Optogenetic visual restoration using chrimson

A use, vision technology, applied to the use of exogenous light-activated ion channels in cells and target subjects, Chrimson polypeptides, reactivation of mammalian retinal neurons in the field of cells, able to solve unproven, no opsin activity improvement, expression Issues such as differences in level or membrane positioning

Active Publication Date: 2019-09-20
GENSIGHT BIOLOGICS SA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] However, this finding was not confirmed for the tdTomato fluorescent label, as no clear differences in expression levels or membrane localization were found in transgenic animals expressing type II channelrhodopsin fused to tdTomato (Madisen et al., 2012, Nat Neurosci., 15(5):793-802)
Furthermore, so far there are no reports of improvements in opsin activity associated with changes in the localization or expression levels of fusion proteins

Method used

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  • Optogenetic visual restoration using chrimson
  • Optogenetic visual restoration using chrimson
  • Optogenetic visual restoration using chrimson

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Example 1: Validation of rdl and P23H Denatured Rodent Models

[0169] Retinal dystrophies are associated with retinal cell dysfunction and degeneration, impairing the flow of visual information and ultimately leading to severe vision loss and blindness. Retinitis pigmentosa (RP) is the most common retinal dystrophy, causing 1 in 4000 vision loss worldwide. RP is composed of more than 60 genes that are inherited as autosomal dominant (30%-40% of cases), autosomal recessive (50%-60%) or X-linked (5%-15%) caused by a change in any one gene.

[0170] In most common forms of RP, rod photoreceptors degenerate first, followed by cone degeneration. Therefore, early symptoms of RP are often night blindness and tubular vision due to peripheral vision loss. All RP conditions are progressive, with varying characteristics of visual deterioration across patients, but blindness is the ultimate result. There is no known treatment for RP.

[0171] Because RP is caused by multiple ...

Embodiment 2

[0209] Example 2: Activation of Retinal Ganglion Cell Populations in Non-Human Primates Below Safe Radiation Limits

[0210]In the above studies, we have demonstrated that ChrimsonR (ChrR), a red-shifted opsin, can induce photoactivation of retinal ganglion cells (RGCs) in blind rodents (rdl mice and P23H rats). In addition, we observed that an extended form of ChrR fused to the fluorescent protein tdTomato appeared to provide greater functional utility in terms of the number of light-responsive cells and the amplitude of their responses. It is known that AAV2 transduces only RGCs in the underside of the concave ring in nonhuman primates compared to rodents (Yin et al., 2011). AAV2-7m8, extends beyond the foveal ring, leading to islands of expression in peripheral regions (Dalkara et al., 2013). Similar transduction features of AAV2 vectors are expected to occur in humans. Therefore, to further assess the translational potential of this therapeutic intervention, we here asse...

Embodiment 3

[0237] Embodiment 3: the effect of fluorescent protein tdTomato in the expression and localization of optogenetic protein ChrimsonR

[0238] AAV2.7m8-CAG-ChrimsonR-tdTomato was much more potent than a similar construct lacking tdTomato (AAV2.7m8-CAG-ChrimsonR) in nonhuman primates and rdl mice with retinitis pigmentosa. Therefore, our goal was to understand its underlying mechanism. To this end, we performed in vitro studies in HEK293 cells focusing on the expression and delivery of ChrimsonR alone or fused to tdTomato. method

[0239] Human HEK293 cells were seeded in 24-well plates in DMEM medium supplemented with 10% fetal bovine serum. The confluence of the cells (Confluence) was 10% to 70%, used between the 3rd and 20th passages. Cell transfection of pssAAV-CAG-ChrimsonR-tdTomato, pssAAV-CAG-ChrimsonR and pssAAV-CAG-ChrimsonR-GFP plasmids was performed using As a transfection agent (1 μl in 50 μl buffer mixed with 0.5 μg of plasmid DNA).

[0240] ChrimsonR, Chrims...

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Abstract

Disclosed are, among other methods, methods for reactivating retinal ganglion cells in mammals by administering an effective amount of channelrhodopsins (such as ChrimsonR), or an effecti ve amount of such channelrhodopsins (such as ChrimsonR) fused to a fluorescent protein, in the form of protein or nucleic acids, and compositions thereof. The methods may include a light stimuli level inducing RGCs response that is below radiation safety limit. The methods may include delivery by an adenoassociated virus vector. The methods may include use of a CAG promoter. The methods may result in a long term expression of an effective amount of the channelrhodopsins (such as ChrimsonR protein).

Description

[0001] Related Application Cross Reference [0002] This application claims priority to U.S. Provisional Application No. 62 / 329,692 filed on April 29, 2016, and the content of this earlier application is fully incorporated into this application by reference. [0003] sequence listing [0004] This application contains a Sequence Listing, which was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. A copy of the file in ASCII format was created on April 28, 2017, named "12295_0006-00304.txt", and 31 bytes in size. technical field [0005] The present invention provides, including but not limited to, compositions and methods for altering transmembrane conductance, cell activity, and cell function, and relates to the use of exogenous light-activated ion channels in cells and target subjects. Specifically, an aspect according to an embodiment of the present invention relates to a method for reactivating retinal neuronal cells (RGC) in a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16C07K14/405C12N15/62C12N15/86
CPCA61K38/16C07K14/405C12N15/625C07K2319/60A61K48/005C12N2750/14143A61P25/00A61P25/02A61P27/02A61P43/00A61P9/10A61K41/00A61K9/0019A61K9/0048A61K35/761A61K38/168A61K38/1709A61K38/1767A61K48/00C12N15/8645C12N15/62A61K36/06
Inventor 迪迪尔·普陆瑙安妮·杜瓦丹尼兹·达尔卡拉简斯·度贝尔罗梅因·卡普莱特格利高里·高文梅莉萨·德罗歇尔斯何塞·萨赫勒塞尔吉·皮科
Owner GENSIGHT BIOLOGICS SA
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