CN118389554A - AAV recombinant virus for improving the sustained expression level and stability of FGF-21 and its application - Google Patents

Abstract

The present invention discloses an AAV for improving the sustained expression level and stability of FGF-21 and an application thereof. ABD is connected to FGF-21 by genetic engineering technology, and then recombined to an AAV2 core plasmid to construct a recombinant plasmid pAAV-ABD-FGF-21-Luciferase. The recombinant plasmid is transfected and packaged to obtain a recombinant AAV2-ABD-FGF-21 adeno-associated virus. The virus can mediate the transduction of the ABD-FGF-21-Luciferase gene into the mouse liver and continuously secrete and express ABD-FGF-21, significantly increasing the level and stability of FGF-21 in the peripheral blood of the mice, significantly enhancing the blood sugar and blood lipid lowering effects of FGF-21 in type II diabetic mice, and the effect can be maintained for more than 7 weeks. At the same time, the liver damage of type II diabetic mice caused by insulin resistance and a high-fat diet (HFD) is significantly reduced.

Description

An AAV recombinant virus for improving the sustained expression level and stability of FGF-21 and its application

Technical Field

The present invention belongs to the technical field of genetic engineering, and specifically relates to an AAV recombinant virus for improving the sustained expression level and stability of FGF-21 and an application thereof.

Background technique

In recent years, the incidence of type 2 diabetes (T2D) has increased rapidly. Type 2 diabetes and its complications such as cataracts and diabetic nephropathy have become a major health problem in my country and around the world. Hyperglycemia, insulin resistance and hepatic steatosis are important characteristics of type 2 diabetes, which significantly increase the risk of diabetic patients suffering from heart disease, immune dysfunction, hypertension, arthritis, neurodegenerative diseases and certain types of cancer. The decline in the hypoglycemic effect of drugs caused by insulin resistance is the main dilemma faced by the current clinical treatment of type 2 diabetes, so new treatment technologies for type 2 diabetes are urgently needed.

Fibroblast growth factor 21 (FGF-21) is a peptide hormone mainly secreted by liver cells. It can bind to FGF-21 receptors through the auxiliary factor β-klotho to act on liver, fat and pancreatic cells, improve insulin sensitivity, promote brown fat heating and lower blood sugar levels, thereby regulating glucose and lipid metabolism. A large number of clinical studies have shown that exogenous supplementation of FGF-21 peptide or structural analogs of FGF-21 peptide can lower blood sugar and blood lipid levels, suggesting that FGF-21 peptide has application prospects in the treatment of type II diabetes and obesity. However, the molecular weight of natural FGF-21 is only 21KDa, and its ten C-terminal amino acids can be cleaved by serine proteases FAP and dipeptidyl peptidase IV. Therefore, intravenously injected FGF-21 peptide is easily metabolized by the kidneys through glomerular filtration, and on the other hand, due to enzymatic cleavage, its half-life in vivo is only 0.5 to 1.5 hours, which cannot exert a sustained hypoglycemic and hypolipidemic effect, severely limiting its application effect as a peptide drug.

However, type II diabetes is usually caused by long-term relative lack of insulin secretion or persistent insulin resistance. Supplementation of exogenous hypoglycemic drugs can cause transient drug metabolism peaks, and it is often necessary to continuously adjust the dosage and interval time, and use the drugs multiple times to maintain the effective concentration of hypoglycemic drugs. At the same time, long-term repeated administration not only increases the pain of patients, but also increases the cost of treatment, and also causes a series of side effects. Li Deshan et al. used monomethoxypolyethylene glycol-propionaldehyde (mPEG-ALD) with a molecular weight of 20ku to modify the N-terminal of mouse FGF-21 to improve the properties of Mfgf-21, increase the half-life in vivo, and reduce immunogenicity. However, the molecular weight of the polyethylene glycol modifier has a great influence on the modification results, and there are problems such as difficulty in purification and low purification efficiency. The antibody FC segment can increase the molecular weight of the fusion protein, making it less likely to pass through glomerular filtration, and can also increase the plasma stability and expression level of FGF-21. However, the molecular weight of the FC segment is relatively large (50-60KDa), which has a significant impact on the biological functions of proteins such as FGF-21 (molecular weight 21 KDa).

Adeno-associated virus (AAV2) is a gene delivery vector with the advantages of good safety, low immunogenicity, continuous expression and long-term stability. Currently, a variety of gene drugs have been developed as delivery vectors for the clinical treatment of genetic diseases such as congenital amaurosis, hemophilia, and spinal muscular atrophy. ABD (Albumin-binding domain), that is, albumin binding domain (molecular weight of 14KDa), has an affinity for binding to serum albumin, which can increase the serum stability of ABD-containing proteins and prolong their half-life. Compared with the use of antibody FC segment fusion protein, the ABD-FGF-21 fusion protein solution can increase the molecular weight of FGF-21 on the one hand, making it difficult to be filtered out through the kidneys; on the other hand, the binding of the ABD domain to albumin can protect FGF-21 from protease degradation in the serum and increase the stability of the FGF-21 molecule peptide. In addition, the molecular weight of the ABD domain used in the present invention is only 14KDa, which is about one-third of the molecular weight of the antibody Fc segment, which significantly reduces the effect of the fusion protein on the biological function of FGF-21. Therefore, using adeno-associated virus vectors to deliver the coding DNA sequence of FGF-21 peptide and ABD fusion protein is expected to achieve sustained long-term expression of ABD-FGF-21 peptide fusion protein in vivo, thereby increasing the serum stability and half-life of FGF-21 and improving the hypoglycemic function of FGF-21. Thus, a gene therapy method for the chronic disease of type II diabetes can be established.

Summary of the invention

In view of the above technical problems, the present invention provides an AAV recombinant virus and its application for improving the sustained expression level and stability of FGF-21. The AAV plasmid carrying the ABD-FGF-21 fusion gene is constructed by molecular cloning technology, and then co-transfected with the AAV capsid protein encoding plasmid and the auxiliary plasmid into 293T cells for packaging to obtain the adeno-associated virus AAV2-ABD-FGF-21-Luciferase. AAV2-ABD-FGF-21-Luciferase significantly increased the peripheral blood FGF-21 level of mice, and after being injected into type II diabetic mice, it showed the effect of continuously lowering blood sugar and blood lipids.

In order to achieve the above object, the present invention provides an ABD-FGF-21 fusion gene, wherein the 5' end of the FGF-21 gene is connected to the ABD gene, and the sequence of the fusion gene is SEQ ID NO:1.

Preferably, the ABD-FGF-21 fusion gene is connected to the coding sequence of the reporter gene Luciferase via the self-cleavage sequence P2A to form an open reading frame.

The present invention also provides an application of an FGF-21 fusion gene in preparing a drug for treating type II diabetes.

The present invention also provides an AAV recombinant virus for improving the sustained expression level and stability of FGF-21, comprising an adeno-associated virus capsid and the above-mentioned ABD-FGF-21 fusion gene.

Preferably, the adeno-associated virus capsid is AAV2, AAV5 or AAV8.

Further preferably, the adeno-associated virus capsid is AAV2.

Preferably, the AAV recombinant virus also includes a Flag sequence, P2A and Luciferase.

Preferably, the promoter in the AAV recombinant virus is the CMV promoter.

The present invention also provides an application of an AAV recombinant virus for improving the sustained expression level and stability of FGF-21 in the preparation of a drug for treating type II diabetes.

The beneficial effects of the present invention are:

1. ABD is fused with FGF-21. After the resulting fusion gene is translated into a peptide, the exogenous ABD-FGF-21 polypeptide quickly binds to albumin after entering the blood circulation, thereby avoiding kidney filtration and protease degradation, and prolonging the half-life of the FGF-21 peptide in the body and the time it circulates in the body.

2. Utilizing the high transduction and long-term stable expression capabilities of the gene therapy vector AAV2, the ABD-FGF-21 fusion gene is delivered to achieve long-term (more than 7 weeks) and sustained expression of FGF-21 in the liver in vivo, effectively increasing the plasma level of FGF-21.

3. The ABD-FGF-21 fusion gene was recombined with the AAV expression plasmid to obtain the recombinant plasmid pAAV-ABD-FGF-21-Luciferase, and then three plasmids were packaged to obtain the recombinant virus AAV2-ABD-FGF-21-Luciferase, which has the efficient gene transduction ability of AAV2 serotype II in liver tissue.

4. The recombinant virus AAV2-ABD-FGF-21-Luciferase was injected into type 2 diabetic mice through the tail vein. It had efficient gene transduction and secretory expression effects on mouse liver tissue, and could significantly increase the level of FGF-21 in the peripheral blood of mice. After a single injection, it showed effects of lowering blood sugar and triglycerides, promoting lipolysis and decreasing insulin resistance, alleviating fatty degeneration of hepatocytes, and improving body weight for more than 7 weeks. In the 7th week of the experiment, the FGF-21 content in the peripheral blood of T2DM mice could still be increased by 1.83 times.

5. The ABD-FGF-21 fusion gene, recombinant plasmid pAAV-ABD-FGF-21-Luciferase and recombinant virus AAV2-ABD-FGF-21-Luciferase prepared by the present invention can be used to prepare gene drugs for treating type II diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the ABD-FGF-21 recombinant expression cassette constructed in Example 1; in the figure, A is pAAV2-Null-Luciferase, B is pAAV2-FGF-21-Luciferase, and C is pAAV2-ABD-FGF-21-Luciferase.

Figure 2 is a gel electrophoresis diagram of the double enzyme digestion products in Example 2, in which A is the double enzyme digestion electrophoresis diagram of the recombinant plasmid pAAV2-FGF-21-Luciferase, and B is the double enzyme digestion electrophoresis diagram of the recombinant plasmid pAAV2-ABD-FGF-21-Luciferase.

Figure 3 is a sequence comparison diagram of ABD-FGF-21 and FGF-21 in Example 3, in which A is a sequence comparison diagram of FGF-21 and B is a sequence comparison diagram of ABD-FGF-21. Figure 4 is a Western Blot analysis diagram of the protein expression of FGF-21 and ABD-FGF-21 after plasmid transfection in Example 6.

FIG5 is a diagram showing the expression of AAV2 viral capsid proteins VP1, VP2, and VP3 detected by Western Blot in Example 7.

FIG6 is a flow chart of the construction of the T2DM mouse model in Example 8.

FIG. 7 is an in vivo imaging image of T2DM mice after injection of recombinant adeno-associated virus in Example 9.

Figure 8 shows the changes in physiological and biochemical indicators of T2DM mice after injection of recombinant adeno-associated virus in Example 10; Figure A is a photo of the mice at the end of the experiment, B is a line graph of the weight changes of the mice during the experiment, C is a line graph of the blood glucose level changes of the mice during the experiment, D is the fasting serum insulin (INS) level of the mice at the end of the experiment, E is the insulin resistance level in the serum of the mice at the end of the experiment (HOME-IR insulin resistance index), and F is a bar graph of the insulin sensitivity of the mice at the end of the experiment (HOMR-IS is the insulin sensitivity index).

Figure 9 shows the effect of the recombinant adeno-associated virus in Example 10 on the biochemical parameters of T2DM mice; in the figure, A is a line graph of mouse glucose tolerance, B is a bar graph of the area under the glucose tolerance curve, C is a bar graph of glycated serum protein content, and D is a bar graph of the circulating content of FGF-21 in serum.

Figure 10 shows the effect of the recombinant adeno-associated virus in Example 11 on the blood lipid levels of T2DM mice; in the figure, A is a bar graph of total cholesterol content, B is a bar graph of triglyceride content, C is a bar graph of low-density lipoprotein cholesterol content, and D is a bar graph of high-density lipoprotein cholesterol content.

Figure 11 shows the effect of the recombinant adeno-associated virus on liver damage in T2DM mice in Example 12; Figure A is a photo of liver morphology, B is a liver index bar graph, C is a serum AST content bar graph, D is a serum ALT content bar graph, and E is a H&E stained liver section photo; Figure E a is a 200x image, and b is a 400x image.

Figure 12 shows the effect of the recombinant adeno-associated virus in Example 13 on brown adipose tissue (BAT) and white adipose tissue (WAT) in T2DM mice; Figure A is H&E staining of brown adipose tissue (BAT) sections, and Figure B is H&E staining of white adipose tissue (WAT) sections.

Detailed ways

The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

Cells, mice and plasmids:

HEK-293T cells: provided by Professor Lu Yafeng and stored in the Basic Medical College of China Three Gorges University;

pAAV-Luciferase plasmid: provided by Professor Lu Yafeng, stored in the Basic Medical College of Three Gorges University;

pHelper plasmid: provided by Professor Lu Yafeng and stored in the School of Basic Medical Sciences of Three Gorges University;

pAAV2 Capsid plasmid: provided by Professor Lu Yafeng and stored in the Basic Medical College of Three Gorges University;

SPF male 6-week-old C57BL/6 mice were purchased from the Experimental Animal Center of China Three Gorges University.

Reagents:

Fetal bovine serum: Biological Industries;

DMEM medium: Thermo Fisher Scientific;

Imafect transfection reagent: Beijing Mayin Technology Co., Ltd.;

Universal nuclease: Shanghai Yisheng Biotechnology Co., Ltd.;

60% iodixanol: Stem cell company;

Protein preparation buffer: Thermo Fisher Scientific;

PVDF membrane: Merck Millipore;

FGF-21 antibody (1:1000): American Research Products;

Anti-mouse IgG (1:2000): Proteintech;

Anti-Capsid (capsid protein) antibody (1:1000): American Research Products;

STZ buffer: pH 4.2-4.5, 0.1 mol/L sodium citrate solution;

Luciferin: Shanghai Yisheng Biotechnology Co., Ltd.;

Elisa kit: Hangzhou Lianke Biotechnology Co., Ltd.;

PBS: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ were dissolved in 800 mL distilled water, the solution was adjusted to 7.4 with HCl, and finally the volume was made up to 1 L with distilled water, and stored at 4°C after autoclaving;

10×PBS MK: 10×100mL PBS solution + 0.2g MgCl ₂ •6H ₂ O + 0.19g KCl, dissolved;

0.5% phenol red: weigh 0.1 g of phenol red in 20 mL of 50% ethanol and heat to dissolve;

1×TBST: 2.42 g Tris base, 8.80 g NaCl dissolved in 900 mL ddH ₂ O, add 500 μL Tween 20, adjust pH to 7.40, and make up to 1000 mL.

Example 1 Construction of recombinant adeno-associated virus vector

The ABD-FGF-21 nucleotide sequence was artificially synthesized and connected to the pAAV2-luciferase vector. The specific method is as follows:

(1) The protein coding nucleotide sequence of mouse FGF-21 was obtained from the website of the National Center for Biotechnology Information (NCBI) of the United States, and then the coding nucleotide sequence of mouse ABD protein was added to its 5' end to enable it to express ABD-FGF-21, and then the EcoR1 restriction site and its protective base were added to the 5' end of ABD-FGF-21, and the P2A sequence, Hind III restriction site and its protective base were added to the 3' end to obtain the ABD-FGF-21 nucleotide sequence, whose sequence is SEQ ID NO: 1;

(2) The ABD-FGF-21 nucleotide sequence obtained in step (1) was sent to Suzhou Jinweizhi Technology Co., Ltd. for synthesis, and then cloned into the vector pAAV2-luciferase (Figure 1A) by double restriction digestion with EcoR I and Hind III to construct the recombinant plasmid pAAV2-ABD-FGF-21-luciferase (Figure 1C);

(3) According to the method described in step (2), the ABD-FGF-21 nucleotide sequence was double-digested with EcoR I and Hind III and mixed with the pAAV2-luciferase plasmid that had been treated with the same double enzyme digestion. The ligation reaction was catalyzed by the DNA ligase method at 16°C, and the conjugate was then transformed into Escherichia coli DH5. The recombinant plasmid was screened on an ampicillin-resistant agar plate and identified by DNA sequencing to complete the construction of the recombinant plasmid pAAV2-FGF-21-luciferase (Figure 1B). In addition, the pAAV2-luciferase vector was used as pAAV-Null-Luciferase as a blank control (Figure 1A).

Example 2 Double enzyme digestion detection of recombinant plasmid

The two recombinant plasmids constructed in Example 1 were double-digested with Hind III and EcoR I to detect whether the size of the digested fragments was consistent with the designed results. The specific steps are as follows:

(1) The two recombinant plasmids constructed in Example 1 were transformed into Escherichia coli, and the plasmids were extracted after amplification by shaking;

(2) Use Hind III and EcoR I and the plasmid extracted in step (1) to prepare a double enzyme digestion system (Table 1), and digest in a constant temperature water bath at 37°C for 1 hour;

(3) After the enzyme digestion is completed, perform agarose gel electrophoresis to observe the electrophoresis bands after enzyme digestion.

Table 1 Double enzyme digestion system

The results are shown in Figure 2. After double enzyme digestion, pAAV2-FGF-21-Luciferase has two bands visible under the gel imaging system after electrophoresis, one is about 5900 bp and the other is about 630 bp (shown in lane 2 in Figure 2), which are consistent with the size of the pAAV2 plasmid fragment and the FGF-21 fragment, respectively, indicating that pAAV2-FGF-21-Luciferase has been correctly identified after enzyme digestion; after double enzyme digestion, plasmid pAAV-ABD-FGF-21-Luciferase has two bands visible under the gel imaging system after electrophoresis, one is about 5900 bp and the other is about 760 bp (shown in lane 5 in Figure 2), which are consistent with the size of the pAAV2 plasmid vector fragment and the ABD-FGF-21-P2A fragment, respectively, indicating that pAAV2-ABD-FGF-21-Luciferase has been correctly identified after enzyme digestion.

Example 3 Sequencing Detection of Recombinant Plasmid

(1) Primers were designed based on the promoter CMV sequence and P2A sequence of the pAAV2-luciferase plasmid, and then synthesized at Suzhou Jinweizhi Biotechnology Co., Ltd.; the forward primer (F) sequence was SEQ ID NO: 2, and the rear primer (R) sequence was SEQ ID NO: 3;

(2) The company used the synthesized primers to perform DNA sequencing of the target fragments of the two recombinant plasmids pAAV2-ABD-FGF-21-luciferase and pAAV2-FGF-21-luciferase constructed in Example 1;

(3) Use DNAMAN software to compare the sequencing results with the expected DNA sequence in the recombinant plasmid.

The results are shown in Figure 3. The ABD-FGF-21 gene sequence inserted in the pAAV2-ABD-FGF-21-luciferase recombinant plasmid was aligned correctly, and the FGF-21 gene sequence inserted in the pAAV2-FGF-21-luciferase recombinant plasmid was aligned correctly, indicating that the gene open reading frame in the recombinant plasmid was docked correctly.

Example 4 Preparation of recombinant adeno-associated virus

The recombinant plasmid pAAV2-ABD-FGF-21-Luciferase and the recombinant plasmid pAAV2-FGF-21-luciferase were co-transfected with the Helper plasmid and the AAV2 capsid protein plasmid into HEK-293T cells, respectively, to prepare AAV viruses AAV2-ABD-FGF-21-luciferase and AAV2-FGF-21-luciferase, respectively, as follows:

(1) HEK-293T cells were adherently cultured in DMEM medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% _CO2 until the cell density reached 80% cell confluence. The cells were then collected and counted.

(2) The HEK-293T cells obtained in step (1) were subcultured into a 10 cm diameter cell culture dish (1×10 ⁶ cells/dish) in DMEM medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% CO ₂ for 20 hours until the cell density reached 90% cell confluence. Then, fresh DMEM medium was replaced 4 hours before plasmid transfection;

(3) Take 10 μg of pAAV2-ABD-FGF-21-Luciferase, 8 μg of pHelper, and 8 μg of pAAV2 capsid plasmid respectively for packaging recombinant AAV2-ABD-FGF-21-Luciferase adeno-associated virus; replace pAAV2-ABD-FGF-21-Luciferase with pAAV2-FGF-21-Luciferase for packaging recombinant AAV2-FGF-21-Luciferase adeno-associated virus; replace pAAV2-Null-Luciferase with pAAV2-ABD-FGF-21-Luciferase for packaging AAV2-Luciferase adeno-associated virus;

(4) Add the three groups of plasmids in step (3) to 500 μL serum-free DMEM medium, mix gently and let stand at room temperature for 5 minutes to obtain liquid A; add 13 μL of Imafect transfection reagent to 500 μL serum-free DMEM medium, mix gently and let stand at room temperature for 5 minutes to obtain liquid B;

(5) Gently add droplets of liquid B to liquid A and let it stand for a few seconds before gently mixing. After the mixture stands at room temperature for 15 minutes, add it dropwise to the cell culture dish obtained in step (2) and gently mix. After 6 hours, replace it with fresh cell culture medium. After the cell culture medium turns yellow, replace the medium and collect the supernatant. After 72 hours of culture after transfection, use 600 μL PBS to gently blow off the adherent cells in the dish and store them in a -80°C refrigerator for later use.

Example 5 Virus extraction and purification

The packaged AAV2-ABD-FGF-21-Luciferase, AAV2-FGF-21-Luciferase and pAAV2-Null-Luciferase viruses were extracted by freeze-thaw method, and then purified by iodixanol density gradient centrifugation. The specific steps are as follows:

(1) The virus-packaged HEK 293T cells obtained in Example 4 were repeatedly frozen and thawed three times between -80°C and 37°C, and the precipitate was vortexed each time to allow the virus to be better released from the cells;

(2) Incubate the cells that have been frozen and thawed three times at 37°C for 2 hours using 100 U of universal nuclease, then centrifuge at 10,000 rpm for 10 minutes to collect the supernatant;

(3) Gently add 15% (3 mL), 25% (2 mL), 40% (2 mL) and 60% (1 mL) density gradient iodixanol (Table 2) from top to bottom in a 10 mL ultracentrifuge tube. After each layer is stable, slowly add the supernatant obtained in step (2) to the top layer and centrifuge at 40,000 rpm for 4 hours.

(4) Pipette the target virus layer (40% iodixanol) and add it to a 10000 kDa filter tube. Fill the filter tube with PBS and centrifuge at 3000 rpm for 4 minutes. Repeat the centrifugation to remove the iodixanol and concentrate the target virus. Store in a -80°C refrigerator for later use.

Table 2 Iodixanol solution formulas of different concentrations

Example 6 Western Blot detection of target protein expression

HEK-293T cells were infected with the purified viruses (AAV2-ABD-FGF-21-Luciferase, AAV2-FGF-21-Luciferase and AAV2-Null-Luciferase) in Example 5, and then the expression of the target protein was detected by Western Blot. The specific method is as follows:

(1) Take 20 μL of the purified virus in Example 5 to infect HEK-293T cells, collect the HEK-293T cell culture fluid after 36 hours of infection, add 5 μL of protein preparation buffer, boil the sample in 100°C boiling water for 7 minutes, quickly cool it on ice, obtain the sample, and store it at 20°C;

(2) Take 20 μL PBS (phosphate buffered saline) and infect HEK-293T cells (HEK-293T cells that have not been infected with the virus) using the same method as step (1) to obtain a sample as a blank control;

(3) The protein samples obtained in step (1) were subjected to 10% SDS-PAGE electrophoresis at a constant voltage of 80 V. After the electrophoresis, the PAGE gel was removed and transferred at a constant current of 300 mA for 90 minutes;

(4) Place the PVDF membrane after the protein sample transfer in 5% milk prepared with 1×TBST and shake it at room temperature for 1 hour to block it. After blocking is complete, discard the milk and add TBST several times and shake it at room temperature for 7 minutes to wash away the residual milk;

(5) Place the PVDF membrane in 3% BSA-prepared FGF-21 antibody (1:1000) and incubate on a shaker at 4°C overnight. After incubation, rinse with TBST several times to remove the FGF-21 antibody. Then place the PVDF membrane in 3% BSA-prepared goat anti-mouse IgG (1:2000) and incubate on a shaker at room temperature for 1 hour. Rinse three times with TBST and observe the results after ECL color development.

The results are shown in Figure 4. FGF-21 protein bands were detected in the culture fluid of HEK-293T cells infected with AAV2-FGF-21-Luciferase and the culture fluid of HEK-293T cells transfected with AAV2-ABD-FGF-21-Luciferase, while no FGF-21 protein bands were detected in the culture fluid of blank control HEK-293T cells.

Example 7 Western Blot Detection of Capsid Protein Expression

Western Blot was used to detect the expression of AAV2 capsid VP1, VP1 and VP3 capsid proteins. The method and steps were the same as in Example 1, except that the FGF-21 antibody (1:1000) in step (5) was replaced by anti-Capsid (capsid protein) antibody (1:1000).

The results are shown in Figure 5. The protein samples prepared by adeno-associated virus AAV2-ABD-FGF-21-Luciferase, AAV2-FGF-21-Luciferase and AAV2-Null-Luciferase all showed obvious AAV capsid protein bands VP1, VP2 and VP3. The results show that the plasmids pAAV2-FGF-21-Luciferase and plasmids pAAV2-ABD-FGF-21-Luciferase prepared in this study can be assembled into AAV2 viruses in HEK-293T cells.

Example 8 Construction of T2DM mouse model

C57BL/6 mice were used to construct a type II diabetes mouse model T2DM. The construction process is shown in Figure 6 and is as follows:

(1) Thirty SPF male C57BL/6 mice aged 6 weeks were selected and raised in a certified standard laboratory animal center in an environment with 12-h reverse light-dark cycle (temperature 22±2°C, humidity 60±5%) with free access to food. They were fed adaptively for 1 week before the experiment.

(2) The 30 mice obtained in step (1) were randomly divided into 5 groups, each with 6 mice, namely, normal feed group (Normal), model control group (Model), AAV2-Null group, AAV2-FGF-21-Luciferase group, and AAV2-ABD-FGF-21-Luciferase group, where:

Model control group (Model): mice fed a high-fat diet (HFD) were prepared to develop insulin resistance.

Normal feed group (Normal): fed with normal feed,

AAV2-Null group: fed with a high-fat diet (HFD),

AAV2-FGF-21-Luciferase group: fed with a high-fat diet (HFD),

AAV2-ABD-FGF-21-Luciferase group: fed with a high-fat diet (HFD),

(3) The mice fed for 8 weeks in step (2) were starved for 12 hours, and then freshly prepared STZ (streptozotocin) buffer was intraperitoneally injected into the model control group (Model), AAV2-Null group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group within 30 minutes. The normal diet group (Normal) was injected with the same volume of sodium citrate solution; the injection dose was 80 mg/kg;

(4) After 72 hours, the fasting blood glucose (FBG) level of the mice was measured using a glucose analyzer. When the FBG level of the mice exceeded 11.1 mmol/L, it indicated that the T2DM mouse model was successfully established. The body weight of the mice was also recorded.

Example 9 Targeting of AAV2 Adeno-Associated Virus to Mouse Liver Tissue

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into the T2DM mouse model constructed in Example 8 by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus, while the normal feed group (Normal) and model control group (Model) were injected with the same volume of PBS;

(2) After 7 days, each mouse was intraperitoneally injected with luciferase substrate Luciferin at a dose of 1.5 mg/10 g, i.e., a 20 g mouse was intraperitoneally injected with 100 μL (30 mg/mL) Luciferin;

(2) Within 10-35 minutes after Luciferin injection, the expression and distribution of AAV2 virus-transduced genes in mice were observed using a small animal in vivo imaging device.

The results are shown in Figure 7. Compared with the Normal group and the Model group, the AAV2-Null-Luciferase group, the AAV2-FGF-21-Luciferase group and the AAV2-ABD-FGF-21-Luciferase group were all able to target liver tissue, indicating that the modification of the AAV2 plasmid did not change its tropism for liver tissue. In other words, AAV2-ABD-FGF-21-Luciferase maintained its original tissue tropism for the liver.

Example 10 Effects of secretory overexpression of FGF-21 and ABD-FGF-21 in the liver on T2DM mice

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into the T2DM mouse model constructed in Example 8 by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus, while the normal feed group (Normal) and model control group (Model) were injected with an equal volume of 100 μL PBS; the body weight of the mice was measured every two days, and the blood glucose level was measured once a week;

(2) After 8 weeks of feeding, the mice were killed, and tissues and blood were collected. Tissue specimens were prepared and immediately stored at -80°C.

(3) The serum in the blood was separated, and the morphology of liver, kidney and adipose tissue was examined by H&E staining, and the expression level of serum FGF-21 was detected by ELISA.

The results are shown in Figure 8. Compared with the mice in the Normal group, the average body weight of the mice in the Model group and the AAV2-Null control group decreased significantly during the entire experimental period ( P < 0.05), which was consistent with the symptoms of type II diabetic mice. The average body weight of the mice in the AAV2-FGF-21-Luciferase group decreased significantly in the first and second weeks after virus injection ( P < 0.01), was alleviated in the second and fifth weeks, and continued to decrease in the fifth and seventh weeks. The average body weight of the mice in the AAV2-ABD-FGF-21-Luciferase group showed a downward trend in the first week after virus injection, but the body weight recovered rapidly in the second and seventh weeks, and returned to the level consistent with normal mice in the seventh week (Figure 8A and B). Compared with the AAV2-FGF-21-Luciferase group, the injection of AAV2-ABD-FGF-21-Luciferase significantly improved the weight loss symptoms of type II diabetic mice ( P < 0.01).

During the entire experiment, firstly, compared with the Normal group, the fasting blood glucose level of mice in the Model group was significantly increased ( P <0.01), indicating that the model was successfully prepared. Secondly, compared with the Model group, there was no significant difference in the fasting blood glucose level of mice in the AAV2-Null group, indicating that the tail vein injection of AAV2 adeno-associated virus had no significant effect on the blood glucose level of type II diabetic mice. The results of the experimental group showed that the fasting blood glucose level of mice in the AAV2-FGF-21-Luciferase group was significantly reduced from 1 to 5 weeks ( P <0.01), but began to increase in the 5th week, indicating that the use of AAV2 to deliver FGF-21 can significantly reduce the blood glucose level of type II diabetic mice, but its hypoglycemic effect decreases with time; while the fasting blood glucose level of mice in the AAV2-ABD-FGF-21-Luciferase group was significantly lower than that in the Model group ( P <0.01), and no hypoglycemia occurred (Figure 8C).

At the end of the experiment, compared with the Normal group, the INS and HOMA-IR levels of the Model group were significantly increased, while the HOMA-IS level was significantly decreased ( P <0.01); compared with the Model group, there were no statistical differences in the INS, HOMA-IR and HOMA-IS levels of the AAV2-Null group, while the INS and HOMA-IR levels of the AAV2-FGF-21 group were decreased, and the HOMA-IS level was increased ( P < 0.05). The INS and HOMA-IR levels of the AAV2-ABD-FGF-21-Luciferase group were significantly decreased, and the HOMA-IS level was significantly increased (p < 0.01) (Figure 8D and F).

The above results indicate that overexpression of FGF-21 in the liver via gene delivery by recombinant AAV2 vector can reduce fasting blood glucose in T2DM mice, but its hypoglycemic effect weakened in the fifth week and fasting blood glucose levels began to rise; whereas administration of ABD-FGF-21-Luciferase can continuously overexpress ABD-FGF-21 in the liver of mice for a long time, significantly reducing fasting blood glucose, restoring body weight, and significantly improving basal insulin resistance and insulin sensitivity in T2DM mice throughout the 8-week experiment.

Embodiment 11

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into the T2DM mouse model constructed in Example 8 by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus. The normal feed group (Normal) and the model control group (Model) were injected with an equal volume of 100 μL PBS and raised under the conditions of the aforementioned Example 8;

(2) After 7 weeks, each mouse was intraperitoneally injected with glucose solution (concentration of 20%), and blood glucose levels were measured at different time points after administration: 0, 15, 30, 60 and 120 minutes to test the glucose tolerance (GTT assay) of each group of mice. The injection amount of glucose was 1 g/kg, and the glycosylated serum protein was measured using an ELISA kit according to the instructions;

(3) After 7 weeks, the mice were killed and blood was collected. The serum was then separated and the level of circulating FGF-21 in the serum was detected using an Elisa kit.

The results are shown in Figure 9. After intraperitoneal injection of glucose, the fasting blood glucose of each group of mice increased, and the blood glucose value reached the highest peak at 15 minutes. The blood glucose levels of the Model group and AAV2-Null group were significantly higher than those of the Normal group, and lasted until 120 minutes after the injection of glucose. The blood glucose level of mice in the AAV2-FGF-21-Luciferase group was lower than that in the Model group ( P <0.05); the blood glucose level of mice in the AAV2-ABD-FGF-21-Luciferase group was significantly lower than that in the Model group ( P < 0.01) (Figure 9A).

The measured blood glucose data were analyzed using the prism program with the formula AUC=7.5*(G0+2G15+G30)+15*(G30+2G60+2G90+G120), and the area under the glucose curve (AUC) of each group was calculated. It can be seen that the blood glucose AUC of mice in the Model group and AAV2-Null group was significantly higher than that in the Normal group ( P <0.01), while the blood glucose AUC of mice in the AAV2-FGF-21-Luciferase group was lower than that in the Model group ( P <0.05). Compared with the AAV2-FGF-21-Luciferase group, the blood glucose AUC of mice in the AAV2-ABD-FGF-21-Luciferase group was significantly decreased (Figure 9B).

Compared with the Model group, the average GSP level of mice in the AAV2-Null group and the AAV2-FGF-21-Luciferase group did not change significantly; however, the average GSP level of mice in the AAV2-ABD-FGF-21-Luciferase group was significantly decreased compared with the Model group, AAV2-Null group, and AAV2-FGF-21-Luciferase group ( P < 0.01) (Figure 9C).

The ELISA test results showed that the average serum FGF-21 level of mice in the AAV2-ABD-FGF-21-Luciferase group was significantly higher than that in the AAV2-FGF-21-Luciferase group and the AAV2-Null group, indicating that the ABD-FGF-21 fusion protein significantly increased the serum level of FGF-21 (Figure 9D).

The above results show that AAV2-ABD-FGF-21-Luciferase adeno-associated virus can significantly increase the serum FGF-21 level of type II diabetic mice, reduce their glycosylated hemoglobin level, improve glucose tolerance, and reduce fasting blood sugar. In addition, the above effects of AAV2-ABD-FGF2-Luciferase adeno-associated virus are better than those of AAV2-FGF-21-Luciferase adeno-associated virus, indicating that AAV2-mediated liver ABD-FGF-21 expression has a better hypoglycemic effect in type II diabetic mice.

Example 12

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into the T2DM mouse model constructed in Example 8 by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus. The normal feed group (Normal) and the model control group (Model) were injected with an equal volume of 100 μL PBS and raised under the conditions of the aforementioned Example 8;

(2) After 7 weeks, the mice were killed and blood was collected. The serum was then separated and the levels of TC (total cholesterol), TG (triglycerides), LDL-C (low-density lipoprotein cholesterol), and HDL-C (high-density lipoprotein cholesterol) in the serum of each group of mice were detected using Elisa kits.

The results are shown in Figure 10. Compared with those in the Normal group, the serum TC, TG and LDL-C levels of the mice in the Model group were significantly increased ( P < 0.01), and the HDL-C content was significantly decreased ( P <0.01); compared with the Model group, the TC, TG, LDL-C and HDL-C levels of the mice in the AAV2-Null group were almost unchanged; while the TC, TG and LDL-C levels of the mice in the AAV2-FGF-21-Luciferase group were decreased ( P < 0.05), and the HDL-C content was increased ( P <0.05); the TC, TG and LDL-C levels of the mice in the AAV2-ABD-FGF-21-Luciferase group were significantly decreased ( P < 0.01), and the HDL-C content was significantly increased ( P < 0.01) (as shown in Figure 10).

The above results show that overexpression of FGF-21 in the liver can reduce TC, TG and LDL-C levels, increase HDL-C levels, and improve blood lipid levels in T2DM mice. The above effects of recombinant AAV2-ABD-FGF-21-Luciferase adeno-associated virus injection are better than recombinant AAV2-FGF-21-Luciferase adeno-associated virus injection, indicating that recombinant AAV2-mediated liver ABD-FGF-21 expression has a better lipid-lowering effect in type II diabetic mice.

Example 13

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into the T2DM mouse model constructed in Example 8 by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus. The normal feed group (Normal) and the model control group (Model) were injected with an equal volume of 100 μL PBS and raised under the conditions of the aforementioned Example 8;

(2) After 7 weeks, the weight of the mice was recorded, and then the mice were killed and their livers were removed. The morphology of the livers was observed and the weight was recorded. The livers were then sliced and stained with HE to observe the pathological changes inside the livers and the morphology of the hepatocytes.

(3) Blood was collected during the sacrifice process, serum was separated, and serum AST and ALT activities were tested.

The livers of the mice in the Normal group were reddish brown, soft, and smooth. The liver tissue morphology of the mice in the Model group and the AAV2-Null group was almost the same, with an enlarged size, lighter color, granular feel, tight capsule, and less softness. The livers of the mice in the AAV2-FGF-21-Luciferase group were slightly larger, partially lighter in color, and less granular. The livers of the mice in the AAV2-ABD-FGF-21-Luciferase group were normal in size, similar in color to those of the mice in the Normal group, and soft in texture (Figure 11A).

Compared with the Normal group, the liver index, serum AST and ALT activities of the mice in the Model group were significantly increased; compared with the Model group, the liver index, serum AST and ALT activities of the mice in the AAV2-Null group were almost unchanged; while the liver index, serum AST and ALT activities of the mice in the AAV2-FGF-21-Luciferase group were decreased ( P <0.05); compared with the AAV2-Null group and the Model group, the liver index, serum AST and ALT activities of the mice in the AAV2-ABD-FGF-21 group were significantly decreased ( P < 0.01) (Figure 11B-D).

H&E staining results showed that the liver lobule structure of the Normal group mice was good, the hepatocytes had regular morphology and good arrangement, and there was no fatty degeneration or necrosis of the hepatocytes; while the liver lobule structure of the Model group and AAV2-Null group mice was disordered, the hepatocytes had irregular morphology, obvious swelling, fatty degeneration, and multiple inflammatory cell infiltration; compared with the Model group, the hepatocyte structure of the AAV2-FGF-21-Luciferase group mice was relatively clear and orderly, the fatty degeneration of hepatocytes was improved, and the infiltration of inflammatory cells was reduced; and the effect of the AAV2-ABD-FGF-21-Luciferase group mice was better than that of the AAV2-FGF-21 group mice (Figure 11E).

The above results indicate that AAV2-ABD-FGF-21-Luciferase adeno-associated virus can reverse hepatocyte fatty degeneration in type II diabetic mice, and its effect is better than AAV2-FGF-21-Luciferase adeno-associated virus.

Embodiment 14

(1) 1×10 ¹¹ vg of the virus (obtained in Example 5) was injected into mice by tail vein injection. The mice were divided into AAV2-Null-Luciferase group, AAV2-FGF-21-Luciferase group and AAV2-ABD-FGF-21-Luciferase group according to the type of virus. The normal feed group (Normal) and model control group (Model) were injected with an equal volume of 100 ul PBS and fed under the conditions of the aforementioned Example 8;

(2) Seven weeks later, the mice were killed and their inguinal white adipose tissue (WAT) and scapular brown adipose tissue (BAT) were removed to prepare immune sections. The sections were then stained with hematoxylin-eosin (H&E) to observe the morphology of the white adipose tissue and brown adipose tissue of the mice.

The results are shown in Figure 12. Compared with the Normal group mice, the adipocytes of the Model group mice were enlarged in both WAT and BAT. The fat morphology of the AAV2-Null group mice was similar to that of the Model group. The adipocyte hypertrophy of the AAV2-FGF-21-Luciferase group mice was improved, the size of the adipocytes was redistributed, and the proportion of small adipocytes was larger. Moreover, the improvement effect of adipocyte hypertrophy in the AAV2-ABD-FGF-21-Luciferase group mice was better than that in the AAV2-FGF-21-Luciferase group mice.

The above results indicate that administration of AAV2-ABD-FGF-21-Luciferase adeno-associated virus can inhibit fat accumulation in type II diabetic mice, and its effect is better than that of AAV2-FGF-21-Luciferase adeno-associated virus.

Claims (7)

1. An FGF-21 fusion gene, characterized in that: the 5' -end of the FGF-21 gene is connected with an ABD gene, and the sequence is SEQ ID NO. 1.

2. The use of an FGF-21 fusion gene according to claim 1 for the preparation of a medicament for the treatment of type II diabetes.

3. An AAV recombinant virus for increasing the sustained expression level and stability of FGF-21, comprising: comprising an adeno-associated viral capsid and the FGF-21 fusion gene of claim 1.

4. The AAV recombinant virus of claim 3, wherein the AAV recombinant virus increases the sustained expression level and stability of FGF-21, wherein: the adeno-associated viral capsid is AAV2, AAV5 or AAV8.

5. The AAV recombinant virus of claim 3, wherein the AAV recombinant virus increases the sustained expression level and stability of FGF-21, wherein: the AAV recombinant virus also comprises a Flag sequence, a P2A sequence and Luciferase.

6. The AAV recombinant virus of claim 3, wherein the AAV recombinant virus increases the sustained expression level and stability of FGF-21, wherein: the promoter in AAV recombinant viruses is CMV promoter.

7. The use of an AAV recombinant virus according to any one of claims 3-6 for increasing the sustained expression level and stability of FGF-21 for the preparation of a medicament for the treatment of type II diabetes.