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Medicament for gene therapy of diabetes type I

A technology of gene therapy and gene expression, applied in the field of gene therapy drugs for type 1 diabetes, which can solve the problems of reducing the probability of immune response

Inactive Publication Date: 2018-06-01
BEIJING GENECRADLE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since miR-142-3p is highly expressed in cells derived from hematopoietic stem cell lines [70] , the homogeneous differentiation of immune cells is derived from hematopoietic stem cell lines, so the principle of using miRNA to inhibit gene expression [71] , the expression of genes carrying miR-142-3p target sequences will be significantly inhibited in immune cells, thereby reducing the probability of the body producing an immune response against gene expression products [72]

Method used

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  • Medicament for gene therapy of diabetes type I
  • Medicament for gene therapy of diabetes type I
  • Medicament for gene therapy of diabetes type I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Plasmid vector construction

[0053] In order to construct the pscAAV-CAM-OFat-1 and pscAAV-CAM-OFat-1-142T plasmids required for packaging recombinant AAV viruses, we first used the pAAV2neo preserved by the company as the basis and used the self-designed CAM promoter (SEQ IDNo. 1) Replace the CMV promoter in the pAAV2neo vector, and replace one of the pAAV2neo vectors with a mutated ITR sequence (named ΔITR) (SEQ ID No.2) that deletes the trs (terminal resolution site) and D sequences in the AAV2 ITR flanking ITR sequences to obtain the pscAAV-CAM vector. Next, the artificially synthesized OFat-1 (SEQ ID No.3) and OFat-1-142T (SEQ ID No.4) sequences were respectively cloned into the KpnI and EcoRI and KpnI and BglII restriction sites of the pscAAV-CAM vector Between, pscAAV-CAM-OFat-1 and pscAAV-CAM-OFat-1-142T vectors were obtained.

[0054] (1) Construction of pscAAV-CAM vector

[0055] The human cytomegalovirus early gene enhancer sequence, the chicken...

Embodiment 2

[0061] Example 2 Preparation and assay of recombinant AAV virus

[0062] References [102] , using the three-plasmid packaging system to package and purify the recombinant AAV virus. Briefly, the AAV vector plasmid (pscAAV-CAM-OFat-1 or pscAAV-CAM-OFat-1-142T), the helper plasmid (pHelper), and the AAV Rep and Cap protein expression plasmids (pAAV-R2C1, pAAV-R2C8 or pAAV -R2C9) mixed according to the molar ratio of 1:1:1, transfect HEK293 cells by calcium phosphate method, after 48 hours of transfection, harvest the cells and culture supernatant, and use cesium chloride density gradient centrifugation to separate and purify the recombinant AAV virus . Packaged and purified to obtain scAAV1-CAM-OFat-1, scAAV1-CAM-OFat-1-142T, scAAV8-CAM-OFat-1, scAAV8-CAM-OFat-1-142T, scAAV9-CAM-OFat-1 and scAAV9-CAM - 6 kinds of recombinant viruses such as OFat-1-142T.

[0063] Quantitative PCR method was used to measure the genome titer of the prepared AAV virus. The specific process is a...

Embodiment 3

[0068] Example 3 Intravenous administration for the treatment of type 1 diabetes

[0069] 70 8-week-old female NOD / LtJ (hereafter abbreviated as NOD) mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., cultured under SPF conditions until 12 weeks, blood was collected from the tail vein, and a blood glucose meter (Accu-Chek , Roche) measured the non-fasting blood glucose concentration of each mouse, and regarded the mice with non-fasting blood glucose concentration higher than 13mM as type 1 diabetic mice, and obtained 51 mice in total. 40 of them were randomly divided into 5 groups with 8 mice in each group. Among the 5 groups of mice, 4 groups of mice were injected with scAAV9-CAM-OFat-1, scAAV9-CAM-OFat-1-142T, scAAV8-CAM-OFat-1 or scAAV8-CAM-OFat-1- 142T recombinant virus, the injection dose is 2×10 10 vg / only, and the remaining 1 group of mice were used as controls injected with scAAV9-CAM-OFat-1 or scAAV9-CAM-OFat-1-142T and mice injected with scAAV8-CAM...

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PUM

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Abstract

The invention provides a medicament for gene therapy of recombinant adeno-associated virus mediated diabetes type I. The recombinant adeno-associated virus vector carries an oFat-1(optimized Fat-1, for short oFat-1) gene cassette containing human a miR-142-3p target sequence. An in vivo experiment shows that the recombinant adeno-associated virus vector can be efficiently introduced into bodies aswell as continuously and stably express Fat-1 protein, in order to increase content of omega-3 polyunsaturated fatty acid, increase content of insulin in vivo, and maintain stabilization of blood sugar. A result shows that the recombinant adeno-associated virus vector is hopeful to be developed into a novel medicament for treating diabetes type I.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene therapy drug for type 1 diabetes carried by a recombinant adeno-associated virus vector carrying an oFat-1 gene expression cassette. Background technique [0002] Type 1 diabetes (T1D) is a polygenic organ-specific autoimmune disease. During the disease, a specific subset of T lymphocytes in the body will attack its own pancreatic beta cells [1-2] , resulting in a decrease in the number of β cells, insufficient insulin secretion, and elevated blood sugar in the body. Currently, T1D is mainly treated with daily insulin injections. However, C-peptide is not included in injected insulin. C-peptide is a by-product produced when proinsulin is processed into insulin, but it has a negative effect on the microvasculature in the human body. [3] ,Neurons [4] and kidney [5] have an important protective role. Therefore, although the blood sugar concentration of diabetic patients ca...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/864A61K48/00A61P3/10
CPCA61K9/0019A61K48/0008C12N9/001C12N15/86C12N2750/14143
Inventor 田文洪董小岩吴小兵马思思
Owner BEIJING GENECRADLE PHARM CO LTD
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