WO2023045996A1 - Nucleic acid construct for gene therapy of carbohydrate metabolism-related diseases - Google Patents

Abstract

A nucleic acid construct for gene therapy of carbohydrate metabolism-related diseases. The nucleic acid construct comprises a polynucleotide encoding GLP-1 or an analogue thereof, the total number of GLP-1 or the analogue thereof is two or more, and GLP-1 and GLP-1, GLP-1 and the analogue thereof, or the GLP-1 analogue and the GLP-1 analogue are linked by means of a polynucleotide encoding a linker peptide. The nucleic acid construct can be used for high-efficiency expression of active GLP-1 and the analogue thereof in vivo and in vitro, and can be used for drug development of carbohydrate metabolism-related diseases such as type 2 diabetes, body metabolic disorder, diabetes-related complications, and obesity.

Description

A nucleic acid construct for gene therapy of sugar metabolism-related diseases technical field

The invention relates to the technical field of gene therapy or biomedicine, in particular to a nucleic acid construct for gene therapy of diseases related to carbohydrate metabolism.

Background technique

Diabetes is a disease in which the body cannot produce fully functional insulin, or cannot properly use and store glucose. Glucose remaining in the blood causes hyperglycemia and a series of complications are the basis of diabetes. Type 2 diabetes, or adult-onset diabetes and non-insulin-dependent diabetes, accounts for about 90% of diabetic patients. Good blood sugar control is crucial for reversing and alleviating complications of type 2 diabetes, reducing morbidity and mortality, and improving patients' quality of life.

Some progress has been made in the treatment of diabetes based on insulin injection, but it is still difficult to achieve optimal blood sugar control. The accompanying hypoglycemia and further weight gain in insulin injection therapy will aggravate the patient's body's metabolic disorders and diabetes-related complications, and lead to related deaths risk.

Glucagon-like peptide-1 (GLP-1)-based diabetes treatment works through mechanisms such as increasing glucose-dependent insulin secretion, slowing gastric emptying, reducing postprandial hyperglycemia, and reducing food intake. To control blood sugar safely and effectively, and at the same time obtain various clinical benefits such as weight loss and cardiovascular protection. GLP-1-based diabetes treatment is expected to replace insulin as the first-line drug for the treatment of diabetes.

At present, the main GLP-1-based diabetes treatments include GLP-1 receptor agonists and dipeptidyl peptidase-4 (dipeptidyl peptidase-4, DPP-4) inhibitors, both of which aim to increase the effective concentration of GLP-1. Among them, the new long-acting glucagon-like peptide-1 (GLP-1) receptor agonists represented by Liraglutide (Liraglutide), Semaglutide (Semaglutide) and Dulaglutide (Dulaglutide) have clinical efficacy. It has outstanding performance in terms of patient compliance and other aspects, and has entered the diabetes guidelines of European and American countries as a first-line drug.

However, GLP-1 and its analogues are quickly degraded by DPP-4 in the body and have a short half-life. Although GLP-1 analogues significantly prolong the duration of action of GLP-1 in vivo, frequent (1-7 days) subcutaneous administration is still required. And because the development, production, and use costs of peptide drugs are much higher than those of small molecule diabetes drugs, GLP-1 analogue diabetes treatment does not have a cost advantage. Diabetic patients with blood sugar control have realistic and urgent clinical needs.

The GLP-1 analog gene therapy technology route delivered by viral and non-viral vectors theoretically solves the goal of long-term or even life-long expression of GLP-1, and can increase the effective concentration of GLP-1 once and for all. Great value for long-term clinical benefit. However, the expression efficiency of polypeptide gene expression vectors is much lower than that of macromolecular protein gene expression vectors. As GLP-1 and its analogs with about 30 amino acids, the small expression frame makes it suitable for any viral and non-viral vectors. Effective expression is not a small difficulty. At the same time, the short plasma half-life further increases the barriers to effective blood concentrations of GLP-1 and its analogs. So far, no GLP-1 analogue gene therapy technology route delivered by viral and non-viral vectors has achieved effective clinical progress.

In view of the above problems, the drug development of gene therapy technology routes based on viral and non-viral vector delivery mainly requires breakthroughs in the following two aspects:

How to use the gene carrier delivered by recombinant virus or non-viral vector to stably and efficiently express GLP-1 and its analogues in the body for a long time in the form of genome integration or non-integration, so that patients with type 2 diabetes and related diseases can be treated for a long time or even Lifetime benefits while reducing production and usage costs.

Contents of the invention

In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a nucleic acid construct for gene therapy of diseases related to carbohydrate metabolism, which is used to solve the problems in the prior art.

To achieve the above-mentioned purpose and other related purposes, the present invention provides a nucleic acid construct comprising a polynucleotide encoding GLP-1 or an analog thereof, and the total amount of said GLP-1 or an analog thereof is More than two, between GLP-1 and GLP-1, between GLP-1 and its analogues or between GLP-1 analogues and GLP-1 and analogues are linked by a polynucleotide encoding a linker peptide.

The present invention also provides a lentivirus, which is packaged from the nucleic acid construct.

The present invention also provides a lentiviral vector system, which includes the nucleic acid construct and a helper plasmid.

The present invention also provides an adeno-associated virus vector system, which includes the nucleic acid construct and a helper plasmid.

The present invention also provides an adeno-associated virus, which is packaged by the adeno-associated virus vector system.

The present invention also provides a cell line, which is a cell line infected by the lentivirus or adeno-associated virus.

The present invention also provides the use of the nucleic acid construct, lentivirus, lentiviral vector system, adeno-associated virus, adeno-associated virus vector system, and cell line in the preparation of products for the prevention and treatment of diseases related to carbohydrate metabolism.

As mentioned above, the nucleic acid constructs of the present invention for gene therapy of diseases related to carbohydrate metabolism have the following beneficial effects: the present invention creates GLP-1 or its analogues in the form of tandem covalent dimers or multimers The nucleic acid construct of the expression framework, which can be used to highly express active GLP-1 and its analogs in vivo and in vitro, which is a great potential for type 2 diabetes, metabolic disorders, diabetes-related complications and obesity A technical route has been developed for gene therapy drugs for diseases related to glucose metabolism such as diabetes. The scope of application of the present invention includes various forms of gene therapy for sugar metabolism-related diseases based on GLP-1 and its analogs, for example, the construct can be used for gene therapy drugs for sugar metabolism-related diseases delivered by viral vectors or non-viral vectors Clinical research and new drug development and production.

Description of drawings

Figure 1A shows the pKL-kan-lenti-EF1α-WPRE plasmid map of the present invention.

Figure 1B shows the pAAV-MCS-CMV-EGFP (reverse) plasmid map of the present invention.

Figure 2 shows the design of an expression framework for a nucleic acid construct expressing a GLP1 receptor agonist.

Figure 3A shows the design of a nucleic acid construct (lentiviral vector) for expression of a GLP1 receptor agonist.

Figure 3B shows the design of a nucleic acid construct (adeno-associated virus vector) for expression of a GLP1 receptor agonist.

Figure 4A shows the GLP1 receptor agonist expression (Westernblotting) reduction gel electrophoresis detection results after transduction of cells with lentivirus expressing GLP1 receptor agonist.

Fig. 4B shows the non-reducing gel electrophoresis detection results of GLP1 receptor agonist expression (Western blotting) after cells are transduced with lentivirus expressing GLP1 receptor agonist.

Figure 5 shows the activation effect of cell culture supernatants transduced with lentiviruses expressing GLP1 receptor agonists on cells with GLP1 receptors. A1-A4 (i.e. the first row) are GLP1(7-37) 8nM, 40nM, 200nM, 1000nM respectively; B1-B4 (i.e. the second row) are the different transduction volumes of KLDi02: 2μL, 5μL, 10μL, 20μL; C1-C4 (that is, the third row) are the different transduction volumes of KLDi01: 2 μL, 5 μL, 10 μL, and 20 μL, respectively.

Figure 6 shows the blood glucose concentration of DB/DB mice after receiving AAV expressing GLP1 receptor agonist.

Figure 7 shows the glucose tolerance of C57BL/6 mice after receiving adeno-associated virus expressing GLP1 receptor agonist.

Detailed ways

Unless otherwise defined below, all technical and scientific terms mentioned herein have the meanings commonly understood by those skilled in the art to which this invention belongs.

The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue, and the nucleic acid construct can be a lentiviral vector or an adeno-associated viral vector, which includes a vector The backbone is the empty vector and expression framework.

The term "expression framework" refers to a sequence that has the potential to encode a protein.

The present invention provides a nucleic acid construct, which comprises a polynucleotide encoding GLP-1 or its analogues, the total number of GLP-1 or its analogues is more than two, GLP-1 and GLP -1, between GLP-1 and its analogs, or between GLP-1 analogs and GLP-1 and analogs through polynucleotides encoding linker peptides.

In one embodiment, the GLP-1 analog is a 7-36 or 7-37 amino acid peptide chain at the N-terminus of GLP-1. This part of amino acids is the biologically active part of GLP-1, so drug research and development are all focused on these amino acid sequences. GLP-1 analogs can also be the substitution, deletion, addition of individual amino acids in the 7-36 or 7-37 amino acids at the N-terminal of GLP-1, or the linking of individual amino acids with different compounds.

The total number of GLP-1 or its analogues can be, for example, two, three, four, five or more.

The meaning of the total amount of the GLP-1 or its analogs is selected from any of the following:

1) When the construct contains GLP-1 but does not contain its analogs, the total number of GLP-1 is more than two;

2) When the construct contains GLP-1 analogs but does not contain GLP-1, the total number of GLP-1 analogs is more than two;

3) When the construct contains both GLP-1 and GLP-1 analogs, the number of GLP-1 and GLP-1 analogs is more than two.

The connection mode between GLP-1 or its analogs in the nucleic acid construct is as follows: signal peptide——GLP-1 or GLP-1 analogs——linking peptide——GLP-1 or GLP-1 analogs— - connecting peptide - GLP-1 or GLP-1 analogue, wherein the number of GLP-1 or GLP-1 analogue - connecting peptide - GLP-1 or GLP-1 analogue unit is one or more, For example, there may be two, three, four, five or more.

The connecting peptide consists of amino acids with small side chains. For example: (Gly)4, GSGGSG, GSGGSGG GSGGSGGG, GGGGSGGG, (GGGGS)3.

The nucleic acid construct can effectively express glucagon-like peptide 1 (glucagon-like peptide-1, GLP-1) or its analogs in vivo. The GLP1 or its analogs expressed by the nucleic acid construct act as an agonist of the GLP1 receptor, so the nucleic acid construct can also be referred to as a construct expressing a GLP1 receptor agonist.

The nucleic acid construct is a nucleic acid construct used for gene therapy of diseases related to carbohydrate metabolism.

Specifically, diseases related to glucose metabolism include diabetes or its complications, and obesity.

In one embodiment, the diabetes is type 2 diabetes.

The nucleic acid construct comprises an expression framework as shown in SEQ ID NO:4 or SEQ ID NO:6. The GLP1 or its analog expressed by the expression framework acts as an agonist of the GLP1 receptor. Or include nucleosides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO.4 or SEQ ID NO.6 acid sequence.

Polynucleotides capable of expressing proteins or polypeptides other than GLP1 or its analogs are not included in the nucleic acid construct. That is, the only protein or polypeptide that can be effectively expressed by the nucleic acid construct is GLP1 or its analogues.

The amino acid sequence of the polypeptide encoded by the nucleic acid construct is shown in SEQ ID NO:3 or SEQ ID NO:5.

The nucleic acid construct is used to produce virus-based gene therapy vectors, preferably the gene therapy vectors are lentiviral vectors or adeno-associated viral vectors.

The nucleic acid construct is a viral vector or a non-viral vector.

In some embodiments, the nucleic acid construct is a lentiviral vector. The vector skeleton in the lentiviral vector can be the vector skeleton in the prior art.

In one embodiment, the nucleotide sequence of the lentiviral vector is shown in SEQ ID NO.12 or SEQ ID NO.13. Or include nucleosides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO.12 or SEQ ID NO.13 acid sequence.

In some embodiments, the nucleic acid construct is an adeno-associated viral vector. The vector skeleton in the adeno-associated virus vector can be the vector skeleton in the prior art.

In one embodiment, the nucleotide sequence of the adeno-associated virus vector is shown in SEQ ID NO.17 or SEQ ID NO.18. Or include nucleosides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to SEQ ID NO.17 or SEQ ID NO.18 acid sequence.

The present invention also provides a lentiviral vector system, which includes the nucleic acid construct and a helper plasmid.

Further, the helper plasmid encodes one or more nucleotide sequences of gag and pol proteins and other essential viral packaging component nucleotide sequences, and the helper plasmid may include packaging plasmids and envelope plasmids.

Further, the lentiviral vector system also includes host cells. The host cell carries a lentiviral vector. The host cells can be selected from various applicable host cells in the art, as long as the purpose of the present invention is not limited. Specific applicable cells may be cells that produce lentivirus, such as 293T cells.

The present invention also provides a lentivirus, which is packaged from the lentiviral vector system.

The present invention also provides an adeno-associated virus vector system, which includes the nucleic acid construct and a helper plasmid.

Further, the helper plasmid encodes one or more nucleotide sequences of gag and pol proteins and other essential viral packaging component nucleotide sequences, and the helper plasmid may include packaging plasmids and envelope plasmids.

Further, the adeno-associated virus vector system also includes host cells. The host cell carries an adeno-associated virus vector. The host cells can be selected from various applicable host cells in the art, as long as the purpose of the present invention is not limited. Specific applicable cells may be cells producing adeno-associated virus, such as 293T cells.

The present invention also provides an adeno-associated virus, which is packaged by the adeno-associated virus vector system.

The present invention also provides a cell line, which is a cell line infected by the lentivirus or adeno-associated virus.

The cell line can be used as a biological agent to prepare products for preventing or treating neurodegenerative diseases.

The present invention also provides the use of the nucleic acid construct, lentivirus, lentiviral vector system, adeno-associated virus, adeno-associated virus vector system, and cell line in the preparation of products for the prevention and treatment of diseases related to carbohydrate metabolism.

Specifically, diseases related to glucose metabolism include diabetes or its complications, and obesity.

In one embodiment, the diabetes is type 2 diabetes.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

The following examples include: constructing a series of nucleic acid constructs capable of expressing GLP1 receptor agonist polypeptides and fusion polypeptides, and cloning them into recombinant adeno-associated virus or recombinant lentiviral vectors. In 293T cells, the corresponding adeno-associated virus and lentiviral vectors were packaged, and the 293T cells, as well as differentiated and undifferentiated muscle cell lines, were infected with a certain biological titer of the virus. The polypeptides and fusion polypeptides produced in the cell supernatant are used for quantitative and qualitative detection, and their binding and neutralizing activities to the GLP-1 receptor (GLP-1R) are tested in the infection activity analysis of the reporter gene cell line. Then, the above-mentioned adeno-associated virus and lentiviral vectors containing polypeptides and fusion polypeptide expression frameworks were purified and delivered to wild-type or diabetic model mice by intravenous or intramuscular injection, and the tested mice were detected at different time points. Plasma concentrations of GLP-1 polypeptides and fusion polypeptides, anti-GLP-1 polypeptide antibodies, and postprandial blood glucose. The specific experimental steps are as follows:

Embodiment 1: the structural design of the expression framework of the nucleic acid construct expressing GLP1 receptor agonist

As shown in Figure 2, the GLP1 receptor agonist carries a signal peptide sequence, and the overall sequence shown in Figure 2 is translated in the cell as a complete precursor molecule and secreted out of the cell. The precursor molecule includes GLP1, a connecting peptide composed of three flexible unit amino acids in series, and an auxiliary peptide; wherein the auxiliary peptide can be GLP1 or GLP1-connecting peptide-GLP1, or human antibodies IgG1CH2 and IgG1CH3 or other structures. The structure of the GLP1 receptor agonist molecule used in the present invention is:

1. Monomer gene expression framework (GLP-1), consisting of N-terminal signal peptide MALLTNLLPLCCLALLALPAQS (SEQ ID NO: 30) and a single GLP-1 (7-37) gene HAEGTFTSDVSSYLE GQAAKEFIAWLVKGRG (SEQ ID NO: 31). The constructed GLP1 receptor agonist expression framework GLP1 protein sequence is shown in SEQ ID NO: 1, and the DNA sequence is shown in SEQ ID NO: 2.

2. Double GLP-1 fusion polypeptide gene expression framework (GLP1-GLP1), composed of N-terminal signal peptide MALLTNLLPLCCLALLALPAQS (SEQ ID NO: 30), GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31), three The connecting peptide GGGGSGGGGSGGGGS (SEQ ID NO: 32) composed of two flexible unit amino acids in series, and the GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31). The protein sequence of the constructed GLP1 receptor agonist expression framework KLDi02 is shown in SEQ ID NO:3, and the DNA sequence is shown in SEQ ID NO:4.

3. Three GLP-1 fusion polypeptide gene expression framework (GLP1-GLP1-GLP1), composed of N-terminal signal peptide MALLTNLLPLCCLALLALPAQS (SEQ ID NO: 30), GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31) , connecting peptide GGGGSGGGGSGGGGS (SEQ ID NO: 32), GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31), connecting peptide GGGGSGGGGSGGGGS (SEQ ID NO: 32), GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31). The protein sequence of the constructed GLP1 receptor agonist gene expression framework KLDi03 is shown in SEQ ID NO:5, and the DNA sequence is shown in SEQ ID NO:6.

4. GLP-1-Fc fragment fusion protein gene expression framework (GLP-1-Fc), consisting of N-terminal signal peptide MXXXX, single GLP-1 (7-37) gene HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO: 31) and human antibody IgG1CH2 and IgG1CH3 composition. The protein sequence of the constructed GLP1 receptor agonist gene expression framework KLDi01 is shown in SEQ ID NO: 7, and the DNA sequence is shown in SEQ ID NO: 8. This construct connects a GLP-1 analog gene to the Fc fragment coding sequence of the antibody, utilizes the formation of antibody-like molecular dimers, optimizes the expression efficiency of the GLP-1 analog gene and increases the plasma half-life of the macromolecular drug. This construct is prior art and is included as a control in the application.

Embodiment 2: Construction of the nucleic acid construct expressing GLP1 receptor agonist

As shown in Figure 3A, the above-mentioned GLP1 receptor agonist gene expression framework was cloned into the latest generation of lentiviral vector backbone pKL-kan-lenti-EF1α-WPRE (Figure 1A, nucleotide Sequence as in SEQ ID NO:9). The lentiviral vector backbone includes: 5'LTR, wherein the promoter region of LTR is replaced with CMV promoter; ψ packaging signal; retroviral export element RRE; cPPT; promoter CBH; polynucleotides; post-transcriptional regulatory elements are wPRE; PPT; ΔU3 3'LTR; and poly A signal. The gene expression frameworks GLP1, GLP1-Fc, GLP1-GLP1 and GLP1-GLP1-GLP1 designed in Example 1 were synthesized by General Biosystems (Anhui) Co., Ltd., and then cloned into lentivirus by homologous recombination methods well known in the art Between the multiple cloning sites EcoRI/EcoRV on the vector backbone pKL-kan-lenti-EF1α-WPRE, the sequence information was confirmed by sequencing after cloning, and the plasmids were named pKL-Kan-lenti-EF1α-GLP1 (see SEQ ID NO:10), pKL-Kan-lenti-EF1α-KLDi01 (see SEQ ID NO:11 for DNA sequence), pKL-Kan-lenti-EF1α-KLDi02 (see SEQ ID NO:12 for DNA sequence) and pKL-Kan- lenti-EF1α-KLDi03 (see SEQ ID NO: 13 for the DNA sequence).

As shown in Figure 3B, the CBH promoter derived from pX261 (see SEQ ID NO: 14 for the DNA sequence) and present in pKL-Kan-lenti-EF1α-KLDi01, pKL-Kan-lenti-EF1α-KLDi02 and pKL-Kan -Lenti-EF1α-KLDi03 GLP1 receptor agonist gene expression frameworks KLDi01, KLDi02, KLDi03 were cloned into the latest generation of currently applied adeno-associated virus vector backbone pAAV-MCS-CMV-EGFP (reverse) ( The sequence is shown between the multiple cloning site MluI/SalI of SEQ ID NO: 15) (see Figure 1B). The adeno-associated virus vector backbone includes: AAV2 ITR, promoter CBH; polynucleotide encoding GLP1 receptor agonist; SV40 poly A signal, AAV2 ITR. The plasmids are respectively named as pAAV-CBH-KLDi01 (see SEQ ID NO:16 for DNA sequence), pAAV-CBH-KLDi02 (see SEQ ID NO:17 for DNA sequence), pAAV-CBH-KLDi03 (see SEQ ID NO:18 for DNA sequence) ).

Example 3: Packaging and purification of viruses expressing GLP1 receptor agonists

In 293T cell line with lentiviral vectors (pKL-Kan-lenti-EF1α-GLP1, pKL-Kan-lenti-EF1α-KLDi01, pKL-Kan-lenti-EF1α-KLDi02 and pKL-Kan-lenti-EF1α-KLDi03) Packaging of lentiviral vectors for antibody gene therapy. The antibody gene lentiviral vectors constructed in Implementation 2 (pKL-Kan-lenti-CBH-GLP1, pKL-Kan-lenti-EF1α-KLDi01, pKL-Kan-lenti-EF1α-KLDi02 and pKL-Kan-lenti-EF1α- KLDi03), envelope plasmid (pKL-Kan-Vsvg, its nucleotide sequence is shown in SEQ ID NO:19) and packaging plasmid (pKL-Kan-Rev, its nucleotide sequence is shown in SEQ ID NO:20 ; pKL-Kan-GagPol, whose nucleotide sequence is shown in SEQ ID NO: 21) mixed and co-transfected 293T cells simultaneously (purchased from American Type Culture Collection Center (ATCC), ATCC preservation number is CRL-3216) , Packaging of HIV neutralizing antibody gene therapy lentivirus in the 293T cell line. The transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer, the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), and the transfection operation refers to the production The standard operation recommended by the manufacturer was carried out, and the transfection scale was 15cm cell culture dish. After 48 hours of transfection, harvest the lentiviral vector (transfected cell culture supernatant). First, centrifuge at 4000rpm at room temperature for 5 minutes to remove cell debris, and then centrifuge at 10000g at 4 degrees for 4 hours to obtain viral particles. After the supernatant, add 1 mL of DMEM complete medium to the virus particle pellet, resuspend the virus particle with a micro-injector, and divide the prepared virus suspension into freezer at -80 degrees for later use.

Antibody gene therapy AAV vectors were packaged in 293T cell line with AAV expression vectors (pAAV-CBH-KLDi01, pAAV-CBH-KLDi02, pAAV-CBH-KLDi03). The antibody gene AAV vector (pAAV-CBH-KLDi01, pAAV-CBH-KLDi02, pAAV-CBH-KLDi03) and envelope plasmid (AAV2/8) constructed in Example 2, its nucleotide sequence is as SEQ ID NO:22 shown) and packaging plasmid (pHelper, whose nucleotide sequence is shown in SEQ ID NO: 23) were mixed and co-transfected 293T cells at the same time, and the GLP1 receptor agonist gene therapy vector AAV was packaged in the 293T cell line . The transfection method is the transient transfection of eukaryotic cells mediated by PEI cationic polymer, the PEI cationic polymer is the PEI-Max transfection reagent purchased from Polysciences (purchased from Polysciences, catalog number: 24765-1), and the transfection operation refers to the production The standard operation recommended by the manufacturer was carried out, and the transfection scale was 15cm cell culture dish. 7 hours after transfection, the supernatant was discarded and replaced with 25ml of toxin-producing medium. After 120 hours of transfection, collect the supernatant and cells, centrifuge at 4200rpm for 10min, separate the supernatant and cells after centrifugation, add lysate and ribozyme to the cells, lyse and digest for 1h, centrifuge at 10000g for 10min, and take the supernatant of the lysate. The lysate supernatant and the culture medium supernatant were purified by affinity chromatography and then frozen and stored at -80°C for later use.

Example 4: Cytological Expression of GLP1 Receptor Agonists

The packaged lentivirus was used to infect 293T cells. The culture supernatant of cells infected with lentivirus was loaded on SDS-PAGE, GLP-1 antibody (6F117): sc-71150 was used as the primary antibody, and the labeled goat anti-mouse antibody was used as the secondary antibody. The results of Western blotting (Figure 4A and Figure 4B) showed that different GLP1 receptor agonist lentiviruses can effectively express GLP1 receptor agonists after in vitro transduction of cells, and secrete mature agonist proteins into the cell culture Qingzhong.

Example 5: Functional verification of GLP1 receptor agonist expressed in supernatant after administration of lentivirus expressing GLP1 receptor agonist

After the GLP1 receptor is activated by GLP1, it translocates from the cell membrane surface into the cell. Cellular translocation of EGFP-tagged proteins in response to drug compounds or other stimuli. GLP1R-EGFP stably transfected U2OS cell line can help to identify the activity of GLP1 receptor agonists. If the secreted and expressed GLP1 receptor agonist is fully functional in the supernatant of cells transduced with the GLP1 receptor agonist gene expression vector, it will be observed that the green fluorescence in GLP1R-EGFP stably transfected U2OS cells is concentrated around the nucleus. The specific functional verification experimental steps are as follows:

1. Collect lentivirus-infected 293T cells, wash the cells with PBS at 4200rpm, 5min, collect the cells by centrifugation, resuspend with 20 μL of QE DNA Extraction Solution, and run the following program with a PCR machine to lyse the cells and extract the total DNA.

Table 1 Cell QE lysis PCR program

temperature	time
65°C	15min
68°C	15min
95°C	10min

The lentivirus copy number (Vector Copy Number, VCN) of 293T cells infected by quantitative PCR was calculated by methods well known in the art.

The primer probe sequences used in quantitative PCR are:

LV Forward primer 5'-AGTAAGACCACCGCACAGCA-3' (SEQ ID NO:24)

LV Reverse primer 5'-CCTTGGTGGGTGCTACTCCT-3' (SEQ ID NO:25)

LV probe 5'-CCTCCAGGTCTGAAGATCAGCGGCCGC-3' (SEQ ID NO: 26)

HK Forward primer 5'-GCTGTCATCTCTTGTGGGCTGT-3' (SEQ ID NO:27)

HK probe 5'-CCTGTCATGCCCACACAAATCTCTCC-3' (SEQ ID NO:28)

HK Reverse primer 5'-ACTCATGGGAGCTGCTGGTTC-3' (SEQ ID NO:29)

Among them, the 5' end of the LV probe has a 6FAM fluorescent group, and the 3' has a TAMRA fluorescent group;

The HK probe has a CY5 fluorophore at the 5' end and a DGB fluorophore at the 3' end.

The running program of quantitative PCR is: 94°C for 5min; 95°C for 10s, 60°C for 30s, 40cyclers.

The VCN results are shown in Table 2.

Table 2 Cell transduction efficiency of lentiviral transduction (unit: ng/mL)

transduction volume	2μL		5μL	10 μL	20 μL
LV-KLDi02	11.96	26.54	43.11	90.51
LV-KLDi01	12.21	12.64	22.78	44.63

2. In this experiment, commercially purchased GLP1(7-37) (GLPBIO, catalog number: GC30058) was used as a positive control. The cell culture supernatant transduced with the GLP1 receptor agonist gene therapy vector and the positive control were incubated with GLP1R-EGFP stably transfected U2OS cells (Northern Biotechnology, Cat. No.: BNCC352040) for a period of time. Fluorescence microscopy identifies the activity of GLP1 receptor agonists by observing the fluorescence of EGFP in cells.

The results showed (Figure 5) that in the in vitro cell test, the GLP1 receptor agonist in the cell culture supernatant transduced with the GLP1 receptor agonist lentivirus had the same activity as the GLP1 positive control (relative molecular weight: 3355.67), and the biological activity Higher than 8nM GLP1 positive control. The activity of the GLP1 receptor agonist in the culture supernatant may exceed 26.85 ng/mL relative to the GLP1 equivalent.

Example 6: Pharmacodynamic data of adeno-associated virus expressing GLP1 receptor agonist on hyperglycemia in diabetic model animals

Since the lentiviral expression framework could not integrate into muscle cells in the case of intramuscular injection in mice, there was little expression in mice (data not shown). Therefore, the present application adopts the GLP1 receptor agonist expression cassette delivered by the adeno-associated virus vector and administered by intramuscular injection. Different types of adeno-associated viruses that can express GLP1 receptor agonists were administered to DB/DB mice by intramuscular injection, and the tail was cut to measure blood sugar with a blood glucose meter (Roche, Excellence Gold) every week, and the blood glucose of different administration groups was compared. blood sugar effect.

The results showed ( FIG. 6 ) that in the in vivo test, the blood glucose of the mice administered with the adeno-associated virus packaged with pAAV-CBH-KLDi02 as a GLP1 receptor agonist was significantly lower than that of the mice in the normal saline group.

Blood was collected at the 4th, 6th and 8th week after the administration, the serum was separated, and the content of the GLP1 receptor agonist in the serum was detected with a human GLP1(7-36) ELISA kit (abcam, ab184857). The concentration of the GLP1 receptor agonist in the serum of the mice in the test group (Table 3) significantly reached the level of GLP1 required by normal people.

Table 3DB/DB mice received serum GLP1 receptor agonist concentration after receiving the adeno-associated virus expressing GLP1 receptor agonist

Example 7: Pharmacodynamic data of polypeptides and fusion polypeptides on hyperglycemia in diabetic model animals

Different doses of GLP1 receptor agonist adeno-associated virus were administered to C57BL/6 mice by intramuscular injection. Glucose tolerance test was performed 5 weeks after administration to evaluate the effect of GLP1 receptor agonist adeno-associated virus on the increase in blood glucose induced by normal meals. Glucose tolerance test: Fasting for 12 hours, intraperitoneal injection of 2.0g/kg body weight of glucose, 0h, 30min, 60min, 120min after glucose injection, tail cut blood was taken to measure blood glucose.

The results showed (Fig. 7) that in the in vivo test, the blood glucose of mice in groups administered with different doses of pAAV-CBH-KLDi02 packaged adeno-associated virus as GLP1 receptor agonists was significantly lower than that of normal saline group mice, and the dose was positive. relevant.

Based on the above embodiments, the present invention connects two or more GLP-1 or its analog gene expression frameworks with a peptide linker (GGGGS)3 composed of flexible unit amino acids to form a tandem covalent dimer or multimer. Experimental results show that the expression efficiency of the expression framework of GLP-1 or its analog in the form of dimer or multimer is much higher than that of the expression framework of monomer GLP-1 analog after being delivered by the recombinant virus vector. At the same time, the experimental results show that the GLP-1 analogues in the form of dimers or multimers have complete GLP-1 receptor binding and agonizing abilities no matter in vitro cytology experiments or in vivo animal experiments. The GLP-1 analog molecules based on the above expression framework showed effective blood drug concentration after being delivered to mice by adeno-associated virus, and also showed positive blood sugar regulation effects in animals.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (17)

1. A nucleic acid construct, characterized in that, the nucleic acid construct comprises polynucleotides encoding GLP-1 or its analogs, the total number of GLP-1 or its analogs is more than two, GLP-1 and Between GLP-1s, between GLP-1 and its analogs, or between GLP-1 analogs and GLP-1 and analogs are linked by polynucleotides encoding linker peptides.
2. The nucleic acid construct according to claim 1, wherein the nucleic acid construct is a nucleic acid construct used for gene therapy of diseases related to carbohydrate metabolism.
3. The nucleic acid construct according to claim 1, wherein the sugar metabolism-related diseases include diabetes or its complications, and obesity; preferably, the diabetes is type 2 diabetes.
4. The nucleic acid construct according to claim 1, wherein the linking mode between GLP-1 or its analogs is as follows: signal peptide——GLP-1 or GLP-1 analog——connecting peptide——GLP-1 or GLP -1 analogue-connecting peptide-GLP-1 or GLP-1 analogue, wherein the number of GLP-1 or GLP-1 analogue-connecting peptide-GLP-1 or GLP-1 analogue unit is one or more.
5. The nucleic acid construct according to claim 1, wherein the nucleic acid construct comprises an expression framework as shown in SEQ ID NO:4 or SEQ ID NO:6.
6. The nucleic acid construct according to claim 1, wherein the polypeptide amino acid sequence encoded by the nucleic acid construct is as shown in SEQ ID NO:3 or SEQ ID NO:5.
7. The nucleic acid construct according to claim 1, wherein the nucleic acid construct is used to produce a virus-based gene therapy vector; preferably, the gene therapy vector is a lentiviral vector or an adeno-associated virus vector.
8. The nucleic acid construct according to claim 7, further comprising any of the following features:

   1) The nucleotide sequence of the lentiviral vector is shown in SEQ ID NO.12 or SEQ ID NO.13;

   2) The nucleotide sequence of the adeno-associated virus vector is shown in SEQ ID NO.17 or SEQ ID NO.18.
9. A lentiviral vector system, characterized in that the lentiviral vector system comprises the nucleic acid construct and helper plasmid according to any one of claims 1-8.
10. The lentiviral vector system according to claim 9, further comprising a host cell carrying the lentiviral vector.
11. A lentivirus, characterized in that the lentivirus is packaged by the lentiviral vector system according to claim 9 or 10.
12. An adeno-associated virus vector system, characterized in that the adeno-associated virus vector system comprises the nucleic acid construct and helper plasmid according to any one of claims 1-8.
13. The adeno-associated virus vector system according to claim 12, wherein the adeno-associated virus vector system further comprises a host cell carrying the adeno-associated virus vector.
14. An adeno-associated virus, characterized in that the adeno-associated virus is packaged by the adeno-associated virus vector system according to claim 12 or 13.
15. A cell line, characterized in that the cell line is a cell line infected by the lentivirus of claim 11 or the adeno-associated virus of claim 14.
16. The nucleic acid construct according to any one of claims 1-8, the lentivirus vector system according to claim 9, the lentivirus according to claim 11, the adeno-associated virus vector system according to claim 12, the vector system according to claim 14 The adeno-associated virus described above and the cell line described in claim 15 are used in the preparation of products for the prevention and treatment of diseases related to glucose metabolism.
17. The use according to claim 16, characterized in that the diseases related to glucose metabolism include diabetes or its complications, obesity; preferably, the diabetes is type 2 diabetes.

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