CN114788876A - mRNA medicinal preparation for treating diabetes, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to an mRNA medicinal preparation for treating diabetes, a preparation method and application thereof. The invention delivers the fusion mRNA of the GLP-1 polypeptide receptor stimulant and the Fc segment of the antibody to the body and utilizes the human body cells to autonomously express the GLP-1 polypeptide receptor stimulant. The GLP-1 polypeptide receptor agonist which is autonomously expressed can be secreted to the outside of cells to stimulate the secretion of insulin, thereby achieving the effect of treating diabetes. The medicinal preparation provided by the invention has the advantages of quick and simple method, high expression amount, long time, high bioavailability, low immunogenicity, short acting time and long duration of the medicament, can quickly stimulate the generation of insulin, and improves the compliance and comfort of patients in medication.

Description

mRNA medicinal preparation for treating diabetes, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to an mRNA medicinal preparation for treating diabetes, a preparation method and application thereof.

Background

Diabetes mellitus is one of three chronic non-infectious diseases threatening human life and health, and occurs 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 it is dependent on insulin. 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 body to utilize insulin effectively, i.e., insulin resistance, or insulin compensatory hyposecretion.

GLP-1 is known as glucagon-like peptide-1, a peptide-like hormone encoded by the human glucagon gene and secreted by intestinal L cells. GLP-1 can act on beta receptor on islet beta cells in a glucose-dependent manner, thereby promoting the transcription of insulin gene and increasing the biosynthesis and secretion of insulin. Meanwhile, GLP-1 can also stimulate the proliferation and differentiation of beta cells, inhibit the apoptosis of the beta cells, further increase the number of pancreatic beta cells, inhibit the secretion of glucagon, inhibit appetite and ingestion, delay the emptying of gastric contents, and is beneficial to the reduction of postprandial blood sugar and the stabilization of blood sugar level. The GLP-1receptor agonist with biological activity in a body is mainly GLP-1, but is extremely easy to be hydrolyzed by dipeptidyl peptidase, and the half-life period is less than 5 minutes, so that the GLP-1receptor agonist with long half-life period is one of the research and development directions of the current diabetes drugs.

mRNA, also known as messenger RNA, is transcribed using one strand of DNA as a template. mRNA carries genetic information and can directly direct protein synthesis. As one of the gene therapies, mRNA has a series of advantages compared to 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 genome, only can transiently express the encoded protein, and has higher safety; finally, mRNA can be formed by in vitro transcription processes, which are relatively inexpensive and can be rapidly applied to a variety of therapies. Meanwhile, theoretically, mRNA can express any protein, so that almost all diseases can be treated, and from the perspective of the pharmaceutical industry, mRNA is a candidate drug with great potential and can meet related requirements of gene personalized treatment, tumor personalized treatment, vaccines 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, the method 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 confirmed that mRNA nano-lipids prepared therefrom could effectively treat diseases. Also disclosed is a polynucleotide encoding a Low Density Lipoprotein Receptor (LDLR), wherein the LDLR comprises at least one mutation in a domain selected from the group consisting of an EGF-A domain, an intracellular domain, and both an EGF domain and an intracellular domain, wherein the polynucleotide comprises (a) a first region of linked nucleosides, the first region encoding a polypeptide of interest for use in reducing cholesterol levels in a patient. There are studies disclosing personalized vaccines for cancer comprising recombinant polypeptides comprising mutation-based neo-epitopes resulting from cancer-specific somatic mutations in tumor samples of cancer patients or nucleic acids encoding said polypeptides.

However, the current mRNA preparations are still concentrated on anti-tumor studies, and little is involved in the mRNA preparation for treating diabetes. Since mRNA formulations still present many challenges, such as instability in nature, chemical degradation of RNA in vitro mainly involves hydrolysis and oxidation, with hydrolysis occurring mainly in the phosphodiester bonds of the mRNA molecular backbone. Naked mRNA is also highly susceptible to degradation and inactivation in vivo, difficult to deliver and effectively express therapeutic amounts of protein in vivo, and potentially associated side effects, are all serious challenges. Thus, there are positive clinical values for providing an mRNA formulation that can be used to treat other than viral vaccines, but many challenges remain.

Disclosure of Invention

In view of this, the present invention aims to provide an mRNA pharmaceutical preparation for treating diabetes, and a preparation method and applications thereof. The medicine for treating diabetes is an mRNA medicine, the mRNA of a coded polypeptide GLP-1receptor stimulant is delivered into the body by using a delivery carrier, and the GLP-1receptor stimulant polypeptide is further secreted out of the body cell after being expressed by a human body cell, so that the GLP-1receptor on the surface of an islet beta cell is activated, insulin is secreted, and the purpose of treating diabetes is achieved.

The present invention provides a pharmaceutical formulation of mRNA comprising a nucleic acid encoding a GLP-1receptor agonist and a nucleic acid encoding an Fc region of an IgG antibody and a delivery lipid.

In the mRNA pharmaceutical preparation, the GLP-1receptor agonist is selected from any one of the following items I) to III):

I) at least one polypeptide of amino acid sequence shown in any one of SEQ ID No. 1-13;

II), a polypeptide having at least 60% identity to the polypeptide of I);

III) in the amino acid sequence of the polypeptide of I), one or more amino acids are substituted, deleted and/or inserted;

the nucleotide sequence of the coding mRNA is the nucleotide sequence optimized by the human codon. In some embodiments, the nucleic acid sequence encoding the GLP-1receptor agonist is selected from any one of i) -iii):

i) at least one of the nucleic acid sequences shown in any one of SEQ ID No. 14-26;

ii) a sequence having at least 60% identity to the sequence of i);

iii) a sequence in which one or more nucleotides are substituted, deleted and/or inserted in the sequence of i);

the Fc sequence of the antibody IgG is selected from the Fc segment 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 present invention, the IgG antibody Fc region 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 in which one or more amino acids are substituted, deleted and/or inserted in the Fc region of A).

The nucleotide sequence of the coding mRNA is the nucleotide sequence optimized by the human codon. In some embodiments, the nucleic acid sequence encoding the Fc region of an IgG antibody is selected from any one of a) to c):

a) and at least one of the nucleic acid sequences shown in any one of SEQ ID No. 27-30;

b) a sequence having at least 60% identity to the sequence of a);

c) a sequence in which one or more nucleotides are substituted, deleted and/or inserted in the sequence of a).

The invention provides a pharmaceutical preparation, wherein the mRNA comprises, sequentially connected from 5 'end to 3' end: 5 ' -CAP, 5 ' -UTR, signalpeptide, nucleic acid encoding a GLP-1receptor agonist, linker, nucleic acid encoding the Fc region of an IgG antibody, 3 ' -UTR and PolyA.

The linker in the medicinal preparation is selected from any one of the following materials: .

The amino acid sequence of the linker is GGGGSGGGGSGGGGSA, GSAGSAAGSGEF, GGGGGGGG, EGKSSGSGSESKST or KESGSVSSEQLAQFRSLD;

② polypeptide which has at least 70% of identity with the amino acid sequence of the linker;

and thirdly, in the sequence of the amino acid, polypeptide of one or more amino acids is substituted, deleted and/or inserted.

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 the sequence.

The protein encoded by the sequence with one or more nucleotide substitutions, deletions and/or insertions in the sequence has the same or similar function with the protein encoded by the original sequence.

In the pharmaceutical formulation provided by the present invention, the 5 'CAP is selected from Cap0(m7Gppp), Cap1(m7GpppmN), Cap2(m7GpppmNmN) or the anti-inversion 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 thymine nucleotides, preferably 100, 120, 150 and 160.

In some embodiments, the amino acid sequence of the signal peptide in the mRNA in the pharmaceutical formulation of the invention is shown in SEQ ID No. 31.

The invention also provides that the delivery vector in the pharmaceutical formulation can be selected from non-viral delivery vectors selected from ionizable liposomes, cationic polymers, lipid nanoparticles and viral delivery vectors. The active ingredient mRNA delivery of the present invention may also include the use of protamine, albumin, or the use of viral delivery vectors including lentiviral vectors, adenoviral vectors, retroviral vectors, adeno-associated viral vectors. In embodiments of the invention, the delivery vehicle is a cationic liposome or a lipid nanoparticle.

In some embodiments, the delivery lipid provided by the present invention consists of a cationic lipid, PEG-lipid, ampholytic phospholipid, and cholesterol:

the cationic lipid is selected from: 3- (N, N-dioleoylamino) -1, 2-propanediol (DOAP), (2, 3-dioleoyl-propyl) -trimethylamine, N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), 2, 3-dioleoyloxy-N- [2 (spermine-carboxamido) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1, 2-dioleyloxy-N, n-dimethylaminopropane (DLinDMA), dioctadecylaminoglycyl spermine (DOGS), 1, 2-di-linolenyloxy-N, N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybut-4-oxy) -1- (cis, cis-9, 12-octadecenyloxy) propane (CLinDMA), preferably 3- (N, N-dioleylamino) -1, 2-propanediol, N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride, 1, 2-dilinonyloxy-N, N-dimethylaminopropane, or a combination of two or more thereof. Wherein the cationic lipid comprises 45% to 55%, preferably 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53% or 54% of the delivery lipid, by mole ratio;

the PEG-lipid is selected from: PEG-phospholipid, PEG-dilauryloxypropyl, PEG-dialkoxypropyl, PEG-diacylglycerol, preferably PEG-diacylglycerol, or PEG-phospholipid. Wherein PEG-lipid comprises 1% to 2%, preferably 1.2%, 1.5% or 1.8% of the delivery lipid, by mole ratio;

the ampholytic phospholipid is selected from: egg yolk phosphatidylcholine (EPC), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dimyristoyl-phosphatidylethanolamine (DMPE), dioelaidoyl-phosphatidylethanolamine (DEPE). Preferably one or a combination of two or more of distearoyl-phosphatidylethanolamine, dipalmitoylphosphatidylcholine, and dioleoylphosphatidylethanolamine. Wherein the amphipathic lipid comprises 9% to 11%, preferably 9.5%, 10% or 10.5% of the delivery lipid, in terms of molar ratio.

The content of cholesterol in the delivery lipid is 33.5 to 42 percent, preferably 35, 36, 37, 38, 39, 40, 41 percent in terms of molar ratio.

In some embodiments, the present invention provides a molar ratio of the cationic lipid, PEG-lipid, ampholytic phospholipid, and cholesterol (45-55): (1-2): (9-11): (33.5 to 42).

The administration mode of the drug of the present invention can be gastrointestinal administration or parenteral administration, and the parenteral administration is selected from sublingual administration preparation, intramuscular administration preparation, intravenous administration preparation, rectal administration preparation, mucosal administration preparation and respiratory administration preparation, and in some embodiments, intramuscular injection and mucosal administration are selected.

The solvent of the pharmaceutical preparation is selected from normal saline, PBS solution with pH value of 7.2-pH value of 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 the mRNA in the pharmaceutical preparation is 100-3000 mu g/mL, and the concentration of the mRNA in the pharmaceutical preparation is preferably 200-2000 mu g/mL.

The invention also provides a preparation method of the pharmaceutical preparation, which comprises the following steps: the 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 a cDNA fragment of fusion mRNA comprising the GLP-1 polypeptide receptor agonist and the antibody Fc, and constructing the cDNA fragment at the downstream of an RNA polymerase promoter to obtain a recombinant plasmid;

s2: transfecting the recombinant plasmid into escherichia coli to obtain escherichia coli expressing an exogenous recombinant plasmid, amplifying the escherichia coli in a large quantity, extracting the recombinant plasmid, and taking the recombinant plasmid as a template to obtain a linearized mRNA fragment template comprising a T7, T3 or SP6 promoter;

s3: constructing mRNA extracellular transcription system reaction to obtain the active component mRNA;

preferably, in step S3, after the reaction system is completed, DNase I reaction is added to remove the DNA template in the reaction system. After obtaining the fusion mRNA of the GLP-1 polypeptide receptor stimulant and the antibody FC from S3, purifying the mRNA by a magnetic bead method or an affinity column chromatography, and subpackaging and storing at-80 ℃, or normal temperature, or 4 ℃ or-20 ℃.

The method for obtaining the linearized mRNA template comprises synthesizing a primer and directly carrying out plasmid PCR or cutting a circular plasmid into linearized fragments by an enzyme cutting method.

The capping method of the mRNA extracellular transcription system of the present invention includes, but is not limited to, the following two methods: 1.) in vitro co-transcription capping with cap structural analogs; 2) after in vitro transcription, capping was performed using the capping enzyme 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, m6A _m M5C, m3C, m7G methylated modified nucleotides and pseudouracil modified and hypoxanthine modified non-methylated modified nucleotides.

In the invention, the preferred in-vitro mRNA synthesis system adopts 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 capped in vitro transcription reaction System

Components	Volume of
		ddH 2 O	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 mRNA is reacted in vitro at 37 ℃ for 24 h.

Preferably, after obtaining said mRNA preparation in step 3), further comprising adding 10. mu.L DNase I to degrade the linearized DNA template.

Preferably, the method for preparing a pharmaceutical preparation for the treatment of diabetes mellitus further comprises encapsulating the mRNA into an ionizable or cationic liposome preparation by a membrane dispersion method.

The invention provides application of the mRNA, the polypeptide, the recombinant protein, the recombinant plasmid or the pharmaceutical preparation in preparing a medicament for treating diabetes.

The invention also provides a medicament for treating diabetes, which comprises the medicinal preparation.

The invention also provides a method of treating diabetes comprising administering a medicament according to the invention.

The drug for treating diabetes based on mRNA provided by the invention is characterized in that the GLP-1receptor stimulant can be effectively expressed by delivering the fusion mRNA of the in vitro synthesized encoded polypeptide GLP-1receptor stimulant and the Fc end of the antibody into the body, so that the normal blood sugar level is effectively reduced. Compared with the prior GLP-1 polypeptide medicament or Fc fusion polypeptide thereof, the mRNA preparation has higher bioavailability, quick in-vivo drug reaction, more stable effect, high expression quantity and long expression duration; on the other hand, the mRNA preparation has low immunogenicity, can quickly stimulate insulin production, has reliable biological safety, and greatly provides the medication comfort and compliance of patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts based on the drawings:

FIG. 1 shows a schematic diagram of the structure of the constructed mRNA;

FIG. 2 shows the effect of mRNA preparations encoding different Fc-segments fused to GLP-1receptor agonists on ADCC effect;

FIG. 3 shows the ability of mRNAs encoding GLP-1, GLP-1-mIgG4 Fc, GLP-1-linker-mIgG4 Fc polypeptides, respectively, to activate the GLP-1 receptor;

FIG. 4 shows IL-8 and TNF-. alpha.expression levels (pg/mL) in rat serum;

FIG. 5 shows the change in blood glucose concentration in rats after injection of mRNA liposome formulation;

FIG. 6 shows the amount of polypeptide GLP-1receptor agonist in rat blood injected with liposomal formulations of mRNA.

Detailed Description

The invention provides an mRNA medicinal preparation for treating diabetes, a preparation method and application thereof, and a person skilled in the art can realize the preparation by properly improving process parameters by referring to the content. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

TABLE 2 amino acid 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 illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.

EXAMPLE 1 construction and amplification of recombinant plasmids

1. The cDNA sequence of the Fc fusion mRNA of the polypeptide GLP-1receptor agonist of SEQ ID No.10 and the antibody IgG4 after partial site mutation is synthesized. The GLP-1receptor agonist is connected with the polypeptide sequence of the antibody Fc through a flexible protein linker of GGGGSGGGGSGGGGSA, and the front end of the GLP-1receptor agonist is connected with a signal peptide sequence shown in SEQ ID No. 31. The fusion polypeptide sequence encoded by the target mRNA is designated GLP-1-linker-mIgG4 Fc.

2. The DNA sequence encoding GLP-1receptor agonist polypeptide and antibody Fc fragment fusion mRNA was ligated to the PRSF vector plasmid by SfiI ligation. First, the vector and the fragment were digested with enzyme. The enzyme digestion reaction system of the vector and the fragment is (50 mu L), and the DNA vector/fragment: 5 mu L of the solution; 10 × NEB Buffer: 5 mu L of the solution; ddH ₂ O: 40 mu L of the solution; uniformly mixing the reaction system, and reacting at 50 ℃ for 2 h; subsequently, ligation of the vector and the fragment was performed. The reaction product after enzyme digestion was recovered as gel, and then diluted with 30. mu.L ddH ₂ And (3) after dissolving the O, using the NanoDrop to measure the concentration of the gel recovery product, wherein the concentration of the product obtained after the carrier is subjected to enzyme cutting and gel recovery is 50 ng/. mu.L, and the concentration of the product obtained after the PCR fragment is subjected to enzyme cutting and gel recovery is 60 ng/. mu.L. The vector and the fragment were added to a reaction system in a molar ratio of 1:3, as shown in Table 3 (20. mu.L system) in this example, under the conditions of 16 ℃ and overnight incubation:

table 3: enzyme digestion ligation reaction system

Components	Volume (mu L)
		10x Buffer	2
Carrier	1
		Fragments	2
T4 ligase	1
		ddH 2 O	44

3. The ligated plasmid was transformed into E.coli and cultured on solid medium overnight. First, commercial sensory state DH5 α was taken out of a-80 ℃ refrigerator and placed on ice to melt; then, 5. mu.L of the post-ligation reaction solution was added to 100. mu.L of the commercially acceptable medium and gently shaken to uniformly distribute the post-ligation reaction solution in the acceptable medium. Placing the competence added with the ligation reaction solution on ice for 30min, thermally shocking at 42 ℃ for 90s, and immediately placing the competence on ice for 2 min; subsequently, 800. mu.L of non-resistant LB medium was added thereto, placed on a shaker, and shaken at 37 ℃ for 50 min; and finally, slightly rotating the amplified competence after heavy shaking for 30s at the rotating speed of 4000rpm, pouring out the supernatant, uniformly coating the sediment at the bottom of the EP tube on LB, and pouring the sediment in an incubator for overnight culture.

4. And carrying out colony PCR, and selecting an escherichia coli colony with a correct strip size for sequencing. First, transformants on an LB plate were randomly picked at room temperature, and single colonies were picked with a sterilized toothpick or a pipette tip, and the picked colonies were marked. Then, the toothpick or tip with the adhered cells was placed in a corresponding PCR reaction plate equipped with a PCR tube or 96 wells, and the colony PCR reaction solution was as shown in Table 4 below (25. mu.L of the reaction system). Finally, the PCR mixture mixed with the thalli is placed in a PCR instrument for amplification.

TABLE 4 colony PCR reaction System

Components	Volume (μ L)
		Taq	47
Primer F	1
		Primer R	1
Bacterial colony

TABLE 5 colony PCR reaction conditions

5. E.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 a correct sequence after sequencing, and shaking the colony in a shaking table at 37 ℃ for 1h for amplification; subsequently, the amplified bacterial liquid is added into 100mL LB culture medium containing kanamycin resistance, and the bacterial liquid is shaken overnight at 37 ℃; finally, according to the TIANGEN kit instruction, extracting endotoxin-free plasmids, measuring the concentration of the extracted plasmids by using NanoDrop, finding that the concentration of the extracted plasmids is 809 ng/. mu.L, and placing the plasmids in a refrigerator at the temperature of 20 ℃ below zero for further storage.

Example 2 In Vitro Transcription (IVT) of target mRNA fragments

1. PCR of the target fragment: after obtaining the purified plasmid in large quantities, the DNA sequence encoding the target mRNA, including the T7 sequence, was amplified using high fidelity Taq enzyme. The PCR amplification system is shown in Table 6 (50. mu.L reaction). The amplified reaction solution was added to 1% agarose gel, and electrophoresed for 20min under constant voltage of 100V.

TABLE 6PCR reaction System

Components	Volume (μ L)
		Taq	47
Primer F	1
		Primer R	1
Plasmid template	1

TABLE 7 PCR reaction conditions for the target fragments

2. Purification of the target PCR fragment: firstly, cutting off fragments with correct sizes observed under an ultraviolet lamp, adding 700 mu L of GSB solution, and dissolving in water bath at 60 ℃ for 10 min; then, cooling the dissolved glue solution together with the target fragment to room temperature, and pouring the glue solution into a glue recovery column; then, centrifuging at the normal temperature for 30s at the rotating speed of 12000rpm, rinsing twice by using a Wash Buffer, and drying the gel recovery column in the air; subsequently, 30. mu.L of ddH having a temperature of 60 ℃ was added thereto ₂ O, standing for 2min at room temperature;finally, centrifuging at 12000rpm for 3min at normal temperature, collecting the centrifugate, and measuring the concentration of the PCR product to be 58 ng/. mu.L by using NanoDrop.

3. The required target mRNA is synthesized in vitro by adopting a one-step capping technology: first, the PCR product was added to 100. mu.L of RNase-Free EP tube together with a commercial Cap analog of mRNA, CleanCap AG, and T7 RNA polymerase, and the reaction system was as shown in Table 8 (10. mu.L); subsequently, the reaction system was left at 37 ℃ for 3 hours.

TABLE 8 one-step in vitro capping transcription System

Components	Volume of
		ddH 2 O	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 was purified by oligo (dT) -cellulose column chromatography as follows: first, the oligo (dT) -cellulose column was pretreated: (1) and 0.5g to 1.0g of oligo (dT) cellulose is suspended by 0.1mol/L NaOH. (2) The suspension was placed in a Pasteur pipette filled with DEPC water-treated, autoclaved glass wool, and the bed was about 0.5mL to 1.0mL, and oligo (dT) cellulose was washed with 3 column volumes of RNase-free sterile double distilled water. (3) Subsequently, the oligo (dT) cellulose was washed with 3-5 times the bed wash buffer I until the effluent pH was less than 8.0. (4) Pouring the treated oligo (dT) cellulose out of the Pasteur pipette, suspending the cellulose in a suitable bed washing buffer I at a concentration of about 0.1g/mL, and storing the cellulose at 4 ℃ for later 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 quickly placed on ice to cool. (6) Adding a certain volume of 5mol/L NaCl to adjust the concentration of salt in the RNA solution to 0.5 mol/L; finally, the mRNA in the reaction solution was purified: (7) resuspending oligo (dT) -cellulose with a pipette and taking the appropriate amountoligo (dT) -cellulose was added to the RNA sample, the lid was closed, and oligo (dT) -cellulose was mixed with the in vitro transcription reaction solution by inverting several times, incubated in a 37 ℃ water bath and gently shaken for 15 min. The volume/mass ratio of oligo (dT) -cellulose to the in vitro transcription reaction solution was 1:1, i.e., 1mL of oligo (dT) -cellulose was required for a total amount of about 0.1mg of mRNA. (8) Taking 15 ml syringe, fastening the front end of the syringe by using a glass wool plug sterilized at high temperature, fixing the syringe on a bracket without RNase, adding oligo (dT) -cellulose/RNA suspension into the syringe, pushing the plug to the bottom, and collecting the effluent liquid, namely collecting the liquid containing the unbound RNA into a centrifuge tube without RNase. (9) The wash buffer was slowly aspirated by syringe, gently shaken, and the mRNA-oligo (dT) -cellulose was thoroughly suspended, the stopper pushed, and the eluate collected using an RNase-Free centrifuge tube. Determining the OD of each tube ₂₆₀ Elution was prepared when the OD value in the eluate was 0. (10) The RNase-Free double distilled water was slowly aspirated into the syringe by syringe, the mRNA-oligo (dT) -cellulose was resuspended thoroughly, the stopper was pushed, and the resulting mixture was dispensed into RNase-Free centrifuge tubes at a bed volume of 1/3 to 1/2. (11) Determining the OD of each tube ₂₆₀ And combining the eluates containing RNA. At 4 ℃, 2500g of the mixture is centrifuged for 2min to 3min, and the supernatant is transferred to a new RNase-Free centrifuge tube. (12) 3mol/L NaAc (pH5.2) in an amount of 1/10 was added to the supernatant, and ice-cold ethanol in an amount of 2.5 times the volume was added thereto, followed by mixing, and then, precipitation was carried out at-20 ℃ for 30min or standing overnight. (13) Centrifugation was carried out at 12000rpm for 15min at 4 ℃ and the supernatant carefully discarded. The precipitate was rinsed with 70% ethanol and centrifuged at 12000g for 5min at 4 deg.C, and the supernatant was carefully discarded. Dissolving the mRNA pellet in the appropriate volume of RNase-Free ddH ₂ And O. The purified and concentrated mRNA was assayed at 1. mu.g/. mu.L using a NanoDrop assay.

The target mRNA is prepared by purification, the 5 ' end of the target mRNA is sequentially connected with the signal peptide, the 5 ' -UTR and the 5 ' -CAP, the 3 ' end is sequentially connected with the 3 ' -UTR and the poly A with the length of 150bp, and the ORF region sequence of the mRNA is shown in Table 2. The polypeptide sequence coded by the target mRNA is GLP-1-linker-mIgG4 Fc.

EXAMPLE 3 preparation of Liposome formulations for fusion of polypeptide GLP-1receptor agonists with the Fc region of antibodies

1. Respectively dissolving cationic lipid, PEG-lipid, amphoteric phospholipid and cholesterol into chloroform in a round-bottom flask according to the formula of the formula shown in the table 8, and performing rotary evaporation at the water bath temperature of 50-70 ℃ to uniformly form a film on the lipid at the bottom of the round-bottom flask;

2. adding the mRNA prepared in the example 2 and a citric acid solution with pH of 5.5 into a round-bottom flask after film formation, and dispersing the film in the mRNA solution by shaking to obtain a multi-chamber liposome;

3. carrying out probe ultrasonic treatment on the obtained multilayer liposome solution for 5-20 min, wherein the ultrasonic working time is 1-5 s, the interval is 2-6 s, and the working power is 220W, so as to obtain a monolayer liposome with relatively uniform particle size;

4. the obtained unilamellar liposome is filtered by a filter membrane of 100nm and extruded at 50-70 ℃ to obtain a liposome preparation which is uniform in particle size and loaded with encoded polypeptide GLP-1receptor agonist and antibody Fc fusion mRNA;

5. and placing the liposome preparation loaded with the target mRNA in a micro gel centrifugal column, centrifuging at the normal temperature and the speed of 2000rpm/min for 5min, concentrating and purifying the prepared 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. Preparing liposome preparations with prescriptions 1-4 respectively.

TABLE 9 lipid material recipe ratio

EXAMPLE 4 characterization of self-amplifying mRNA Liposomal formulations of polypeptide GLP-1receptor agonists

1. Measurement of particle size potential of the prepared liposomes. The liposomes prepared in formulation 1 of example 3 were diluted 1x with PBS a hundred fold. The background of the sample is measured by PBS by a Mastersizer 3000, after the background is measured, the dispersed sample is added, and when the concentration of the dispersion system is stable, the potential and the particle size of the liposome loaded with the target mRNA are measured. Finally, the particle size distribution of the obtained cationic liposome is measured to be between 100nm and 120nm, and the Zeta potential value is about (+10mV) to (+14 mV);

2. and (3) determining the entrapment rate of the prepared liposome to mRNA. The liposome preparation (formula 1-4) prepared in example 3 is added with methanol-chloroform (volume ratio 1:2) in equal amount, and is mixed uniformly to break emulsion, and simultaneously, RiboGreen is adopted to determine the entrapment rate of the liposome to target mRNA. The specific determination steps are as follows: (1) preparing the demulsified liposome and placing the liposome in an ice bath. Then, setting the excitation wavelength of a fluorescence spectrophotometer to be 485nm and the emission wavelength to be 530 nm; (2) melting the regenanta staining solution in the refrigerator kit at-20 ℃ at room temperature, adding 2.5 mu L of regenata into 500 mu L of regenatb diluent, uniformly mixing, wrapping by tinfoil, and placing in a dark room for later use; (3) 5 EP tubes of 1.5mL are labeled separately as tubes No. 1/2/3/4/5, and 100. mu.L of ReagenB are added separately. Subsequently, 100. mu.L of ReagentC was added to tube I, mixed well and serially diluted in gradient so that the total amount of RNA in tube 1/2/3/4/5 became concentrations of 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL and 0. mu.g/mL, respectively. And moving 100 mu L of the prepared mixed solution of Reagent A and Reagent B 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 the RNA standard substances with different gradient concentrations and corresponding RFU; (4) and (4) taking 100 mu L of prepared dyeing working solution of reagentA and reagentB again to 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, wherein the subsequent steps are the same as the step 3. When 2mg/mL of mRNA was added, the encapsulation concentration of mRNA of the liposome prepared was 1.88mg/mL, and the encapsulation efficiency of the liposome to GLP-1receptor agonist-antibody Fc fusion mRNA was finally 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 cellular protein expression level identification of Liposome formulations based on fusion mRNA of polypeptide GLP-1receptor agonist-antibody Fc fragment

1. Inoculating HEK-293T cells into a 6-well plate one day in advance, and adopting high-glucose DMEM containing 10% FBS for overnight culture to adhere to the walls on the next day and grow to 70% confluency;

2. transfecting the adherent cells with the prepared liposome preparation (formula 1 in example 3) loaded with the polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA, standing in an incubator at 37 ℃ for 6h, and replacing with fresh culture solution;

3. and (3) taking the cell culture solution supernatant, adopting an Elisa kit of an antibody Fc segment, operating according to the instructions of the Elisa kit, determining the content of the target GLP-1receptor agonist polypeptide secreted by the cells, and finally determining that the content of the GLP-1receptor agonist polypeptide in the 6-well plate is 300 pg/mL.

Example 6 ADCC Effect of liposome preparation of fusion mRNA based on polypeptide GLP-1receptor agonist-antibody Fc fragment in vitro assay

1. According to the method described in examples 1 to 2, target mRNAs encoding the following fusion polypeptides were prepared, respectively: the polypeptide GLP-1receptor agonist of SEQ ID No.10 is linked to the Fc region of IgG1, the Fc region of IgG4 and the Fc region of anti-IgG 4 after mutation by a flexible protein linker (GGGGSGGGGSGGGGSA) to the fusion polypeptide. 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 fusion polypeptide of GLP-1-linker-mIgG4 Fc is shown in example 2, and the sequence of ORF region of the target mRNA encoding the fusion polypeptide of GLP-1-linker-IgG1 Fc or the fusion polypeptide of GLP-1-linker-IgG4 Fc is shown in Table 2, the target mRNA is different from the sequence of ORF region, and the remaining signal peptide, 5 ' -UTR, 5 ' -Cap, 3 ' -UTR and Poly A sequence are the same as the corresponding portions of the target mRNA in example 2.

Then, the above 3 kinds of target mRNAs were prepared into nanoliposomes by referring to the formulation and preparation method of formulation 1 in example 3.

2. The liposome preparations of the polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA prepared as described above were transfected into cells, respectively, and cell supernatants containing GLP-1-linker-IgG1 Fc, GLP-1-linker-IgG4 Fc, and GLP-1-linker-mIgG4 Fc fusion polypeptides were collected by continuous culture for one week, respectively, and subjected to affinity chromatography using a ProteinA affinity chromatography column, and hydrophobic chromatography was further performed since the protein after affinity chromatography still contained degradation products and multimers. The fusion protein obtained by two-step purification shows that the purity of the protein is higher than 90% by HPLC analysis and SDS-PAGE analysis. The GLP-1-Fc fusion polypeptide obtained by purification is subjected to Western-blotting detection by respectively taking an anti-GLP-1 antibody and an anti-IgG Fc antibody as detection antibodies, and the fusion protein secreted by the cell is found to have better integrity. Adjusting the concentration of the purified GLP-1-Fc fusion polypeptide to 1mg/mL by PBS of pH7.4, and respectively diluting the GLP-1-linker-Fc fusion polypeptide obtained from the three vegetations 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 later use;

3. construction of lentivirus pWSLV07-EF-1 alpha-GLP-1 receptor-T2A-CMV-luThe ciferase vector and the packaging plasmid are transferred into 293T cells together for virus packaging; after 72h, the supernatant broth was collected and 1/5 volumes of PEG were added to it ₈₀₀₀ Standing at 4 ℃ overnight, centrifuging at 4000rpm for 30min, resuspending the centrifuged virus precipitate with PBS, respectively dripping into HeLa cells, sorting GFP positive cells, namely HeLa cell lines of high-expression GLP-1receptor and luciferase, by a flow cytometry sorter after 48h, and further performing amplification culture;

4. co-culturing the high expression GLP-1receptor and luciferase HeLa cell line obtained in the step 3 and human peripheral blood mononuclear cells separated from human blood cells, and respectively adding three groups of fusion polypeptide diluents obtained in the step 2) in the example 6. In the co-culture system, a HeLa cell line highly expressing GLP-1receptor and luciferase is used as a target cell for ADCC, and a human peripheral blood mononuclear cell separated from a human blood cell is used as an effector cell for ADCC. Adding different groups of GLP-1-Fc fusion polypeptides with different concentrations into a co-culture system for a certain time, adding cell lysis solution, lysing cells, adding a substrate D-Luciferin, detecting the OD value of target cells by a microplate luminescence detector, namely HeLa cells highly expressing GLP-1receptor and luciferase, reacting the generated luciferase with the added D-Luciferin to release chemiluminescence intensity, and reacting the luminescence intensity value of the luciferase with the ADCC effect of the GLP-1linker-Fc fusion polypeptides adopting different Fc segments.

The specific results are shown in figure 2, and the results show that the ACDD effect generated by the mRNA encoding the GLP-1-linker-IgG1 Fc fusion polypeptide is stronger than that of the mRNA encoding the GLP-1-linker-IgG4 Fc or GLP-1-linker-mIgG4 Fc fusion polypeptide. The low ADCC effect of the IgG4 Fc fragment fusion polypeptide after partial site mutation 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 in turn means a reduction in dose-dependent cellular cytotoxicity, and mRNAs encoding GLP-1-linker- -IgG4 Fc or GLP-1-linker-mIgG4 Fc fusion polypeptides will have fewer side effects when administered in vivo.

Example 7 Effect of GLP-1receptor agonist and intermediate linker in IgG Fc fragment on GLP-1receptor agonist function

1. Target mRNAs encoding the following polypeptides were prepared and purified, respectively, according to the method described in (1) in example 6: GLP-1 polypeptide, GLP-1-mIgG4 Fc polypeptide which is not connected with a Linker and GLP-1-Linker-mIgG4 Fc polypeptide sequence which is connected with the Linker sequence (GGGGSGGGGSGGGGSA) are respectively marked as GLP-1, GLP-1-mIgG4 Fc and GLP-1-Linker-mIgG4 Fc.

Among them, the target mRNA encoding the fusion polypeptide of GLP-1-linker-mIgG4 Fc is shown in example 2, and the sequences of the ORF regions of the target mRNA encoding the GLP-1 polypeptide or the GLP-1-mIgG4 Fc polypeptide which is not linked by a linker are shown in Table 2, respectively, and the target mRNA is different in the sequence of the ORF regions, and the remaining signal peptide, 5 ' UTR, 5 ' Cap, 3 ' UTR and Poly A sequence are the same as those of the target mRNA in example 2. Then, the above 3 kinds of target mRNAs were prepared into nanoliposomes by referring to the formulation and preparation method of formulation 1 in example 3. The liposome preparations of mRNA prepared as described above were transfected into cells respectively according to the method of step (2) of example 6.

2. Preparing a stable 293T cell line based on CRE-BLAM reporter system of lacZ high expression cAMP response and high expression GLP-1receptor according to the method described in (3) in example 6;

3. the stable transgenic reporter 293T cell line of step (2) above was seeded in 24-well plates and cultured overnight at 27 ℃. After the cells were attached, 10nM of GLP-1, GLP-1-mIgG4 Fc, GLP-1-linker-mIgG4 Fc diluted in serum-free DMEM, respectively, were added thereto. After 5h, 20. mu.L of the substrate lactamase was added thereto, and after 1h of adding the substrate, the fluorescence emitted was measured and used to indicate the amount of activated aAMP, which also reflects the intensity of the signal generated by the action of GLP-1receptor agonist on GLP-1 receptor.

The results are shown in FIG. 3. The experimental results show that when the mRNA encoding the fusion polypeptide in which GLP-1 is directly linked to the Fc segment of IgG is not linked by a suitable linker, the ability of the mRNA to activate the GLP-1receptor by GLP-1 in vivo is rather reduced; when mRNA encoding a fusion polypeptide of GLP-1 and IgG Fc fragments linked by a suitable linker, the mRNA in vivo would have 4 times the ability of GLP-1 to activate the GLP-1receptor as compared to the mRNA encoding GLP-1 alone.

Example 8 in vivo immunogenicity assessment of Liposomal pharmaceutical formulations based on polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA

1. Feeding SD rats under SPF condition in ventilated cages under 12h light and 12h dark circulation conditions;

2. injecting a normal saline injection of mRNA liposome (formula 1 of embodiment 2) of the polypeptide GLP-1receptor stimulant into different rats at the same part, and injecting a normal saline with the same dose and a commercial semaglutide injection into a control group;

3. after 24h, orbital bleeds were performed on different groups of rats, and the serum was separated and the concentrations of IL-8 and TNF- α in the serum were determined using an Elisa kit, respectively.

The determination result is shown in figure 4, and the results show that the immunogenicity of the prepared liposome pharmaceutical preparation of the fusion mRNA of the encoded polypeptide GLP-1receptor agonist-antibody Fc is not greatly different from that of the commercially available semaglutide, and the immunogenicity is not different in statistical significance.

Example 9 in vivo potency evaluation of liposomal pharmaceutical formulations based on mRNA encoding polypeptide GLP-1receptor agonist-antibody Fc fusion

1. Culturing male rats with the weight of 160 g-200 g and the SPF grade of 4W-6W in an aeration cage under the conditions of 12h of illumination and 12h of dark circulation;

2. injecting streptozotocin into abdominal cavity of the rat for 7 days, supplementing with high sugar diet, measuring body weight and blood sugar concentration 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 liposomal mRNA pharmaceutical formulation of formulation 1 of example 2 into model rats to give a final concentration of 200. mu.g/rat of active ingredient mRNA per rat per injection;

4. after injection of the formulation, the rats were bled from the orbit every day, centrifuged at 3000rpm/min for 10min, and the serum was isolated for use. When the blood glucose protein in rat serum is measured, firstly, preparing o-phenylmethylamine, 55.56mmol/L standard glucose storage liquid and 5.56mmol/L standard glucose application liquid; then, taking 10mL of 3 test tubes, adding 5mL of o-toluidine reagent, respectively adding 0.1mL of distilled water, standard glucose application solution and a serum sample into the three test tubes, placing the three test tubes in an electric heating thermostat, boiling for 20min, taking out and cooling; finally, using a 721 type spectrophotometer, zeroing a cuvette with an optical path of 1cm and a blank tube at a wavelength of 630nm, and reading the absorbance values A of the sample tube and the standard tube respectively. Finally, the blood glucose concentration of the sample (mmol/L) ═ a sample/a standard × 5.56 mmol/L.

The results are shown in figure 5, which shows that the blood glucose concentration of rats injected with liposome drug formulation encoding polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA gradually decreased and finally gradually returned to normal steady-state level.

Example 10 in vivo half-life and bioavailability of Liposomal formulations based on mRNA encoding polypeptide GLP-1receptor agonist-antibody Fc fusions

1. Feeding SD rats under the SPF condition in a ventilation cage under the conditions of 12h of light and 12h of dark circulation;

2. injecting 200 mug/mL of normal saline injection of liposome (formula 1 in example 2) encoding polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA into different rats at the same part, and injecting the same amount of commercially available semaglutide injection into a control group;

3. the rats were bled from the orbit every day, and the serum was separated and assayed for GLP-1 content by the Elisa kit according to the instructions of the Elisa kit.

The results are shown in figure 6, the corresponding GLP-1 protein level in the serum of the rat injected with the protein drugs is reduced rapidly and is reduced to below 1/3 at the beginning at 14 days, and the preparation injected with the mRNA for encoding the polypeptide GLP-1receptor agonist-antibody Fc fusion can maintain the expression of the GLP-1receptor agonist at a higher level within 14 days without obvious reduction. Therefore, the mRNA liposome preparation of the present invention can continuously express the GLP-1 protein for a long period of time, and exert long-lasting effects.

The embodiment shows that the medicine based on the polypeptide GLP-1receptor agonist-antibody Fc fusion mRNA has long action duration, short onset time and low side effect, and can obviously reduce the blood sugar level of the patients with type II diabetes.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> Beijing Hospital

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Lys Asn Asp Trp Lys His Asn Leu Thr Gln

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Tyr Ala Glu Gly Thr Phe Ile Ser Asp Tyr Ser Ile Ala Met Asp Lys

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His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

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Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

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Ser Gly Ala Pro Pro Ser Lys Lys Lys Lys Lys Lys

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His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

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Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

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His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu

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Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

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Ser Gly Ala Pro Pro Pro Ser

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His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

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His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

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His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn

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gaatttattg cgtggctggt gcgcggccgc ggc 93

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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acccag 126

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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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<211> 132

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60

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<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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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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catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60

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<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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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 (10)

1. A mRNA pharmaceutical formulation characterized in that it comprises mRNA and a delivery lipid;

   the mRNA includes: a nucleic acid encoding a GLP-1receptor agonist and a nucleic acid encoding an Fc region of an IgG antibody.
2. 2. The mRNA pharmaceutical preparation of claim 1,

   the GLP-1receptor agonist is selected from any one of I) to III):

   I) at least one polypeptide of an amino acid sequence shown in any one of SEQ ID No. 1-13;

   II), a polypeptide having at least 60% identity to the polypeptide of I);

   III) in the amino acid sequence of the polypeptide of I), one or more amino acids are substituted, deleted and/or inserted;

   the IgG antibody Fc region 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 in which one or more amino acids are substituted, deleted and/or inserted in the Fc region of A).
3. 3. The mRNA pharmaceutical preparation of claim 2,

   the nucleic acid sequence of the GLP-1receptor agonist is selected from any one of the following items i) to iii):

   i) at least one of the nucleic acid sequences shown in any one of SEQ ID No. 14-26;

   ii) a sequence having at least 60% identity to the sequence of i);

   iii) a sequence in which one or more nucleotides are substituted, deleted and/or inserted in the sequence of i);

   the nucleic acid sequence encoding the Fc region of an IgG antibody is selected from any one of a) to c):

   a) and at least one of the nucleic acid sequences shown in any one of SEQ ID No. 27-30;

   b) a sequence having at least 60% identity to the sequence of a);

   c) a sequence in which one or more nucleotides are substituted, deleted and/or inserted in the sequence described in a).
4. 4. The pharmaceutical formulation of claim 1, wherein the mRNA comprises, sequentially from 5 'to 3': 5 ' -CAP, 5 ' -UTR, a signal peptide, a nucleic acid encoding a GLP-1receptor agonist, a linker, a nucleic acid encoding an Fc region of an IgG antibody, 3 ' -UTR, and PolyA; wherein, the linker is selected from any one of (i) to (iii):

   the amino acid sequence of the linker is GGGGSGGGGSGGGGSA, GSAGSAAGSGEF, GGGGGGGG, EGKSSGSGSESKST or KESGSVSSEQLAQFRSLD;

   ② polypeptide which has at least 70% of identity with the amino acid sequence of the linker;

   and (c) polypeptide with one or more amino acids substituted, deleted and/or inserted in the sequence of the amino acids.
5. 5. The pharmaceutical preparation of claim 4, wherein the amino acid sequence of the signal peptide in the mRNA is shown in SEQ ID No. 31.
6. 6. The pharmaceutical formulation of claim 1, wherein the delivery lipid consists of a cationic lipid, PEG-lipid, ampholytic phospholipid, 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-carboxamido) ethyl ] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1, 2-dioleyloxy-N, n-dimethylaminopropane (DLinDMA), Dioctadecylaminoglycylspermine (DOGS), 1, 2-dinenyloxy-N, N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2- (cholest-5-en-3- β -oxybut-4-oxy) -1- (cis, cis-9, 12-octadecenyloxy) propane (CLinDMA), preferably 3- (N, N-dioleylamino) -1, 2-propanediol, N- (1- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride, 1, 2-dilinonyloxy-N, N-dimethylaminopropane;

   the PEG-lipid is selected from PEG-phospholipid, PEG-dilauryloxypropyl, PEG-dialkoxypropyl, and PEG-diacylglycerol, preferably one or more of PEG-diacylglycerol and PEG-phospholipid;

   the ampholytic phospholipid is selected from egg yolk phosphatidylcholine (EPC), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylcholine (DSPC), dimyristoyl-phosphatidylethanolamine (DMPE), dioelaidoyl-phosphatidylethanolamine (DEPE).
7. 7. The pharmaceutical formulation of claim 6, wherein the molar ratio of the cationic lipid, PEG-lipid, amphophospholipid and cholesterol is (45-55): (1-2): (9-11): (33.5 to 42).
8. 8. The pharmaceutical preparation according to any one of claims 1 to 7, wherein the concentration of the mRNA in the pharmaceutical preparation is from 100 μ g/mL to 3000 μ g/mL.
9. 9. A process for preparing a pharmaceutical formulation according to any one of claims 1 to 8, comprising: the mRNA is encapsulated into lipid delivery using either a thin film hydration method or an organic solvent evaporation method.
10. 10. Use of the mRNA pharmaceutical formulation according to any one of claims 1 to 8 for the preparation of a medicament for the prophylaxis and/or treatment of diabetes.

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