In vivo self-assembling system targeting central nervous system for treating diabetes

Abstract

The present application belongs to the technical field of biomedicine, and relates to an in vivo self-assembling system targeting the central nervous system for treating diabetes. The present application provides an in vivo self-assembling system targeting the central nervous system. The in vivo self-assembling system targeting the central nervous system is a recombinant plasmid expressing a nucleic acid molecule, wherein the nucleic acid molecule encodes a fibroblast growth factor-based fusion protein, and the fibroblast growth factor-based fusion protein comprises a propeptide, a peptide targeting the central nervous system, a linker peptide, and a fibroblast growth factor variant having an amino acid sequence as set forth in SEQ ID NO.1, which are sequentially linked. The in vivo self-assembling system targeting the central nervous system can cross the blood-brain barrier and specifically reach the central nervous system via routes other than intracranial injection, such as subcutaneous injection and intravenous injection. The system exerts a long-lasting blood glucose-lowering effect, without causing adverse reactions, thereby retaining the anti-diabetic activity of fibroblast growth factors and avoiding the tumorigenic risks thereof.

Description

In vivo self-assembly central targeting system for treating diabetes

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202410897716X, filed on July 5, 2024, and entitled “In vivo self-assembly central targeting system for treating diabetes”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to an in vivo self-assembly central targeting system for treating diabetes, belonging to the field of biomedicine and medical technology. BACKGROUND

[0004] Diabetes is a group of metabolic diseases characterized by chronically elevated blood glucose levels, mainly including type 1 diabetes and type 2 diabetes. Among them, the main cause of type 2 diabetes is insulin resistance or insufficient insulin secretion caused by various reasons. The clinical manifestations of type 2 diabetes are polyuria, polydipsia, polyphagia, weight loss or weight loss. If blood glucose is not controlled for a long time, it will cause chronic progressive lesions, functional decline or even failure of the eyes, kidneys, nerves, hearts, blood vessels and other tissues and organs, and may cause acute severe metabolic disorders, and eventually lead to blindness, amputation and kidney failure and other complications.

[0005] Many drugs have been used in the treatment of diabetes in clinical practice, but at present, no hypoglycemic drug can cure diabetes, and diabetes patients need to take medicine for life, and the existing hypoglycemic drugs cannot achieve sustained remission of type 2 diabetes. At present, the treatment of type 2 diabetes with hypoglycemic drugs mostly needs to be administered at least once a day, and the longest acting semeglutide, tirzepide and other drugs on the market also need to be injected once a week. Therefore, it is urgent to develop a long-term effective treatment method for type 2 diabetes.

[0006] Fibroblast growth factors (FGFs) are polypeptides secreted by the pituitary and hypothalamus, and the family has 22 members in common. Studies have shown that several members of the FGF family, including FGF19, FGF21, FGF1 and FGF4, have great potential in the treatment of type 2 diabetes. These FGF members have strong anti-hyperglycemic effects when administered peripherally, and, notably, a single intracerebroventricular (i.c.v.) administration of FGF1 and FGF4 can cause sustained diabetes remission in type 2 diabetic rodents. Therefore, the development of long-acting type 2 diabetes treatment drugs based on FGF1 and FGF4 is very promising. However, in addition to the anti-diabetic activity, FGF1 and FGF4 also have mitogenic activity, which makes FGF1 / FGF4 administration have the risk of tumorigenesis, which seriously inhibits the development of type 2 diabetes treatment drugs based on FGF1 / FGF4.

[0007] In order to overcome the problem of tumorigenesis caused by FGF1 / FGF4 administration, further studies have found that the mitogenic activity (i.e. cell proliferation activity) and the anti-diabetic activity of FGF1 and FGF4 can be separated, wherein the mitogenic activity is mainly induced by FGFR3 and FGFR4, and the anti-diabetic activity is mainly mediated by FGFR1. Therefore, the mitogenic characteristics of FGF1 / FGF4 can be reduced by developing engineered FGF1 / FGF4 variants to inhibit tumorigenesis. However, whether these FGF1 / FGF4 variants can still maintain anti-diabetic activity in animals has not been tested. In addition, in terms of drug administration, FGF1 and FGF4 need to be injected centrally, and there is a problem of low patient compliance. There are also studies trying to develop intranasal delivery systems for FGF1 and FGF4. However, it is not yet clear whether intranasal delivery is sufficient to provide the same long-term anti-diabetic activity as central injection. SUMMARY

[0008] To solve the above problems, the present application provides an in vivo self-assembly central targeting system for treating diabetes, the in vivo self-assembly central targeting system is a recombinant plasmid expressing a nucleic acid molecule; the nucleic acid molecule encodes a fibroblast growth factor-based fusion protein; the fibroblast growth factor-based fusion protein comprises a propeptide, a central targeting peptide, a linker peptide and a fibroblast growth factor variant connected in sequence; the amino acid sequence of the fibroblast growth factor variant is shown in SEQ ID NO. 1.

[0009] In an embodiment of the present application, the amino acid sequence of the propeptide is shown in SEQ ID NO. 2.

[0010] In an embodiment of the present application, the central targeting peptide comprises a brain-targeting chimeric rabies virus glycoprotein fragment peptide (RVG), a brain-targeting rabies virus glycoprotein derivative peptide (RDP), a glioma-targeting peptide (Angiopep-2), a transferrin-targeting peptide (THR), a cell-penetrating peptide (Peptide-22), and / or an apolipoprotein E fragment peptide (ApoE(159-167)2); the amino acid sequence of the brain-targeting chimeric rabies virus glycoprotein fragment peptide is shown in SEQ ID NO. 3.

[0011] In an embodiment of the present application, the connecting peptide is a flexible connecting peptide (flexible linker); the flexible connecting peptide comprises a GS connecting peptide (GS linker); the amino acid sequence of the GS connecting peptide is shown in SEQ ID NO. 4.

[0012] In an embodiment of the present application, the fibroblast growth factor-based fusion protein consists of a propeptide, a central targeting peptide, a connecting peptide, and a fibroblast growth factor variant connected in sequence.

[0013] In an embodiment of the present application, the amino acid sequence of the fibroblast growth factor-based fusion protein is shown in SEQ ID NO. 5.

[0014] In an embodiment of the present application, the vector of the recombinant plasmid is a pcDNA6.2-GW / EmGFP-miR plasmid and / or a pcDNA3.1 plasmid.

[0015] In an embodiment of the present application, the method for preparing the recombinant plasmid comprises linearizing a vector to obtain a linearized vector; and connecting the linearized vector and a nucleic acid molecule to obtain a recombinant plasmid.

[0016] The present application also provides a fibroblast growth factor-based fusion protein comprising a propeptide, a central targeting peptide, a connecting peptide, and a fibroblast growth factor variant (FGF1 non-mitogenic repressor FGF1 ΔHBS ) connected in sequence; the amino acid sequence of the fibroblast growth factor variant is shown in SEQ ID NO. 1.

[0017] In an embodiment of the present application, the amino acid sequence of the propeptide is shown in SEQ ID NO. 2.

[0018] In an embodiment of the present application, the central targeting peptide comprises a brain-targeting chimeric rabies virus glycoprotein fragment peptide (RVG), a brain-targeting rabies virus glycoprotein-derived peptide (RDP), a glioma-targeting peptide (Angiopep-2), a transferrin-targeting peptide (THR), a cell-penetrating peptide (Peptide-22), and / or an apolipoprotein E fragment peptide (ApoE(159-167)2); the amino acid sequence of the brain-targeting chimeric rabies virus glycoprotein fragment peptide is shown in SEQ ID NO. 3.

[0019] In an embodiment of the present application, the connecting peptide is a flexible connecting peptide (flexible linker); the flexible connecting peptide comprises a GS connecting peptide (GS linker); the amino acid sequence of the GS connecting peptide is shown in SEQ ID NO. 4.

[0020] In an embodiment of the present application, the fibroblast growth factor-based fusion protein consists of a propeptide, a central targeting peptide, a connecting peptide, and a fibroblast growth factor variant connected in sequence.

[0021] In an embodiment of the present application, the amino acid sequence of the fibroblast growth factor-based fusion protein is shown in SEQ ID NO. 5.

[0022] The present application also provides a nucleic acid molecule encoding the in vivo self-assembled central targeting system or the fibroblast growth factor-based fusion protein described above.

[0023] In an embodiment of the present application, when encoding the fibroblast growth factor-based fusion protein described above, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 6.

[0024] In an embodiment of the present application, when encoding the in vivo self-assembled central targeting system described above, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 7.

[0025] The present application also provides a host cell carrying the in vivo self-assembled central targeting system described above; or, the host cell expresses the fibroblast growth factor-based fusion protein described above; or, the genome of the host cell is integrated with the nucleic acid molecule described above.

[0026] In an embodiment of the present application, the host cell comprises Escherichia coli, insect cells, mammalian cells, Bacillus subtilis, and / or yeast.

[0027] The application also provides application of the in-vivo self-assembled central targeting system, the fusion protein based on the fibroblast growth factor, the nucleic acid molecule or the host cell in preparation of a drug for treating diabetes.

[0028] In an embodiment of the application, the diabetes is type 2 diabetes.

[0029] The application also provides a drug for treating diabetes, which comprises the in-vivo self-assembled central targeting system, the fusion protein based on the fibroblast growth factor, the nucleic acid molecule and / or the host cell.

[0030] In an embodiment of the application, the diabetes is type 2 diabetes.

[0031] In an embodiment of the application, the drug for treating diabetes further comprises a drug carrier; the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0032] In an embodiment of the application, the nanoparticles comprise lipid nanoparticles (LNP).

[0033] In an embodiment of the application, the drug for treating diabetes is the in-vivo self-assembled central targeting system wrapped by the lipid nanoparticles.

[0034] In an embodiment of the application, components of the lipid nanoparticles comprise ionizable lipids, cholesterol, helper lipids and polyethylene glycol (PEG) lipids; the ionizable lipids comprise cationic liposome SM-102; the helper lipids comprise distearoylphosphatidylcholine (DSPC); and the PEG lipids comprise PEG-DMG-2000.

[0035] The technical solution of the application has the following advantages:

[0036] 1. The application provides an in-vivo self-assembled central targeting system for treating diabetes, which is a recombinant plasmid expressing a nucleic acid molecule, wherein the nucleic acid molecule encodes a fusion protein based on a fibroblast growth factor, and the fusion protein based on the fibroblast growth factor comprises a pre-peptide, a central targeting peptide, a connecting peptide and a fibroblast growth factor variant (FGF1 non-mitogenic mutant FGF1 ΔHBS);the amino acid sequence of the fibroblast growth factor variant is shown as SEQ ID NO. 1. The fusion protein expressed by the in vivo self-assembly central targeting system has a preposed prepeptide, and researches show that the in vivo self-assembly central targeting system can pass through the blood-brain barrier by means other than intracranial injection (subcutaneous injection, intravenous injection) and specifically reach the central nervous system, play a lasting hypoglycemic effect (at least can last for 13 weeks of stable hypoglycemic effect), and does not cause adverse reactions, both retaining the anti-diabetic activity of fibroblast growth factor and avoiding the tumorigenic risk of fibroblast growth factor, at the same time, solving the problem of low patient compliance caused by intracranial injection, providing more ideas for the development of central targeting drugs, and having great application prospect in the treatment of type 2 diabetes.

[0037] 2. The application provides a fusion protein based on fibroblast growth factor, which comprises, in sequence, a prepeptide, a central targeting peptide, a connecting peptide and a fibroblast growth factor variant (FGF1 non-mitogenic mutant FGF1 ΔHBS );the amino acid sequence of the fibroblast growth factor variant is shown as SEQ ID NO. 1. The fusion protein has a preposed prepeptide, and researches show that after the fusion protein is integrated into an expression vector to form an in vivo self-assembly central targeting system, the in vivo self-assembly central targeting system can pass through the blood-brain barrier by means other than intracranial injection (subcutaneous injection, intravenous injection) and specifically reach the central nervous system, play a lasting hypoglycemic effect (at least can last for 13 weeks of stable hypoglycemic effect), and does not cause adverse reactions, both retaining the anti-diabetic activity of fibroblast growth factor and avoiding the tumorigenic risk of fibroblast growth factor, at the same time, solving the problem of low patient compliance caused by intracranial injection, providing more ideas for the development of central targeting drugs, and having great application prospect in the treatment of type 2 diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1: Plasmid map of recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS .

[0039] Figure 2: Schematic diagram of administration of type 2 diabetes mice by intravenous injection of recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS .

[0040] Figure 3: Schematic diagram of administration of type 2 diabetes mice by subcutaneous injection of LNP-wrapped recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS .

[0041] Figure 4: Drug administration effect of type 2 diabetes based on fusion protein (prepeptide-RVG-linker-FGF1 ΔHBS ).

[0042] Figure 5: Drug administration effects of different fusion proteins in the treatment of type 2 diabetes. Detailed Implementation

[0043] The following embodiments are provided to better understand this application and are not limited to the preferred embodiments described herein. They do not constitute a limitation on the content and scope of protection of this application. Any product that is the same as or similar to this application, derived by anyone under the guidance of this application or by combining features of this application with other prior art, falls within the scope of protection of this application.

[0044] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0045] Example 1-1: A fusion protein based on fibroblast growth factor

[0046] This embodiment provides a fusion protein based on fibroblast growth factor (propeptide-RVG-linker-FGF1). ΔHBS This fibroblast growth factor-based fusion protein is composed of a propeptide, a central targeting peptide, a linker peptide, and a fibroblast growth factor variant linked together in sequence, and its amino acid sequence is shown in SEQ ID NO.5.

[0047] Examples 1-2: An in vivo self-assembled central targeting system for the treatment of type 2 diabetes

[0048] This embodiment provides an in vivo self-assembling central nervous system targeting system for the treatment of type 2 diabetes. This in vivo self-assembling central nervous system targeting system is a recombinant plasmid pcDNA6.2-p-RVG-FGF1. ΔHBS This recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS To carry the fusion protein encoding Example 1-1 (propeptide-RVG-linker-FGF1) ΔHBS The pcDNA6.2-GW / EmGFP-miR plasmid of the gene is shown in SEQ ID NO.7 (see Figure 1 for plasmid map).

[0049] The preparation method of this in vivo self-assembled central targeting system for the treatment of type 2 diabetes is as follows:

[0050] The construction of the recombinant plasmid for treating type 2 diabetes was completed using the BLOCK-iT Pol II miR RNAi Designer of Invitrogen. The designer provided the sequences of two DNA oligos by which the gene (SEQ ID NO. 6) encoding the fusion protein (propeptide-RVG-linker-FGF1 ΔHBS ) of Example 1-1 was cloned into the pcDNA6.2-GW / EmGFP-miR linearized vector to obtain a ligation product (this process was completed by Jin Sui Biotech Co., Ltd.); the ligation product was transformed into E. coli competent cells DH5a (purchased from Tsingke, TSC01) to obtain a transformation product; the transformation product was streaked on LB agar plate medium (purchased from Thermo Fisher) containing 50 μg / mL ampicillin (Amp) and cultured at 37°C for 12 h, and a single colony was picked; the single colony was inoculated into 3 mL of LB liquid medium (purchased from Thermo Fisher) containing 50 μg / mL spectinomycin and cultured at 37°C for 14 h to obtain a bacterial solution; the recombinant plasmid in the bacterial solution was extracted and purified for sequencing, and the recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS was obtained upon successful sequencing. ΔHBS This recombinant plasmid pcDNA6.2-p-RVG-FGF1

[0051] Example 1-3: An LNP drug for treating type 2 diabetes

[0052] This example provides an LNP drug for treating type 2 diabetes, which is a lipid nanoparticle wrapping the recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS of Example 1-2; the lipid nanoparticle is a lipid delivery carrier based on four lipid components, cationic liposome SM-102, cholesterol, distearoyl phosphatidylcholine (DSPC), and PEG-DMG-2000 (the process of LNP wrapping the recombinant plasmid with the four lipid components was performed by Maianna (Shanghai) Instrument Technology Co., Ltd.).

[0053] Comparative Example 1-1: A fusion protein based on fibroblast growth factor

[0054] This comparative example provides a fusion protein based on fibroblast growth factor (targeting peptide RVG-linker-propeptide-FGF1 ΔHBS), which is sequentially connected by a central targeting peptide, a connecting peptide, a propeptide and a fibroblast growth factor variant, and has an amino acid sequence as shown in SEQ ID NO. 8.

[0055] Comparative Example 1-2: An in vivo self-assembly central targeting system for treating type 2 diabetes

[0056] The present comparative example provides an in vivo self-assembly central targeting system for treating type 2 diabetes, which is a recombinant plasmid pcDNA6.2-RVG-FGF1 ΔHBS , for treating type 2 diabetes. ΔHBS , which is a pcDNA6.2-GW / EmGFP-miR plasmid carrying a gene encoding the fusion protein (targeting peptide RVG-linker-propeptide-FGF1 ΔHBS ) of Comparative Example 1-1.

[0057] The preparation method of the in vivo self-assembly central targeting system for treating type 2 diabetes is as follows: on the basis of Example 1-1, the gene encoding the fusion protein (propeptide-RVG-linker-FGF1 ΔHBS ) of Example 1-1 is replaced by a gene encoding the fusion protein (targeting peptide RVG-linker-propeptide-FGF1 ΔHBS ) of Comparative Example 1-1 (nucleotide sequence as shown in SEQ ID NO. 9).

[0058] Comparative Example 1-3: An LNP drug for treating type 2 diabetes

[0059] The present comparative example provides an LNP drug for treating type 2 diabetes, which is a lipid nanoparticle encapsulating the recombinant plasmid pcDNA6.2-RVG-FGF1 ΔHBS of Comparative Example 1-2; the lipid nanoparticle is a lipid delivery carrier constructed based on four lipid components of cationic liposome SM-102, cholesterol, distearoylphosphatidylcholine (DSPC) and PEG-DMG-2000 (the process of LNP encapsulation of the recombinant plasmid using the four lipid components is performed by Maianna (Shanghai) Instrument Technology Co., Ltd.).

[0060] Comparative Example 2-1: A fibroblast growth factor-based fusion protein

[0061] The present comparative example provides a fibroblast growth factor-based fusion protein (propeptide-FGF1 ΔHBSa propeptide, a fibroblast growth factor variant, a linker, and a central targeting peptide RVG, in this order, and has an amino acid sequence as shown in SEQ ID NO. 10.

[0062] Comparative Example 2-2: An in vivo self-assembling central targeting system for treating type 2 diabetes

[0063] The present comparative example provides an in vivo self-assembling central targeting system for treating type 2 diabetes, which is a recombinant plasmid pcDNA6.2-FGF1 ΔHBS -RVG for treating type 2 diabetes, which is a pcDNA6.2-GW / EmGFP-miR plasmid carrying a gene encoding the fusion protein (propeptide-FGF1 ΔHBS -RVG of Comparative Example 2-1. ΔHBS -linker-targeting peptide RVG.

[0064] The preparation method of the in vivo self-assembling central targeting system for treating type 2 diabetes is as follows: on the basis of Example 1-1, the gene encoding the fusion protein (propeptide-RVG-linker-FGF1 ΔHBS ) of Example 1-1 is replaced by a gene encoding the fusion protein (propeptide-FGF1 ΔHBS -linker-targeting peptide RVG) of Comparative Example 2-1 (nucleotide sequence as shown in SEQ ID NO. 11).

[0065] Comparative Example 2-3: An LNP drug for treating type 2 diabetes

[0066] The present comparative example provides an LNP drug for treating type 2 diabetes, which is a lipid nanoparticle encapsulating the recombinant plasmid pcDNA6.2-FGF1 ΔHBS -RVG of Comparative Example 2-2; the lipid nanoparticle is a lipid delivery carrier constructed based on four lipid components, i.e., cationic liposome SM-102, cholesterol, distearoylphosphatidylcholine (DSPC), and PEG-DMG-2000 (the process of LNP encapsulation of the recombinant plasmid using the four lipid components was performed by Maianna (Shanghai) Instrument Technology Co., Ltd.).

[0067] Experimental Example 1: Effects of in vivo self-assembling central targeting system and LNP drug on type 2 diabetic mice

[0068] The present experimental example provides an experiment of effects of in vivo self-assembling central targeting system and LNP drug on type 2 diabetic mice, and the experimental process is as follows:

[0069] Experiment 1: Influence of in vivo self-assembly central target system and LNP drug on type 2 diabetic mice under different administration methods

[0070] Forty ob / ob mice (purchased from Jisui Yakang, weighing ~40 g, 6-7 weeks old) were grouped, and divided into five groups, namely, empty control group (NC-plasmid), intravenous injection drug group (I.V. p-RVG-FGF1 ΔHBS -plasmid), subcutaneous injection drug group A (S.C. LNP p-RVG-FGF1 ΔHBS -plasmid), subcutaneous injection drug group B (S.C. FGF1 ΔHBS -protein), and intracerebral injection drug group (I.C.V. FGF1 ΔHBS -protein), with 8 mice in each group.

[0071] After grouping, the mice in the empty control group were injected intravenously with 10 mg / kg of empty plasmid (i.e., pcDNA6.2-GW / EmGFP-miR plasmid) once, the mice in the intravenous injection drug group were injected intravenously with 10 mg / kg of the in vivo self-assembly central target system (recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS ) of Example 1-2 once, the mice in the subcutaneous injection drug group A were injected subcutaneously with 50 μg of the LNP drug for treating type 2 diabetes of Example 1-3 once, the mice in the subcutaneous injection drug group B were injected subcutaneously with 30 μg of FGF1 protein (consisting of propeptide and fibroblast growth factor variant, i.e., SEQ ID NO. 2+SEQ ID NO. 1) once, and the mice in the intracerebral injection drug group were injected intracerebrally with 3 μg of FGF1 protein (the drug was dissolved in 100 μL of normal saline before injection) once.

[0072] The principle of intravenous injection is as follows: a genetic circuit capable of expressing FGF variant is designed, and the genetic circuit is delivered into the body by intravenous injection; the genetic circuit entering the body uses the liver as a biological reaction generator to express drug protein; since a central target peptide is expressed in the genetic circuit, the expressed FGF variant has the ability to pass through the blood-brain barrier and target the central nervous system, thereby playing a role (see FIG. 2 for details).

[0073] The principle of subcutaneous injection is: using the liver targeting function of LNP, through subcutaneous administration, the genetic circuit capable of expressing FGF variant is efficiently delivered into the liver, and the liver is used as a biological reactor to express the engineered drug protein; since a central targeting peptide is expressed in the genetic circuit, the expressed FGF variant has the ability to target the central nervous system through the blood-brain barrier, thereby exerting an effect (see Figure 3 for details).

[0074] After injection, the blood glucose of the mice was continuously detected at different time points, and the glucose tolerance test (GTT) was performed on the mice every week after a single injection of the drug. The GTT experiment is as follows: the blood glucose value of the mice was measured immediately before (0 min) and 15, 30, 60, and 120 min after intraperitoneal injection of glucose (1 g / kg) in mice fasted for 12 hours to obtain the GTT experiment results. The experimental results are shown in Figure 4 and Table 1 (see the literature: Scarlett JM, Rojas JM, Matsen ME, Kaiyala KJ, Stefanovski D, Bergman RN, Nguyen HT, Dorfman MD, Lantier L, Wasserman DH, Mirzadeh Z, Unterman TG, Morton GJ, Schwartz MW. Central injection of fibroblast growth factor 1 induces sustained remission of diabetic hyperglycemia in rodents. Nat Med. 2016 Jul; 22(7): 800-6.).

[0075] As can be seen from Figure 4 and Table 1, after a single injection of the drug for 13 weeks, the fasting blood glucose of the mice in the empty control group was about 148.5 mg / dL, and the GTT peak was about 493.2 mg / dL; the fasting blood glucose of the mice in the intracerebral injection drug group was about 100.2 mg / dL, and the GTT peak was about 407.4 mg / dL; the fasting blood glucose of the mice in the intravenous injection drug group was about 95.4 mg / dL, and the GTT peak was about 342.0 mg / dL; the fasting blood glucose of the mice in the subcutaneous injection drug group A (S.C.p-RVG-FGF1 ΔHBS -plasmid) was about 103.2 mg / dL, and the GTT peak was about 317.2 mg / dL; the fasting blood glucose of the mice in the subcutaneous injection drug group B (S.C.FGF1 ΔHBSThe ob / ob mice purchased from the above-mentioned company have a fasting blood glucose of about 147.0 mg / dL and a peak value of about 506.4 mg / dL in a glucose tolerance test (GTT). The peripheral administration of FGF1 protein directly by subcutaneous injection is simple and less traumatic, but the effect is maintained for a short time (less than one week), and the intracerebral injection can maintain the effect for a longer time, but is more traumatic. The in vivo self-assembling central targeting system (recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS ) of Example 1-2 has the dual advantages of peripheral administration and central administration, and can maintain a long-term hypoglycemic effect, and is simple and less traumatic to operate.

[0076] Table 1: Results of GTT experiment after single injection of drug for 13 weeks

[0077] Experiment Two: Effects of in vivo self-assembling central targeting system expressing different fusion proteins and LNP drug on type 2 diabetic mice

[0078] Twenty-four ob / ob mice (purchased from the above-mentioned company, weighing about 40 g, and 6-7 weeks old) were divided into three groups, i.e., intravenous injection drug group A, intravenous injection drug group B, and intravenous injection drug group C, with 8 mice in each group.

[0079] After the grouping, the mice in the intravenous injection drug group A were injected intravenously with 10 mg / kg of the in vivo self-assembling central targeting system (recombinant plasmid pcDNA6.2-p-RVG-FGF1 ΔHBS ) of Example 1-2, the mice in the intravenous injection drug group B were injected intravenously with 10 mg / kg of the in vivo self-assembling central targeting system (recombinant plasmid pcDNA6.2-RVG-FGF1 ΔHBS ) of Comparative Example 1-2, and the mice in the intravenous injection drug group C were injected intravenously with 10 mg / kg of the in vivo self-assembling central targeting system (recombinant plasmid pcDNA6.2-FGF1 ΔHBS -RVG) (the drug was dissolved in 100 μL of normal saline before injection).

[0080] After the injection, the blood glucose of the mice was continuously detected at different time points, and a glucose tolerance test (GTT) was performed on the mice every week after the single injection of the drug. The GTT was performed as follows: the mice were injected intraperitoneally with glucose (1 g / kg) after being fasted for 12 hours, and the blood glucose value was measured immediately before (0 minute) and after (15, 30, 60, and 120 minutes) the intraperitoneal injection of glucose to obtain the results of the GTT, which are shown in FIG. 5 and Table 2.

[0081] As shown in Figure 5 and Table 2, the fasting blood glucose of mice in intravenous administration group A was about 102.6 mg / dL, and the peak value of GTT was about 314.1 mg / dL; the fasting blood glucose of mice in intravenous administration group B was about 132.3 mg / dL, and the peak value of GTT was about 368.1 mg / dL; the fasting blood glucose of mice in intravenous administration group C was about 144.5 mg / dL, and the peak value of GTT was about 431.6 mg / dL. The results showed that the self-assembly central targeting system of Example 1-2 was the optimal structure modification mode.

[0082] Table 2. Results of GTT experiment after single injection of drug for 13 weeks

[0083] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. All the embodiments do not need to be exhausted here. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. An in vivo self-assembly, centrally targeted system for the treatment of diabetes, characterized in that, The in vivo self-assembling central targeting system is a recombinant plasmid expressing a nucleic acid molecule; the nucleic acid molecule encodes a fibroblast growth factor-based fusion protein; the fibroblast growth factor-based fusion protein comprises a propeptide, a central targeting peptide, a linker peptide and a fibroblast growth factor variant connected in sequence; the amino acid sequence of the fibroblast growth factor variant is shown as SEQ ID NO. 1; the amino acid sequence of the propeptide is shown as SEQ ID NO.

2.

2. The in vivo self-assembling hub-targeting system of claim 1, wherein, The central targeting peptide comprises a brain-targeting chimeric rabies virus glycoprotein fragment peptide, a brain-targeting rabies virus glycoprotein-derived peptide, a glioma-targeting peptide, a transferrin-targeting peptide, a cell-penetrating peptide and / or an apolipoprotein E fragment peptide; the amino acid sequence of the brain-targeting chimeric rabies virus glycoprotein fragment peptide is shown as SEQ ID NO. 3; the linker peptide is a flexible linker peptide; the flexible linker peptide comprises a GS linker peptide; the amino acid sequence of the GS linker peptide is shown as SEQ ID NO.

4.

3. The in vivo self-assembly hub targeting system of claim 1 or 2, wherein, The fibroblast growth factor-based fusion protein consists of a propeptide, a central targeting peptide, a linker peptide and a fibroblast growth factor variant connected in sequence; the amino acid sequence of the fibroblast growth factor-based fusion protein is shown as SEQ ID NO.

5.

4. A fibroblast growth factor-based fusion protein, characterized by, The fibroblast growth factor-based fusion protein comprises a propeptide, a central targeting peptide, a linker peptide and a fibroblast growth factor variant connected in sequence; the amino acid sequence of the fibroblast growth factor variant is shown as SEQ ID NO. 1; the amino acid sequence of the propeptide is shown as SEQ ID NO.

2.

5. The fusion protein of claim 4, wherein, The central targeting peptide comprises a brain-targeting chimeric rabies virus glycoprotein fragment peptide, a brain-targeting rabies virus glycoprotein-derived peptide, a glioma-targeting peptide, a transferrin-targeting peptide, a cell-penetrating peptide and / or an apolipoprotein E fragment peptide; the amino acid sequence of the brain-targeting chimeric rabies virus glycoprotein fragment peptide is shown as SEQ ID NO. 3; the linker peptide is a flexible linker peptide; the flexible linker peptide comprises a GS linker peptide; the amino acid sequence of the GS linker peptide is shown as SEQ ID NO.

4.

6. The fusion protein of claim 4 or 5, wherein, The fibroblast growth factor-based fusion protein consists of a propeptide, a central targeting peptide, a linker peptide and a fibroblast growth factor variant connected in sequence; the amino acid sequence of the fibroblast growth factor-based fusion protein is shown as SEQ ID NO.

5.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the in vivo self-assembling central targeting system of any one of claims 1-3 or the fusion protein of any one of claims 4-6.

8. A host cell, characterized in that, The host cell carries the in vivo self-assembling central targeting system of any one of claims 1-3; or, the host cell expresses the fusion protein of any one of claims 4-6; or, the genome of the host cell is integrated with the nucleic acid molecule of claim 7.

9. Use of the in vivo self-assembling central nervous system targeting system of any one of claims 1-3 or the fusion protein of any one of claims 4-6 or the nucleic acid molecule of claim 7 or the host cell of claim 8 in the manufacture of a medicament for treating diabetes.

10. A medicament for treating diabetes, characterized by, The medicament for treating diabetes comprises the in vivo self-assembling central nervous system targeting system of any one of claims 1-3, the fusion protein of any one of claims 4-6, the nucleic acid molecule of claim 7 and / or the host cell of claim 8.

11. The medicament according to claim 10 for treating diabetes, wherein The medicament for treating diabetes further comprises a pharmaceutical carrier; the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes; the nanoparticles comprise lipid nanoparticles; the medicament for treating diabetes is a lipid nanoparticle-encapsulated in vivo self-assembling central nervous system targeting system of any one of claims 1-3.

12. A method of treating diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-11. The in vivo self-assembling central nervous system targeting system of any one of claims 1-3 or the fusion protein of any one of claims 4-6 or the nucleic acid molecule of claim 7 or the host cell of claim 8 is administered to a subject in need thereof.

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