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Mutant light-inducible ion channel of channelrhodopsin

An ion channel, light-induced technology, applied in the field of mutant light-induced ion channels, can solve problems such as the applicability of slow closing kinetics

Inactive Publication Date: 2019-03-15
MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN EV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite their excellent photosensitivity, their slow closure kinetics remain a limiting factor for their applicability

Method used

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  • Mutant light-inducible ion channel of channelrhodopsin
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  • Mutant light-inducible ion channel of channelrhodopsin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0159] Example 1 - Identification of mutations that accelerate closing kinetics

[0160] The inventors aimed to identify residues within ChR-2 whose mutation further accelerates the closing kinetics. The inventors focused on the sixth transmembrane domain.

[0161] NG108 heterologously expressing ChR2-YFP, ChR2-YFP F219Y, ReaChR-Citrine, ReaChR-Citrine F259Y, VChR1-YFP and VChR1-YFP F214Y were studied in whole-cell mode by patch clamp measurement of clamp potential at -60mV -15 cells. Bath solution contains 140mM NaCl, 2mM CaCl 2 , 2mM MgCl 2 , 10mM HEPES, pH7.4, the pipette solution contains 110mM NaCl, 2mM MgCl 2 , 10 mM EGTA, 10 mM HEPES, pH 7.4. Response to 500ms light pulse (intensity 23mW / mm 2 , wavelength 594nm) to measure the photocurrent. τ 关闭 Values ​​were determined from the fit of the current after cessation of illumination to a single exponential function.

[0162] In order to evaluate the calcium ion relative to the sodium ion permeability (P Ca / P Na ...

Embodiment 2

[0167] Example 2 - Optogenetic stimulation of the auditory pathway

[0168] Hernandez et al. J Clin Invest. 2014;124(3):1114-29 demonstrated an optogenetic stimulation strategy for the auditory pathway in rodents. In particular, the authors describe an animal model to characterize optogenetic stimulation, which is optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activates auditory pathways, as demonstrated by recording single and population responses of neurons. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by local field potential (LFP) recording of inferior colliculus responses to suprathreshold optical, acoustic, and electrical stimuli suggests that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. ...

Embodiment 3

[0170] Example 3 - An Optogenetic Approach to Restoring Vision

[0171] Macé et al. Mol Ther. 2015;23(1):7-16 is an earlier publication written by some of the inventors describing optogenetics of retinal neurons mediated by adeno-associated virus (AAV) gene therapy Reactivate. Most inherited retinal dystrophies show progressive photoreceptor cell degeneration, resulting in severe visual impairment. Adeno-associated virus (AAV) gene therapy-mediated optogenetic reactivation of retinal neurons has the potential to restore vision regardless of patient-specific mutations. The clinical translatability challenge is to restore vision as close to natural vision as possible while using a surgically safe delivery route for the fragile degenerated retina. To preserve visual processing in the inner retina, ON bipolar cells are targeted, which persist in advanced stages of the disease. For safe gene delivery, recently engineered AAV variants were used that can transduce bipolar cells af...

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Abstract

The invention relates to mutant light-inducible ion channel having improved properties as compared to the parent channel, nucleic acid constructs encoding same, expression vectors carrying the nucleicacid construct, cells comprising said nucleic acid construct or expression vector, and their respective uses, as well as non- human animals comprising the mutant light-inducible ion channel, the nucleic acid construct or the expression vector as disclosed herein.

Description

[0001] The present invention relates to mutant light-induced ion channels with improved properties, nucleic acid constructs encoding them, expression vectors carrying said nucleic acid constructs, cells comprising said nucleic acid constructs or expression vectors, and their respective uses, as defined in the claims. Background technique [0002] Light-gated, inwardly rectifying cation channels, channelrhodopsin-1 (ChR1) and channelrhodopsin 2 (ChR2), have emerged as tools of choice for targeted light activation of neurons in vitro and in vivo 1-5 . Although wild-type (WT) ChR2 can be used for light-induced depolarization, ChR2 mutants with faster kinetics and increased photosensitivity are currently being sought for potential future clinical applications (WO 03 / 084994 and 6-8 ). [0003] Since the first description in 2002 and 2003, a different set of ChR2 variants have been described, including the red-light-absorbing channelrhodopsin. ChRs are modified in terms of kineti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C07K14/405
CPCA61P27/02C07K14/405C07K14/705A61K35/30C12N5/0618C12N2510/00A61K9/0019C12N7/00C12N15/86
Inventor E·班贝格P·伍德T·马格
Owner MAX PLANCK GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN EV