RNAi adeno-associated virus for inhibiting Slc2a1 as well as preparation and application of RNAi adeno-associated virus

A virus and virus vector technology, applied in the field of biotechnology and gene therapy, can solve problems such as no stress-induced cognitive impairment, and achieve the effect of improving the spatial learning and memory dysfunction and reducing the content of mice.

Active Publication Date: 2022-05-10
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The role of microglia Glut1 in stress-induced cognitive impairment has not been reported so far, and there is no related technology to treat stress-induced cognitive impairment by targeting microglia Glut1

Method used

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  • RNAi adeno-associated virus for inhibiting Slc2a1 as well as preparation and application of RNAi adeno-associated virus
  • RNAi adeno-associated virus for inhibiting Slc2a1 as well as preparation and application of RNAi adeno-associated virus
  • RNAi adeno-associated virus for inhibiting Slc2a1 as well as preparation and application of RNAi adeno-associated virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037]Example 1 Construction and identification of Slc2a1 / Glut1 interference adeno-associated virus vector

[0038] (1) Design of interference targets of Slc2a1 / Glut1

[0039] Obtained the transcript sequence of the Glut1-encoding gene Slc2a1 (NM_011400.3) from NCBI, designed interference sites targeting Slc2a1, and screened out the 3 sites with the highest scores. The target sequence is as follows:

[0040] Position 452: 5'-ctctgtcggcctctttgttaa-3' (SEQ ID NO.1)

[0041] Position 963: 5'-atgcgggagaagaaggtcacc-3' (SEQ ID NO.2)

[0042] Position 1166: 5'-cttcactgtggtgtcgctgtt-3' (SEQ ID NO.3)

[0043] (2) Validation of target interference effect.

[0044] 1. Design siRNAs targeting the above three targets respectively, and use INTERFERin transfection reagent (Polyplus) to transfect the mouse microglial cell line BV2.

[0045] 2. After 48 hours of transfection, cells were collected and RNA was extracted.

[0046] (1) Add 1 mL Trizol (Invitrogen) to the culture plate for lys...

Embodiment 2

[0103] Example 2 Slc2a1 interferes with packaging and titer detection of adeno-associated virus

[0104] (1) Culture of AAV-293 cells

[0105]1. Recovery of AAV-293 cells

[0106] (1) Prepare DMEM medium (called complete medium) containing 10% FBS for the cultivation of AAV-293 cells.

[0107] (2) Add 3 mL of complete medium into a 10 mL glass centrifuge tube.

[0108] (3) Take the cells out of the liquid nitrogen tank or -80°C refrigerator, quickly put them into a 37°C water bath, and shake them gently for 1-2 minutes to completely melt them.

[0109] (4) Take the cryopreservation tube to the ultra-clean table, and wipe the surface with alcohol cotton ball for disinfection. Add the cell suspension to the centrifuge tube prepared in advance.

[0110] (5) Centrifuge at 800g×3min, discard the supernatant, add 2mL of new complete medium, blow gently with a dropper to suspend the cells, inoculate into a 10cm culture dish containing 8mL of fresh complete medium, place at 37°C, ...

Embodiment 3

[0163] Example 3 Interference effect detection of AAV-mirSlc2a1

[0164] (1) Targeted injection of intracranial hippocampus and identification of virus transfection

[0165] With the adeno-associated virus expressing GFP protein only as control virus (AAV-GFP, titer: 1.24E+13v.g / ml, purchased from Shanghai Jima Co., Ltd.), and AAV-mirSlc2a1 (titer: 5.27E+12v .g / ml) were injected into the dentate gyrus region of the dorsal hippocampus of mice through a brain stereotaxic apparatus, with an injection volume of 1 μl. Whether the transfection was successful or not was identified by GFP fluorescence in tissue sections: rat brains were made into 30 μm continuous coronal sections of the hippocampus in a cryostat, and DAPI was used to mark the nuclei, and the expression of GFP in the hippocampus was observed ( Figure 5 ). Glut1 expression in the hippocampus of the mice was detected 9 weeks after the injection.

[0166] (2) Add 1 mL Trizol (Invitrogen) to the homogenate of hippocamp...

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Abstract

The invention provides an RNAi adeno-associated virus for inhibiting Slc2a1 and a preparation method of the RNAi adeno-associated virus, wherein nucleic acid used by the RNAi is designed aiming at three interference sites on the Slc2a1. The adeno-associated virus disclosed by the invention can be used for remarkably and specifically inhibiting the expression of microglial cells Glut1; the mouse space learning and memory dysfunction caused by stress is obviously improved; the polarization of stress-induced microglial cells M1 can be inhibited, and the content of inflammatory factors in the hippocampus can be reduced; the compound is further applied to treatment of irritable learning and memory impairment, neuroinflammation and other diseases.

Description

technical field [0001] The application belongs to the field of biotechnology and gene therapy, specifically, the application provides an RNAi adeno-associated virus inhibiting Slc2a1 and its preparation and application. Background technique [0002] With the acceleration of the pace of life in modern society and the intensification of social competition, most people are under different levels of stress load. Long-term excessive stress has been recognized as an important driving factor for many major fatal diseases in humans. The occurrence and development of 70% of diseases are closely related to the body's stress injury. The brain is the regulatory center of the body's induction and response to stress, and it is also a target organ that is easily damaged by stress, especially the cognitive functions involving high-level brain activities are particularly vulnerable in the face of stress. Epidemiological surveys have shown that the prevalence of mild cognitive impairment and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/113C12N15/864A61K31/713A61P25/00A61P3/10C12R1/93
CPCC12N7/00C12N15/113C12N15/86C07K14/47A61K31/713A61P25/00A61P3/10C12N2750/14121C12N2310/141C12N2750/14152C12N2750/14143Y02A50/30
Inventor 王雪谢方钱令嘉田英瑞赵云孙兆炜
Owner ACADEMY OF MILITARY MEDICAL SCI
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