WO2023045996A1 - Construction d'acide nucléique pour la thérapie génique de maladies associées au métabolisme des hydrates de carbone - Google Patents

Construction d'acide nucléique pour la thérapie génique de maladies associées au métabolisme des hydrates de carbone Download PDF

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WO2023045996A1
WO2023045996A1 PCT/CN2022/120427 CN2022120427W WO2023045996A1 WO 2023045996 A1 WO2023045996 A1 WO 2023045996A1 CN 2022120427 W CN2022120427 W CN 2022120427W WO 2023045996 A1 WO2023045996 A1 WO 2023045996A1
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glp
nucleic acid
acid construct
adeno
associated virus
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吴昊泉
孙保贞
党颖
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康霖生物科技（杭州）有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GLP-1 and its analogues are quickly degraded by DPP-4 in the body and have a short half-life.
  • GLP-1 analogues significantly prolong the duration of action of GLP-1 in vivo, frequent (1-7 days) subcutaneous administration is still required.
  • 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.
  • the expression efficiency of polypeptide gene expression vectors is much lower than that of macromolecular protein gene expression vectors.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1A shows the pKL-kan-lenti-EF1 ⁇ -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
  • B1-B4 i.e. the second row
  • C1-C4 that is, the third row
  • KLDi01 2 ⁇ L, 5 ⁇ L, 10 ⁇ L, and 20 ⁇ L, 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.
  • 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.
  • 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.
  • 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:
  • the number of GLP-1 and GLP-1 analogs is more than two.
  • 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.
  • (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.
  • diseases related to glucose metabolism include diabetes or its complications, and obesity.
  • 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.
  • 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.
  • 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.
  • the nucleic acid construct is a lentiviral vector.
  • the vector skeleton in the lentiviral vector can be the vector skeleton in the prior art.
  • the nucleotide sequence of the lentiviral vector is shown in SEQ ID NO.12 or SEQ ID NO.13.
  • 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.
  • the nucleotide sequence of the adeno-associated virus vector is shown in SEQ ID NO.17 or SEQ ID NO.18.
  • the present invention also provides a lentiviral vector system, which includes the nucleic acid construct and a helper plasmid.
  • helper plasmid encodes one or more nucleotide sequences of gag and pol proteins and other essential viral packaging component nucleotide sequences
  • helper plasmid may include packaging plasmids and envelope plasmids.
  • 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.
  • helper plasmid encodes one or more nucleotide sequences of gag and pol proteins and other essential viral packaging component nucleotide sequences
  • helper plasmid may include packaging plasmids and envelope plasmids.
  • 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.
  • diseases related to glucose metabolism include diabetes or its complications, and obesity.
  • the diabetes is type 2 diabetes.
  • 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.
  • 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
  • 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:
  • GLP-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.
  • 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.
  • GLP1-GLP1-GLP1 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.
  • GLP-1-Fc fragment fusion protein gene expression framework consisting of N-terminal signal peptide MXXX, 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
  • the lentiviral vector backbone includes: 5'LTR, wherein the promoter region of LTR is replaced with CMV promoter; ⁇ packaging signal; retroviral export element RRE; cPPT; promoter CBH; polynucleotides; post-transcriptional regulatory elements are wPRE; PPT; ⁇ U3 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-EF1 ⁇ -WPRE, the sequence information was confirmed by sequencing after cloning, and the plasmids were named pKL-Kan-lenti-EF1 ⁇ -GLP1 (see SEQ ID NO:10), pKL-Kan-lenti-EF1 ⁇ -KLDi01 (see SEQ ID NO:11 for DNA sequence), pKL-Kan-lenti-EF1 ⁇ -KLDi02 (see SEQ ID NO:12 for DNA sequence) and pKL-Kan- lenti-EF1 ⁇ -KLDi
  • the CBH promoter derived from pX261 (see SEQ ID NO: 14 for the DNA sequence) and present in pKL-Kan-lenti-EF1 ⁇ -KLDi01, pKL-Kan-lenti-EF1 ⁇ -KLDi02 and pKL-Kan -Lenti-EF1 ⁇ -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
  • the antibody gene lentiviral vectors constructed in Implementation 2 (pKL-Kan-lenti-CBH-GLP1, pKL-Kan-lenti-EF1 ⁇ -KLDi01, pKL-Kan-lenti-EF1 ⁇ -KLDi02 and pKL-Kan-lenti-EF1 ⁇ - 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.
  • ATCC American
  • 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.
  • 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
  • envelope plasmid AAV2/8 constructed in Example 2
  • its nucleotide sequence is as SEQ ID NO:22 shown
  • packaging plasmid pHelper, whose nucleotide sequence is shown in SEQ ID NO: 23
  • 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.
  • 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
  • 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:
  • VCN Vector Copy Number
  • the primer probe sequences used in quantitative PCR are:
  • 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.
  • 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.
  • Example 6 Pharmacodynamic data of adeno-associated virus expressing GLP1 receptor agonist on hyperglycemia in diabetic model animals
  • 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.
  • 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
  • GLP1 receptor agonist adeno-associated virus 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 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.
  • GGGGS peptide linker
  • 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.
  • 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.

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Abstract

L'invention concerne également une construction d'acide nucléique pour la thérapie génique de maladies liées au métabolisme des hydrates de carbone. La construction d'acide nucléique comprend un polynucléotide codant pour le GLP-1 ou un analogue de celui-ci, le nombre total de GLP-1 ou d'analogue de celui-ci est de deux ou plus, et le GLP-1, le GLP-1 et son analogue, ou l'analogue du GLP-1 et l'analogue du GLP-1 sont liés au moyen d'un polynucléotide codant pour un peptide lieur. La construction d'acide nucléique peut être utilisée pour l'expression à haute efficacité du GLP-1 actif et de son analogue in vivo et in vitro, et peut être utilisée pour le développement de médicaments pour les maladies liées au métabolisme des hydrates de carbone, telles que le diabète de type 2, les troubles du métabolisme corporel, les complications liées au diabète et l'obésité.
PCT/CN2022/120427 2021-09-26 2022-09-22 Construction d'acide nucléique pour la thérapie génique de maladies associées au métabolisme des hydrates de carbone WO2023045996A1 (fr)

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