Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Application of astrocyte specific METTL3 overexpressed recombinant adeno-associated virus

A star-shaped glial cell-specific technology, applied in the field of biomedicine, can solve the problems of reducing expression, knocking down, shortening the half-life of YAP1mRNA, etc., to prolong the half-life, promote activation, and improve the function of spinal cord injury

Pending Publication Date: 2022-07-29
JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that METTL3 can bind to the mRNA of YAP1 and promote its m 6 A modifies and regulates its stability, thereby affecting tumor progression
Interestingly, we demonstrated through MeRIP-sequence combined with mRNA-sequence sequencing that knockdown of METTL3 in primary astrocytes can also reduce the m of the 3' untranslated region of YAP1 mRNA. 6 A abundance, shortened YAP1 mRNA half-life, and reduced its expression

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of astrocyte specific METTL3 overexpressed recombinant adeno-associated virus
  • Application of astrocyte specific METTL3 overexpressed recombinant adeno-associated virus
  • Application of astrocyte specific METTL3 overexpressed recombinant adeno-associated virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] A recombinant adeno-associated virus overexpressing astrocyte-specific METTL3, the specific steps are as follows:

[0040] (1) primers were designed, and the full-length sequence of METTL3 gene was obtained by PCR amplification;

[0041] 1) Extract and culture primary astrocytes in vitro: Take 1-3 day old C57BL / 6 suckling mice, sterilize the skin with alcohol, cut the head and peel off the whole brain tissue, separate the prefrontal cortex, then remove the meninges, cut the tissue Minced, digested with DMEM / F12 medium (Gibco) containing 0.125% trypsin (Gibco) and DNase (Sigma) (trypsin:DNase=5:1) for 20 minutes, shaken every 5 minutes to prevent Tissue adhesions. After terminating digestion, centrifugation, discarding the supernatant, resuspending in complete medium (DMEM high-glucose medium containing 10% FBS fetal bovine serum and 1% penicillin-streptomycin), and finally filtering through a 70 μm cell sieve for 3 times to obtain a single Cell suspension was incubate...

Embodiment 2

[0054] Example 2 Modeling of mouse spinal cord injury, virus injection and detection of functional recovery

[0055] 8-week-old C57BL / 6 mice (Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing City) were taken and divided into control group AAV-Con group and experimental group AAV-METTL3 group, with 12 mice in each group. The modeling process and treatment steps are as follows: C57BL / 6 mice were fasted with water 6 hours before surgery, and after inhalation anesthesia with isoflurane (Shenzhen Reward Life Technology Co., Ltd.), skin preparation and iodophor were performed on the back skin of the mice. disinfect. A midline incision was made on the back, and the subcutaneous tissue, fascia, muscle and paravertebral tissue were separated layer by layer, and the T8 and adjacent segments were exposed. The T8 lamina was carefully removed with curved forceps to expose the spinal cord, paying attention to hemostasis. The mice were fixed on the spinal cord striker (Shenzh...

Embodiment 3

[0082] Example 3AAV-METTL3 targeted modification of YAP1 mRNA m 6 A level

[0083] In order to further study the mechanism of the above-mentioned AAV-METTL3 recombinant adeno-associated virus promoting the activation and functional recovery of reactive astrocytes after spinal cord injury in mice, we firstly isolated and cultured primary astrocytes according to the method in Example 1. , and then knocked down METTL3 levels in primary astrocytes (shMETTL3) by short hairpin RNA technology, followed by MeRIP-sequence sequencing combined with mRNA-sequence sequencing. We found that after knockdown of METTL3 levels in primary astrocytes, YAP1 mRNA m 6 Both A methylation levels and mRNA levels were significantly reduced. Visualizing m with IGV 6 After peak A, we found that compared with the control group (shNC), the methylation abundance of the 3' untranslated region of YAP1 mRNA in astrocytes in the shMETTL3 group was significantly reduced. Subsequently, we used MeRIP-qPCR to fu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to an application of astrocyte specific METTL3 overexpressed recombinant adeno-associated virus in preparation of drugs for preventing and treating spinal cord injury. The invention successfully constructs the recombinant adeno-associated virus overexpressed by the astrocyte specificity METTL3, and the virus can specifically overexpress the METTL3 protein in the in-vivo astrocyte, modify the m6A level of downstream YAP1 mRNA in a targeted manner, prolong the half-life period, maintain the stability of the mRNA, up-regulate the expression level of the YAP1 protein, promote the activation of reactive astrocyte, and inhibit the expression of the METTL3 protein in the astrocyte. The spinal cord injury function is improved, and a new way is provided for clinical treatment of the spinal cord injury.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a recombinant adeno-associated virus with astrocyte-specific METTL3 overexpression and its application in the preparation of a spinal cord injury therapeutic drug. Background technique [0002] Spinal cord injury (SCI) is a central nervous system disease in which the integrity and continuity of the spinal cord are damaged due to factors such as trauma, tumor or inflammation, resulting in motor, sensory, and autonomic dysfunction. With the continuous development of the transportation industry, the incidence of spinal cord injury has increased sharply, and the patients with spinal cord injury also tend to be younger. After the occurrence of spinal cord injury, the physiological structure of the spinal cord is destroyed for the first time and further affects the nerve function. This process is called primary spinal cord injury. Secondary spinal cord injury refers to...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): A61K38/45A61K48/00A61P25/00C12N7/01C12N15/864C12N15/54C12N15/66
CPCA61K38/45A61K48/005A61K48/0008A61P25/00C12N9/1007C12N15/86C12N7/00C12N15/66C12N2750/14121C12N2750/14143
Inventor 蔡卫华葛旭辉刘蔚凡进唐鹏宇蒋东冬杨思亭
Owner JIANGSU PROVINCE HOSPITAL THE FIRST AFFILIATED HOSPITAL WITH NANJING MEDICAL UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com