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Nucleic acid construct for gene therapy of glycometabolism-related diseases

A technology for nucleic acid constructs and sugar metabolism, applied in metabolic diseases, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2021-12-28
KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no GLP-1 analogue gene therapy technology route delivered by viral and non-viral vectors has achieved effective clinical progress

Method used

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  • Nucleic acid construct for gene therapy of glycometabolism-related diseases
  • Nucleic acid construct for gene therapy of glycometabolism-related diseases
  • Nucleic acid construct for gene therapy of glycometabolism-related diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: the structural design of the expression framework of the nucleic acid construct expressing GLP1 receptor agonist

[0072] Such as figure 2 As shown, the GLP1 receptor agonist carries a signal peptide sequence, figure 2 The overall sequence shown is translated in the cell as an intact 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:

[0073] 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 frame...

Embodiment 2

[0077] Embodiment 2: Construction of the nucleic acid construct expressing GLP1 receptor agonist

[0078] Such as Figure 3A As shown, 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 , the nucleotide sequence is 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 ...

Embodiment 3

[0080] Example 3: Packaging and purification of viruses expressing GLP1 receptor agonists

[0081] 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 simultaneously co-transfected 293T cells (purchased from the American Type Culture Collection Center (ATCC), ATCC preservation number is CRL-3216), Packaging of HIV neutralizing antibody gene therapy lentivirus was carried out in this 293T cell line. ...

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Abstract

The invention relates to the technical field of gene therapy or biological medicine, in particular to a nucleic acid construct for gene therapy of glycometabolism-related diseases; the nucleic acid construct comprises polynucleotide for coding GLP-1 or an analogue thereof; the total number of the GLP-1 or the analogue thereof is more than two; the GLP-1 and the GLP-1 are connected through polynucleotide for coding a connecting peptide; the GLP-1 and the analogue thereof are connected through the polynucleotide for coding the connecting peptide; or the analogue of the GLP-1, the GLP-1 and the analogue are connected through the polynucleotide for coding the connecting peptide. The nucleic acid construct disclosed by the invention can be used for efficiently expressing the GLP-1 with the activity and the analogue thereof in vivo and in vitro, so that a technical route is provided for developing medicines for treating glycometabolism-related diseases such as type 2 diabetes, body metabolic disorder, diabetes-related complications and obesity which have great potential.

Description

technical field [0001] 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 [0002] 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. [0003] 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 insu...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/16C12N15/864C12N5/10A61K38/26A61P3/04A61P3/10
CPCC07K14/605C12N15/86A61K38/26A61P3/10A61P3/04C12N2740/15043C12N2750/14143
Inventor 吴昊泉孙保贞党颖
Owner KANGLIN BIOTECHNOLOGY (HANGZHOU) CO LTD
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