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

Synthetic microrna mimics

A technology of composition and vehicle, applied in DNA/RNA fragments, recombinant DNA technology, medical preparations containing active ingredients, etc., can solve the problems of weak delivery efficiency, tumor occurrence, and undeveloped VEGFA therapy.

Pending Publication Date: 2021-08-06
原住民股份有限公司
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are several drawbacks to traditional gene delivery: delivery efficiency is often weak, the vector and transgene can elicit an immunological response, usually only one isoform of the gene is delivered and the natural balance of the different isoforms becomes unbalanced, and the vector can Its transgene integrates into tandem sites of chromatin: possible tumorigenesis
However, despite extensive efforts to develop VEGFA therapies, no consistently effective VEGFA therapy has been developed

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
  • Synthetic microrna mimics
  • Synthetic microrna mimics
  • Synthetic microrna mimics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0202] Example 1. Deletion of mmu-miR-466c abolishes Vegfa expression in hypoxic endothelial cells

[0203] To analyze the role of mmu-miR-466 in regulating Vegfa in response to hypoxia, CRISPR-mediated gene editing was used to remove a 97 bp region from intron 10 of Sfmbt2 containing the mmu-miR-466 hairpin. Vegfa expression is known to be upregulated in hypoxia. The normally observed induction of mmu-miR-466 expression under hypoxia was not present after removal of mmu-miR-466 ( Figure 4 A). The expression of the parental gene Sfmbt2 was also no longer induced in response to hypoxic stimulation ( Figure 4 B). Importantly, the upregulation of Vegfa expression in response to hypoxia was also abolished when miR-466 was removed ( Figure 4 C). When miR-466 levels were restored using lentiviral transduction, Vegfa levels increased equally under normoxic and hypoxic conditions, but hypoxia did not further induce Vegfa expression ( Figure 4 D).

Embodiment 2

[0204] Example 2. Generation of mmu-miR-466c-deleted cell lines by CRISPR / Cas9

[0205] To remove mmu-miR-466c from intron 10 of Sfmbt2, the two guide RNAs were cloned into separate expression plasmids (pcDNA-H1-sgRNA, see Figure 9) and transfected into C166 cells using Nucleofector I (Amaxa) together with a Cas9 plasmid co-expressing GFP (PX458, catalog number 48138, Addgene). CRISPR is guided to the side of the deletion by guide oligomers (SEQ ID NO: 9-12) such as disclosed in US8697359B1. Based on GFP positivity, single cells were sorted into 96-well plates using sorting FACS (BDFACSARIA III cell sorter) and established clonal cell populations. Cultures were typed by PCR to identify cells containing the desired deletion, and positive clones were further confirmed by Sanger sequencing.

[0206] Table 1. Forward and reverse guides designed to target miR-466c flanking regions

[0207]

[0208] Table 2. Primers used to type miR-466c deletion

[0209]

Embodiment 3

[0210] Example 3. mmu-miR-466c regulates VEGFA in human cell lines

[0211] To test whether murine miR-466c may have any effect on the mRNA levels of human VEGFA, the inventors transfected human Ea.hy926 and ARPE-19 cells with a synthetic miR-466c mimic.

[0212] A t-Student test in Ea.hy926 samples treated with the hairpin form miR-466c-3p mim revealed that VEGFA mRNA levels were significantly reduced when compared to negative control transfection (Negative Control siRNA Cat# AM4611, Ambion). ( Figure 5 ). The miR-466c-3p mimic was able to downregulate VEGFA by inducing an approximately 3-fold downregulation (0.296-fold change). The same effect was observed when cells were treated with a single-stranded ssRNA-466c-3p mimic that was able to downregulate VEGFA by inducing approximately 1.42-fold downregulation when compared to negative control transfection (0.697-fold change) ( Figure 6 ).

[0213] In contrast, when testing the hairpin mimic 5p (miR-466c-5p mim) and the s...

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 present invention relates to the treatment of cardiovascular diseases. In particular the present invention relates to micro RNA (mi RNA) molecules for use in the regulation of the gene expression of Vascular endothelial growth factor A (VEGFA), Vascular endothelial growth factor D (VEGFD) and / or Hypoxia-inducible factor 1-alpha (HIF1A) in a variety of applications, including use in therapeutic and diagnostic applications. VEGFA has diverse functions in both developing and mature individuals. VEGFA is a well-known critical regulator of angiogenesis and is also involved in the development and metastasizing of cancer.

Description

technical field [0001] The present invention relates to the treatment of cardiovascular diseases. In particular, the present invention relates to microRNAs useful in regulating gene expression of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor D (VEGFD) and / or hypoxia-inducible factor 1-alpha (HIF1A) in various applications (miRNA) molecules, said use includes use in therapeutic and diagnostic applications. VEGFAs function differently in both developing and mature individuals. VEGFA is a well-known key regulator of angiogenesis and is also involved in the development and metastasis of cancer. Background technique [0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide, and it is predicted that the number of deaths will increase in the future due to population aging and average life expectancy increase (top 10 causes of death, WHO, 2017). CVD includes a broad group of diseases involving the heart and blood vess...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7105
CPCC12N15/1136C12N2310/141G01N33/6893
Inventor M·P·图鲁宁T·A·图鲁宁S·伊拉-赫图拉P·莱蒂宁M-A·瓦纳能
Owner 原住民股份有限公司
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