgg cagctttttt ctgtatagca aactgaccgt ggataaaagc 600
cgctggcagc agggcaacgt gtttagctgc agcgtgatgc atgaagcgct gcataaccat 660
tatacccaga aaagcctgag cctgagcccg ggcaaa 696
<210> 28
<211> 684
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 28
gaacgcaaat gctgcgtgga atgcccgccg tgcccggcgc cgccggtggc gggcccgagc 60
gtgtttctgt ttccgccgaa accgaaagat accctgatga ttagccgcac cccggaagtg 120
acctgcgtgg tggtggatgt gagccatgaa gatccggaag tgcagtttaa ctggtatgtg 180
gatggcgtgg aagtgcataa cgcgaaaacc aaaccgcgcg aagaacagtt taacagcacc 240
tttcgcgtgg tgagcgtgct gaccgtggtg catcaggatt ggctgaacgg caaagaatat 300
aaatgcaaag tgagcaacaa aggcctgccg gcgccgattg aaaaaaccat tagcaaaacc 360
aaaggccagc cgcgcgaacc gcaggtgtat accctgccgc cgagccgcga agaaatgacc 420
aaaaaccagg tgagcctgac ctgcctggtg aaaggctttt atccgagcga tattagcgtg 480
gaatgggaaa gcaacggcca gccggaaaac aactataaaa ccaccccgcc gatgctggat 540
agcgatggca gcttttttct gtatagcaaa ctgaccgtgg ataaaagccg ctggcagcag 600
ggcaacgtgt ttagctgcag cgtgatgcat gaagcgctgc ataaccatta tacccagaaa 660
agcctgagcc tgagcccggg caaa 684
<210> 29
<211> 837
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 29
gaactgaaaa ccccgctggg cgataccacc catacctgcc cgcgctgccc ggaaccgaaa 60
agctgcgata ccccgccgcc gtgcccgcgc tgcccggaac cgaaaagctg cgataccccg 120
ccgccgtgcc cgcgctgccc ggaaccgaaa agctgcgata ccccgccgcc gtgcccgcgc 180
tgcccggcgc cggaactgct gggcggcccg agcgtgtttc tgtttccgcc gaaaccgaaa 240
gataccctga tgattagccg caccccggaa gtgacctgcg tggtggtgga tgtgagccat 300
gaagatccgg aagtgcagtt taaatggtat gtggatggcg tggaagtgca taacgcgaaa 360
accaaaccgc gcgaagaaca gtataacagc acctttcgcg tggtgagcgt gctgaccgtg 420
ctgcatcagg attggctgaa cggcaaagaa tataaatgca aagtgagcaa caaagcgctg 480
ccggcgccga ttgaaaaaac cattagcaaa accaaaggcc agccgcgcga accgcaggtg 540
tataccctgc cgccgagccg cgaagaaatg accaaaaacc aggtgagcct gacctgcctg 600
gtgaaaggct tttatccgag cgatattgcg gtggaatggg aaagcagcgg ccagccggaa 660
aacaactata acaccacccc gccgatgctg gatagcgatg gcagcttttt tctgtatagc 720
aaactgaccg tggataaaag ccgctggcag cagggcaaca tttttagctg cagcgtgatg 780
catgaagcgc tgcataaccg ctttacccag aaaagcctga gcctgagccc gggcaaa 837
<210> 30
<211> 687
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 30
gaaagcaaat atggcccgcc gtgcccgagc tgcccggcgc cggaatttct gggcggcccg 60
agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa 120
gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat 180
gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc 240
acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa 300
tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa 360
gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg 420
accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg 480
gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg 540
gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag 600
gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag 660
aaaagcctga gcctgagcct gggcaaa 687
<210> 31
<211> 21
<212> PRT
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 31
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp
20

Claims (6)

  1. An mRNA pharmaceutical formulation characterized in that it comprises mRNA and a delivery lipid;
    The mRNA comprises: nucleic acids encoding GLP-1 receptor agonists, linker and nucleic acids encoding the Fc region of IgG antibodies;
    The amino acid sequence of the linker is GGGGSGGGGSGGGGSA;
    The nucleic acid sequence for encoding the GLP-1 receptor agonist is shown as SEQ ID No.23,
    The nucleic acid sequence of the Fc region of the encoded IgG antibody is shown as SEQ ID No. 30;
    The delivery lipid consists of cationic lipids, PEG-lipid, ampholytic phospholipids and cholesterol:
    The cationic lipid is selected from: 3- (N, N-dioleylamino) -1, N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethylammonium chloride or 1, 2-dioleyloxy-N, N-dimethylaminopropane;
    the PEG-lipid is selected from: PEG-phospholipid or PEG-diacylglycerol;
    The ampholytic phospholipids are selected from: distearoyl-phosphatidylethanolamine or dipalmitoyl phosphatidylcholine;
    The molar ratio of the cationic lipid to the PEG-lipid to the amphoteric phospholipid to the cholesterol is (45-55): (1-2): (9-11): (33.5-42).
  2. 2. The pharmaceutical formulation of claim 1, wherein the mRNA comprises, in order from the 5 'end to the 3' end: 5' -CAP, 5' -UTR, signal peptide, nucleic acid encoding GLP-1 receptor agonist, linker, nucleic acid encoding IgG antibody Fc region, 3' -UTR and PolyA.
  3. 3. The pharmaceutical formulation of claim 2, wherein the amino acid sequence of the signal peptide in the mRNA is shown in SEQ ID No. 31.
  4. 4. A pharmaceutical formulation according to any one of claims 1 to 3, wherein the concentration of mRNA in the pharmaceutical formulation is 100 μg/mL to 3000 μg/mL.
  5. 5. The method for preparing a pharmaceutical preparation according to any one of claims 1 to 4, comprising: mRNA is encapsulated into lipid delivery using either thin film hydration or organic solvent evaporation.
  6. 6. The use of the mRNA pharmaceutical preparation according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of diabetes.
CN202210176697.2A 2022-02-24 MRNA (messenger ribonucleic acid) medicinal preparation for treating diabetes and preparation method and application thereof Active CN114788876B (en)

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CN114788876B true CN114788876B (en) 2024-07-05

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111108117A (en) * 2017-09-22 2020-05-05 瑞泽恩制药公司 Glucagon-like peptide 1 receptor agonists and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111108117A (en) * 2017-09-22 2020-05-05 瑞泽恩制药公司 Glucagon-like peptide 1 receptor agonists and uses thereof

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