CN118910160A - In vivo self-assembled targeting system for effectively secreting target protein - Google Patents

Abstract

The present invention relates to an in vivo self-assembly targeting system for effectively secreting a target protein, and belongs to the field of biotechnology. The present invention provides an in vivo self-assembly targeting system, which is a recombinant plasmid expressing a nucleic acid molecule, wherein the nucleic acid molecule encodes a fusion protein based on the target protein, and the fusion protein based on the target protein comprises a propeptide, a targeting peptide and a target protein connected in sequence. The fusion protein expressed by this in vivo self-assembly targeting system places the propeptide in front, and studies have shown that this in vivo self-assembly targeting system can achieve long-term effective expression of the target protein in mammals, and has great application prospects in the preparation of gene therapy drugs based on the expression of the target protein.

Description

An in vivo self-assembling targeting system for efficient secretion of target proteins

Technical Field

The invention relates to an in vivo self-assembly targeting system for effectively secreting a target protein, and belongs to the field of biotechnology.

Background Art

Gene therapy is the process of introducing target genes into specific tissues and cells of patients for proper expression, in order to correct or compensate for diseases caused by gene defects or abnormalities, thereby achieving the purpose of treating diseases. The correction approach can be either to repair defective genes in situ, or to transfer functional normal genes into a certain part of the patient's body to replace defective genes to play a role. Compared with traditional treatment methods, gene therapy can correct abnormal genes that cause diseases from the root, which has significant advantages.

Since naked genes have defects such as being easily degraded by ribozymes in tissues or cells and having poor cell targeting ability, certain technical methods or vectors must be used to introduce exogenous genes into patients. Currently, commonly used gene therapy drugs are usually composed of vectors or delivery systems containing target genes, which further express target proteins or edit cell genes in patient cells using "cell factories" to exert their functions.

If different gene therapy drugs can be designed for different diseases, the therapeutic effect and efficiency of the disease will be effectively improved. Among them, gene therapy drugs based on target protein expression need to be able to effectively secrete the target protein in the patient's body for a long time. However, due to the current design level, most gene therapy drugs based on target protein expression still cannot meet this requirement.

Summary of the invention

To solve the above problems, the present invention provides an in vivo self-assembly targeting system for effectively secreting a target protein, wherein the in vivo self-assembly targeting system is a recombinant plasmid expressing a nucleic acid molecule; the nucleic acid molecule encodes a fusion protein based on the target protein; the fusion protein based on the target protein comprises a propeptide, a targeting peptide and the target protein connected in sequence; the targeting peptide is used to deliver the target protein to its target site.

In one embodiment of the present invention, the fusion protein based on the target protein further includes a connecting peptide.

In one embodiment of the present invention, the connecting peptide is connected between the targeting peptide and the target protein.

In one embodiment of the present invention, the target protein includes fibroblast growth factor, fibroblast growth factor variant, platelet-derived growth factor (PDGF), interleukins (ILs) and/or tumor necrosis factor (TNF).

In one embodiment of the present invention, the fibroblast growth factor includes FGF1 and/or FGF4; the fibroblast growth factor variant includes FGF1 variant and/or FGF4 variant.

In one embodiment of the present invention, the interleukin includes one or more of interleukin-1 (IL-1) to interleukin-35 (IL-35).

In one embodiment of the present invention, the tumor necrosis factor includes tumor necrosis factor-α (TNF-α) and/or tumor necrosis factor-β (TNF-β).

In one embodiment of the present invention, the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ); the amino acid sequence of the FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) is shown in SEQ ID NO.1.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ), the fusion protein based on the target protein includes a propeptide, a targeting peptide, a connecting peptide and an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) connected in sequence.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the amino acid sequence of the propeptide is as shown in SEQ ID NO.2.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the targeting peptide is a central targeting peptide; the central targeting peptide includes a brain-targeted chimeric rabies virus glycoprotein fragment peptide (RVG), a brain-targeted 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 apolipoprotein E fragment peptide (ApoE (159-167) 2); the amino acid sequence of the brain-targeted chimeric rabies virus glycoprotein fragment peptide is shown in SEQ ID NO.3.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the connecting peptide is a flexible connecting peptide (flexible linker); the flexible connecting peptide includes a GS connecting peptide (GSlinker); the amino acid sequence of the GS connecting peptide is shown in SEQ ID NO.4.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ), the fusion protein based on the target protein is composed of a propeptide, a central targeting peptide, a connecting peptide and an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) connected in sequence.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the amino acid sequence of the fusion protein based on the target protein is as shown in SEQ ID NO.5.

In one embodiment of the present invention, the vector of the recombinant plasmid is pcDNA6.2-GW/EmGFP-miR plasmid and/or pcDNA3.1 plasmid.

In one embodiment of the present invention, 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.

The present invention also provides a fusion protein based on a target protein, wherein the fusion protein based on the target protein comprises a propeptide, a targeting peptide and a target protein which are connected in sequence; the targeting peptide is used to deliver the target protein to its target site.

In one embodiment of the present invention, the fusion protein based on the target protein further includes a connecting peptide.

In one embodiment of the present invention, the connecting peptide is connected between the targeting peptide and the target protein.

In one embodiment of the present invention, the target protein includes fibroblast growth factor, fibroblast growth factor variant, platelet-derived growth factor (PDGF), interleukins (ILs) and/or tumor necrosis factor (TNF).

In one embodiment of the present invention, the fibroblast growth factor includes FGF1 and/or FGF4; the fibroblast growth factor variant includes FGF1 variant and/or FGF4 variant.

In one embodiment of the present invention, the interleukin includes one or more of interleukin-1 (IL-1) to interleukin-35 (IL-35).

In one embodiment of the present invention, the tumor necrosis factor includes tumor necrosis factor-α (TNF-α) and/or tumor necrosis factor-β (TNF-β).

In one embodiment of the present invention, the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ); the amino acid sequence of the FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) is shown in SEQ ID NO.1.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ), the fusion protein based on the target protein includes a propeptide, a targeting peptide, a connecting peptide and an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) connected in sequence.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the amino acid sequence of the propeptide is as shown in SEQ ID NO.2.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the targeting peptide is a central targeting peptide; the central targeting peptide includes a brain-targeted chimeric rabies virus glycoprotein fragment peptide (RVG), a brain-targeted 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 apolipoprotein E fragment peptide (ApoE (159-167) 2); the amino acid sequence of the brain-targeted chimeric rabies virus glycoprotein fragment peptide is shown in SEQ ID NO.3.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the connecting peptide is a flexible connecting peptide (flexible linker); the flexible connecting peptide includes a GS connecting peptide (GSlinker); the amino acid sequence of the GS connecting peptide is shown in SEQ ID NO.4.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ), the fusion protein based on the target protein is composed of a propeptide, a central targeting peptide, a connecting peptide and an FGF1 variant (FGF1 non-mitogenic modified form FGF1 ^ΔHBS ) connected in sequence.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the amino acid sequence of the fusion protein based on the target protein is as shown in SEQ ID NO.5.

The present invention also provides a nucleic acid molecule, which encodes the in vivo self-assembly targeting system or the fusion protein based on the target protein.

In one embodiment of the present invention, when encoding the above-mentioned fusion protein based on the target protein, and the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.6.

In one embodiment of the present invention, when encoding the above-mentioned in vivo self-assembly targeting system, and the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.7.

The present invention also provides a host cell, wherein the host cell carries the in vivo self-assembly targeting system; or the host cell expresses the fusion protein based on the target protein; or the genome of the host cell is integrated with the nucleic acid molecule.

In one embodiment of the present invention, the host cell comprises Escherichia coli, insect cells, mammalian cells, Bacillus subtilis and/or yeast.

The present invention also provides the use of the in vivo self-assembly targeting system or the target protein-based fusion protein or the nucleic acid molecule or the host cell in the preparation of drugs.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the drug is a drug for treating diabetes.

In one embodiment of the present invention, the diabetes is type 2 diabetes.

In one embodiment of the present invention, the drug further comprises a drug carrier; the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.

In one embodiment of the invention, the nanoparticles comprise lipid nanoparticles (LNPs).

In one embodiment of the present invention, the drug is the above-mentioned in vivo self-assembly targeting system encapsulated by lipid nanoparticles.

In one embodiment of the present invention, the components of the lipid nanoparticles include ionizable lipids, cholesterol, auxiliary lipids and polyethylene glycol (PEG) lipids; the ionizable lipids include cationic liposomes SM-102; the auxiliary lipids include distearoylphosphatidylcholine (DSPC); and the polyethylene glycol lipids include PEG-DMG-2000.

The present invention also provides a medicine, which comprises the above-mentioned in vivo self-assembly targeting system, the above-mentioned fusion protein based on the target protein, the above-mentioned nucleic acid molecule and/or the above-mentioned host cell.

In one embodiment of the present invention, when the target protein is an FGF1 variant (FGF1 non-mitogenic variant FGF1 ^ΔHBS ), the drug is a drug for treating diabetes.

In one embodiment of the present invention, the diabetes is type 2 diabetes.

In one embodiment of the present invention, the drug further comprises a drug carrier; the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.

In one embodiment of the invention, the nanoparticles comprise lipid nanoparticles (LNPs).

In one embodiment of the present invention, the drug is the above-mentioned in vivo self-assembly targeting system encapsulated by lipid nanoparticles.

In one embodiment of the present invention, the components of the lipid nanoparticles include ionizable lipids, cholesterol, auxiliary lipids and polyethylene glycol (PEG) lipids; the ionizable lipids include cationic liposomes SM-102; the auxiliary lipids include distearoylphosphatidylcholine (DSPC); and the polyethylene glycol lipids include PEG-DMG-2000.

The technical solution of the present invention has the following advantages:

1. The present invention provides an in vivo self-assembly targeting system, which is a recombinant plasmid expressing a nucleic acid molecule, wherein the nucleic acid molecule encodes a fusion protein based on a target protein, and the fusion protein based on the target protein comprises a propeptide, a targeting peptide and a target protein connected in sequence. The fusion protein expressed by this in vivo self-assembly targeting system places the propeptide in front, and studies have shown that this in vivo self-assembly targeting system can achieve long-term effective expression of the target protein in mammals, and has great application prospects in the preparation of gene therapy drugs based on the expression of the target protein.

Further, the target protein is a fibroblast growth factor variant; the amino acid sequence of the fibroblast growth factor variant is shown in SEQ ID NO.1; the fusion protein based on fibroblast growth factor includes a propeptide, a targeting peptide, a connecting peptide and a fibroblast growth factor variant (FGF1 non-mitogenic modified body FGF1 ^ΔHBS ) connected in sequence; the targeting peptide is a central targeting peptide. The fusion protein expressed by this in vivo self-assembly targeting system places the propeptide in front. Studies have shown that this in vivo self-assembly targeting system can pass through the blood-brain barrier and specifically reach the central nervous system by means other than intracranial injection (subcutaneous injection, intravenous injection), exert a lasting hypoglycemic effect (at least 13 weeks of continuous and stable hypoglycemic effect), and does not cause adverse reactions. It not only retains the anti-diabetic activity of fibroblast growth factor, but also avoids the tumorigenic risk of fibroblast growth factor. At the same time, it solves the problem of low patient compliance caused by intracranial injection, provides more ideas for the development of central targeted drugs, and has great application prospects in the treatment of type 2 diabetes.

2. The present invention provides a fusion protein based on a target protein, wherein the fusion protein based on the target protein comprises a propeptide, a targeting peptide and a target protein connected in sequence. This fusion protein places the propeptide in front, and studies have shown that after the fusion protein is integrated into an expression vector to form an in vivo self-assembly targeting system, it can achieve long-term effective expression of the target protein in mammals, and has great application prospects in the preparation of gene therapy drugs based on the expression of the target protein.

Further, the target protein is a fibroblast growth factor variant; the amino acid sequence of the fibroblast growth factor variant is shown in SEQ ID NO.1; the fusion protein based on fibroblast growth factor includes a propeptide, a targeting peptide, a connecting peptide and a fibroblast growth factor variant (FGF1 non-mitogenic modified body FGF1 ^ΔHBS ) connected in sequence; the targeting peptide is a central targeting peptide. This fusion protein places the propeptide in front. Studies have shown that after the fusion protein is integrated into the expression vector to form an in vivo self-assembly targeting system, it can pass through the blood-brain barrier and specifically reach the central nervous system by means other than intracranial injection (subcutaneous injection, intravenous injection), exert a lasting hypoglycemic effect (at least 13 weeks of continuous and stable hypoglycemic effect), and does not cause adverse reactions. It not only retains the anti-diabetic activity of fibroblast growth factor, but also avoids the tumorigenic risk of fibroblast growth factor. At the same time, it solves the problem of low patient compliance caused by intracranial injection, provides more ideas for the development of central targeted drugs, and has great application prospects in the treatment of type 2 diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Plasmid map of the recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS .

Figure 2: Schematic diagram of drug administration of recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS to type 2 diabetic mice via intravenous injection.

Figure 3: Schematic diagram of the administration of LNP-encapsulated recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS to type 2 diabetic mice via subcutaneous injection.

FIG. 4 : The administration effect of the fusion protein (propeptide-RVG-linker-FGF1 ^ΔHBS ) in treating type 2 diabetes.

Figure 5: Drug administration effects of different fusion proteins in the treatment of type 2 diabetes.

DETAILED DESCRIPTION

The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

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

This embodiment provides a fusion protein based on fibroblast growth factor (propeptide-RVG-linker-FGF1 ^ΔHBS ), which is obtained by sequentially connecting the propeptide, the central targeting peptide, the linker peptide and the fibroblast growth factor variant, and the amino acid sequence is shown in SEQ ID NO.5.

Example 1-2: An in vivo self-assembly targeting system for treating type 2 diabetes

This embodiment provides an in vivo self-assembly targeting system for treating type 2 diabetes. This in vivo self-assembly targeting system is a recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS . This recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS is a pcDNA6.2-GW/EmGFP-miR plasmid carrying a gene encoding the fusion protein (propeptide-RVG-linker-FGF1 ^ΔHBS ) of Example 1-1, and the nucleotide sequence is shown in SEQ ID NO.7 (see Figure 1 for the plasmid map).

The preparation method of the in vivo self-assembly targeting system for treating type 2 diabetes is as follows:

The construction of a recombinant plasmid for the treatment of type 2 diabetes was completed using the BLOCK-iT Pol II miR RNAi designer of Invitrogen. The designer provides two DNA oligos sequences, through which the gene (SEQ ID NO.6) encoding the fusion protein (propeptide-RVG-linker-FGF1 ^ΔHBS ) of Example 1-1 is cloned into the pcDNA6.2-GW/EmGFP-miR linearized vector to obtain a ligation product (this process was completed by GenScript Biotech); the ligation product was transformed into Escherichia coli competent cells DH5α (purchased from Tsingke, TSC01) to obtain a transformation product; the transformation product was streaked on an LB agar plate medium (purchased from Thermo Fisher) containing 50 μg/mL ampicillin (Amp), cultured at 37°C for 12 hours, 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. Fisher), cultured at 37°C for 14 hours 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 after successful sequencing. This recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS is the in vivo self-assembly targeting system.

Example 1-3: A LNP drug for treating type 2 diabetes

This embodiment provides an LNP drug for treating type 2 diabetes. The LNP drug for treating type 2 diabetes is a lipid nanoparticle encapsulating the recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS of Example 1-2; the lipid nanoparticle is a lipid delivery vector constructed based on four lipid components: 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 Myanna (Shanghai) Instrument Technology Co., Ltd.).

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

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

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

This comparative example provides an in vivo self-assembly targeting system for treating type 2 diabetes. The in vivo self-assembly targeting system is a recombinant plasmid pcDNA6.2-RVG-FGF1 ^ΔHBS . The recombinant plasmid pcDNA6.2-RVG-FGF1 ^ΔHBS for treating type 2 diabetes 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.

The preparation method of the in vivo self-assembly 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 with the gene encoding the fusion protein (targeting peptide RVG-linker-propeptide-FGF1 ^ΔHBS ) of Comparative Example 1-1 (the nucleotide sequence is shown in SEQ ID NO.9).

Comparative Example 1-3: A LNP drug for treating type 2 diabetes

This comparative example provides an LNP drug for treating type 2 diabetes, which is a lipid nanoparticle that encapsulates the recombinant plasmid pcDNA6.2-RVG-FGF1 ^ΔHBS of comparative example 1-2; the lipid nanoparticle is a lipid delivery vector constructed based on four lipid components: 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 Myanna (Shanghai) Instrument Technology Co., Ltd.).

Comparative Example 2-1: A fusion protein based on fibroblast growth factor

This comparative example provides a fusion protein based on fibroblast growth factor (propeptide-FGF1 ^ΔHBS -linker-targeting peptide RVG), which is obtained by sequentially connecting the propeptide, fibroblast growth factor variant, linker peptide and central targeting peptide, and the amino acid sequence is shown in SEQ ID NO.10.

Comparative Example 2-2: An in vivo self-assembly targeting system for treating type 2 diabetes

This comparative example provides an in vivo self-assembly targeting system for treating type 2 diabetes. The in vivo self-assembly targeting system is a recombinant plasmid pcDNA6.2-FGF1 ^ΔHBS -RVG. The recombinant plasmid pcDNA6.2-FGF1 ^ΔHBS -RVG for treating type 2 diabetes is a pcDNA6.2-GW/EmGFP-miR plasmid carrying a gene encoding the fusion protein (propeptide-FGF1 ^ΔHBS -linker-targeting peptide RVG) of comparative example 2-1.

The preparation method of the in vivo self-assembly 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 with the gene encoding the fusion protein (propeptide-FGF1 ^ΔHBS -linker-targeting peptide RVG) of Comparative Example 2-1 (nucleotide sequence is shown in SEQ ID NO.11).

Comparative Example 2-3: A LNP drug for treating type 2 diabetes

This comparative example provides an LNP drug for treating type 2 diabetes, which is a lipid nanoparticle that encapsulates the recombinant plasmid pcDNA6.2-FGF1 ^ΔHBS -RVG of comparative example 2-2; the lipid nanoparticle is a lipid delivery vector constructed based on four lipid components: 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 carried out by Myanna (Shanghai) Instrument Technology Co., Ltd.).

Experimental Example 1: Effects of in vivo self-assembly targeting system and LNP drugs on type 2 diabetic mice

This experimental example provides an experiment on the effects of the in vivo self-assembled targeting system and LNP drugs on type 2 diabetic mice. The experimental process is as follows:

Experiment 1: Effects of in vivo self-assembled targeting system and LNP drugs on type 2 diabetic mice under different drug administration methods

Forty ob/ob mice (purchased from Jicui Yaokang, weighing ~40 g, 6-7 weeks old) were divided into five groups, including empty control group (NC-plasmid), intravenous injection group (IVp-RVG-FGF1 ^ΔHBS -plasmid), subcutaneous injection group A (SCLNP p-RVG-FGF1 ^ΔHBS -plasmid), subcutaneous injection group B (SCFGF1 ^ΔHBS -protein) and intracerebral injection group (ICVFGF1 ^ΔHBS -protein), with 8 mice in each group.

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

Among them, the principle of intravenous injection is: design a genetic circuit that can express FGF variants, and deliver the genetic circuit into the body through intravenous injection; the genetic circuit that enters the body uses the liver as a biological reaction generator to express drug proteins; because a fused central targeting peptide is also expressed in the genetic circuit, the expressed FGF variant has the ability to cross the blood-brain barrier and target the central nervous system, thereby exerting its effect (see Figure 2 for details).

The principle of subcutaneous injection is: utilizing the liver targeting function of LNP, the genetic circuit that can express FGF variants is efficiently delivered to the liver through subcutaneous administration, and the liver is used as a bioreactor to express the engineered drug protein; since a fused central targeting peptide is also expressed in the genetic circuit, the expressed FGF variant has the ability to cross the blood-brain barrier and target the central nervous system, thereby exerting its effect (see Figure 3 for details).

After the injection, the blood glucose changes of the mice were continuously detected at different time points, and the glucose tolerance test (GTT) test was performed on the mice every week after a single injection of the drug. The GTT test was specifically as follows: glucose (1 g/kg) was intraperitoneally injected into the mice that had fasted for 12 hours, and the blood glucose levels were immediately measured before (0 minute) and 15, 30, 60, and 120 minutes after the intraperitoneal injection of glucose to obtain the GTT test results. The experimental results are shown in Figure 4 and Table 1 (for GTT experiments, 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 inrodents. NatMed. 2016Jul; 22(7):800-6.).

As shown in Figure 4 and Table 1, 13 weeks after a single injection of the drug, 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 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 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 group A (SCp-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 group B (SCFGF1 ^ΔHBS -protein) was about 147.0 mg/dL, and the GTT peak was about 506.4 mg/dL. Consistent with literature reports, although direct subcutaneous peripheral administration of FGF1 protein is easy to operate and less invasive, the effect is short-lived (less than one week), and although intracerebral injection can maintain the effect for a longer time, it is more traumatic. The in vivo self-assembly targeting system of Example 1-2 (recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS ) has the dual advantages of peripheral administration and central administration, which can maintain a long-term hypoglycemic effect, is easy to operate, and is less traumatic.

Table 1 GTT test results 13 weeks after single injection of drug

Experiment 2: Effects of in vivo self-assembly targeting system expressing different structural fusion proteins and LNP drugs on type 2 diabetic mice

Twenty-four ob/ob mice (purchased from Jicui Yaokang, weighing 40 g, 6-7 weeks old) were divided into three groups, namely intravenous medication group A, intravenous medication group B and intravenous medication group C, with 8 mice in each group.

After the grouping was completed, the mice in the intravenous injection medication group A were given a single intravenous injection of 10 mg/kg of the in vivo self-assembly targeting system of Example 1-2 (recombinant plasmid pcDNA6.2-p-RVG-FGF1 ^ΔHBS ), the mice in the intravenous injection medication group B were given a single intravenous injection of 10 mg/kg of the in vivo self-assembly targeting system of Comparative Example 1-2 (recombinant plasmid pcDNA6.2-RVG-FGF1 ^ΔHBS ), and the mice in the intravenous injection medication group C were given a single intravenous injection of 10 mg/kg of the in vivo self-assembly targeting system of Comparative Example 2-2 (recombinant plasmid pcDNA6.2-FGF1 ^ΔHBS -RVG) (the drug was dissolved in 100 μL of normal saline for injection).

After the injection, the blood glucose changes of the mice were continuously monitored at different time points, and the glucose tolerance test (GTT) test was performed on the mice every week after a single injection of the drug. The GTT test was as follows: glucose (1 g/kg) was injected intraperitoneally into mice that had fasted for 12 hours, and the blood glucose levels were measured immediately before (0 minute) and 15, 30, 60, and 120 minutes after the intraperitoneal injection of glucose to obtain the GTT test results. The experimental results are shown in Figure 5 and Table 2.

As shown in Figure 5 and Table 2, the fasting blood glucose of mice in the intravenous injection group A was about 102.6 mg/dL, and the GTT peak was about 314.1 mg/dL; the fasting blood glucose of mice in the intravenous injection group B was about 132.3 mg/dL, and the GTT peak was about 368.1 mg/dL; the fasting blood glucose of mice in the intravenous injection group C was about 144.5 mg/dL, and the GTT peak was about 431.6 mg/dL. This result shows that the in vivo self-assembly targeting system of Example 1-2 is the optimal structural modification method.

Table 2 GTT test results 13 weeks after single injection of drug

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims (10)

1. An in vivo self-assembled targeting system for efficient secretion of a protein of interest, wherein the in vivo self-assembled targeting system is a recombinant plasmid expressing a nucleic acid molecule; the nucleic acid molecule encodes a fusion protein based on a protein of interest; the fusion protein based on the target protein comprises a propeptide, a targeting peptide and the target protein which are connected in sequence; the targeting peptides are useful for delivering a protein of interest to its target of action.

2. The in vivo self-assembled targeting system according to claim 1, wherein said protein-based fusion protein of interest further comprises a linking peptide; the linker peptide is linked between the targeting peptide and the protein of interest.

3. The in vivo self-assembled targeting system according to claim 1 or 2, wherein said protein of interest comprises a fibroblast growth factor, a fibroblast growth factor variant, a platelet-derived growth factor, an interleukin and/or a tumor necrosis factor.

4. A fusion protein based on a target protein, which is characterized by comprising a propeptide, a targeting peptide and the target protein which are connected in sequence; the targeting peptides are useful for delivering a protein of interest to its target of action.

5. The fusion protein of claim 4, wherein the protein-based fusion protein of interest further comprises a linker peptide; the linker peptide is linked between the targeting peptide and the protein of interest.

6. The fusion protein of claim 4 or 5, wherein the protein of interest comprises a fibroblast growth factor, a fibroblast growth factor variant, a platelet-derived growth factor, an interleukin, and/or a tumor necrosis factor.

7. A nucleic acid molecule encoding the in vivo self-assembled targeting system according to any one of claims 1 to 3 or the fusion protein according to any one of claims 4 to 6.

8. A host cell carrying the in vivo self-assembled targeting system of any one of claims 1 to 3; or the host cell expresses the fusion protein of any one of claims 4-6; or the genome of the host cell has integrated the nucleic acid molecule of claim 7.

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

10. A medicament comprising the in vivo self-assembled targeting system according to any one of claims 1 to 3, the fusion protein according to any one of claims 4 to 6, the nucleic acid molecule according to claim 7 and/or the host cell according to claim 8.