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Promoter for the expression of genes

a gene and promoter technology, applied in the field of nuclear medicine, can solve the problems of lack of extensive functional characterization of most cellular promoters, and achieve the effect of being useful in addressing problems

Pending Publication Date: 2022-06-16
FRIEDRICH MIESCHER INST FOR BIOMEDICAL RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The identified nucleic acid sequence enables targeted gene expression in retinal endothelial cells, facilitating research and therapeutic interventions for neurodegenerative disorders and vision-related conditions by ensuring high specificity and efficiency, addressing the limitations of existing systems.

Problems solved by technology

Since expression of eukaryotic genes is controlled by a complex machinery of cis- and transacting regulatory elements, most cellular promoters suffer from a lack of extensive functional characterization.

Method used

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  • Promoter for the expression of genes

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Experimental program
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Effect test

examples

Gene Construct

[0110]The synthetic promoter ID NO:1 used in this study consists of 794 bp. ChR2-eGFP coding sequence was inserted immediately after this promoters and the optimized Kozak sequence (GCCACC), and followed by a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and SV40 polyadenylation site. Retinal neurons were targeted using AAVs serotype 2 / 8 with a titer of 1.75E+12 GC / mL.

Viral Transfection and Tissue Preparation

[0111]For AAV administration, the eyes of anesthetized animals were punctured in the sclera close to the lens by a sharp 30 gauge needle. 2 microL of AAV particle suspension were injected subretinally by a Hamilton syringe. After 3 weeks, the isolated retinas were fixed for 30 min in 4% PFA in PBS, followed by a washing step in PBS at 4° C. Whole retinas were treated with 10% normal donkey serum (NDS), 1% BSA, 0.5% Triton X-100 in PBS for 1 h at room temperature. Treatment with monoclonal rat anti-GFP Ab (Molecular Probes Inc.; 1:500) in 3...

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PUM

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Abstract

The present invention provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:1 or a nucleic acid sequence of at least 600 bp having at least 80% identity to said sequence of SEQ ID NO:1, wherein said isolated nucleic acid molecule specifically leads to the expression in retinal endothelial cells of a gene when operatively linked to a nucleic acid sequence coding for said gene.

Description

FIELD OF THE INVENTION[0001]The present invention relates to a nucleic acid sequence leading to the expression of genes specifically in retinal endothelial cells.BACKGROUND OF THE INVENTION[0002]For expression purposes recombinant genes are usually transfected into the target cells, cell populations or tissues, as cDNA constructs in the context of an active expression cassette to allow transcription of the heterologous gene. The DNA construct is recognized by the cellular transcription machinery in a process that involves the activity of many trans-acting transcription factors (TF) at cis-regulatory elements, including enhancers, silencers, insulators and promoters (herein globally referred to as “promoters”).[0003]Gene promoter are involved in all of these levels of regulation, serving as the determinant in gene transcription by integrating the influences of the DNA sequence, transcription factor binding and epigenetic features. They determines the strength of e.g. transgene expres...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/11A61K48/00C12N15/85C07K14/515C12N15/86
CPCC12N15/11A61K48/0058C12N2830/008C07K14/515C12N15/86C12N15/85
Inventor JUETTNER, JOSEPHINEROSKA, BOTOND
Owner FRIEDRICH MIESCHER INST FOR BIOMEDICAL RES