rexresearch
Zhou-Hua PAN
Channelrhodopsin 2 vs Blindness
http://singularityhub.com/2015/09/20/meet-the-mind-controlling-algae-protein-that-could-cure-blindness/
20 September 2015
Meet the Mind-Controlling Algae Protein That Could Cure Blindness
by Shelly Fan
It sounds completely crazy: as early as next year, using gene therapy scientists hope to restore sight in the blind by giving their eyes additional “light sensors.”
We’re not talking about bionic eyes: instead of implantable electronics, scientists are turning to a protein called channelrhodopsin-2. You’ve probably heard of this protein before — it’s the same magical switch that, in response to light, can turn a gentle mouse aggressive, shut down obsessive grooming behavior, and implant false memories in unsuspecting mice.
What does a mind-controlling protein have to do with restoring vision?
Meet the protein that sparked a neuroscience revolution
The answer lies in how channelrhodopsin-2 works. The protein comes from the lowly green algae, which uses it to seek out sunlight for photosynthesis.
At its core, channelrhodopsin-2 is a light-sensitive protein tunnel that sits on the surface of cells. Normally the tunnel is completely cinched up, which allows a cell to maintain a steady interior environment.
However, when a certain wavelength of light hits the protein, the tunnel temporarily flashes open, much like a camera shutter. When open, the protein is like a highway, shuttling ions into the cell — the same biophysical process that makes a neuron burst with activity.
About a decade ago, neuroscientists realized that they could stick the protein into mouse neurons that were previously impervious to light. By using sophisticated genetic tools, the protein could be restricted to certain types or populations of neurons, rather than huge chunks of the brain.
Then, by shining light through an implanted fiber optic laser, researchers can artificially activate select networks of neurons. The results are nothing short of science fiction: a flash of light, and a mouse — going about its business as usual — might, for example, suddenly freeze in place as if terrorized. Turn the light off, and it’ll revert back to its normal happy-go-lucky state, seemingly unaware that anything strange just happened. Hence, the “mind control” part.
The scientists dubbed this powerful new technique optogenetics, and hundreds — if not thousands — of labs around the world are now using this technique to explore the intricate neural connections in the brain.
Adding backup hardware to the human eye
The brain-bending powers of channelrhodopsin-2 are so mind-boggling that it’s easy to forget the simple nature of the protein: it senses light, and transmits that information through electricity to higher processing centers.
Broadly speaking, human eyes work similarly. Light passes through the length of our eyeballs and falls on the back of the retina, activating light-sensitive proteins called photoreceptors (these are shaped like rods and cones). The photoreceptors transmit light information through two filter layers — ganglion and bipolar cells — that process the electric signals and send them to visual areas of the brain.
In many eye disorders, such as retinitis pigmentosa or macular degeneration, the rods and cones gradually die off. This leads to progressively failing vision, and — without a cure — eventually turns one out of four sufferers legally blind.
These are cold, brutal diseases, but there is one silver lining: they leave ganglion and bipolar cells intact and still able to still communicate with the brain.
The obvious treatment would be to introduce human hardware back into the human retina through gene therapy. Yet, human light-sensitive proteins are notoriously hard to engineer. To function normally, they have to be tightly coupled to many other supporting proteins. This means scientists would have to get multiple genes at the right ratio and levels into the retina — a feat that is currently impossible.
Channelrhodopsin-2, on the other hand, works just by itself.
In 2006, Dr. Zhou Hua Pan, a researcher at Wayne State University, decided to stick the protein into mice that were genetically engineered for photoreceptor degeneration. It worked on the first try; in less than three months after a single treatment, the mice passed every vision test the scientists could throw at them.
“It worked perfectly, even in the very beginning,” Pan told Wired. “That was basically just really, really lucky.”
Pan’s success did not escape the notice of the biotech industry. In 2009, RetroSense Therapeutics, a startup located a short drive away from Wayne State, leased the eye-wiring tech from Pan in a bid to bring it to human trials. Last month, the FDA gave its nod of approval: as early as this fall, the company will start installing channelrhodopsin-2 into the retinas of 15 patients blinded by retinitis pigmentosa through gene therapy.
The company is aiming for retinal ganglion cells, which are spared by the disease even in advanced stages. In essence, by giving these “middle men” the ability to sense light, scientists hope to circumvent the need for rods and cones.
A long road ahead to color
To be clear, as promising at it is, the algae protein can’t restore human vision to all its colorful vividness.
The photoreceptors in our eyes, optimized by eons of evolution, operate over a wide range of light intensities and wavelengths — we get to see everything from pale starlight blinking in an indigo sky to glaring sunlight above white hot sand, and all the colors in between.
In contrast, channelrhodopsin-2 paints a dim monochromatic landscape. The protein is 2,000 times less sensitive to light than our retinal cones. It only responds to a very narrow set of wavelengths — and thus colors — of light, a far cry from our normal eyesight. In a way, going from rods and cones to channelrhodopsin-2 is like going from an expensive 20-gear road bike to a fixie. It works, but it’s not optimal.
Ganglion cells also don’t normally deal with light — they generally process electrical signals that come from rods and cones. Whether raw light signals work as well as pre-processed electrical information from photoreceptors is still up in the air. If the gene therapy successfully delivers channelrhodopsin-2 to these cells, the brain will have some serious rewiring to do before it can interpret these strange new signals.
Even with these caveats, the therapy may be a game changer. A decade ago, channelrhodopsin-2 dramatically changed the face of neuroscience — here’s to hoping it’ll spark another revolution soon.
http://news.yahoo.com/scientists-may-finally-discovered-cure-154544209.html
21 September 2015
Scientists May Have Finally Discovered a Cure for Blindness
by Trace William Cowen
When Wayne State University researcher Dr. Zhou-Hua Pan placed a light-sensitive green algae protein into blind mice in 2006, he was amazed to find that it restored the subjects' vision almost immediately. Fast forward to 2015, the year of many great things, and that protein is now the subject of a forthcoming set of human trials aimed at unveiling a potential cure for blindness in humans.
RetroSense Therapeutics, the company who leased the research from Dr. Zhou-Hua Pan and recently received approval from the Food and Drug Administration to administer human trials, is expected to begin testing the protein on 15 patients by the end of the year. According to Singularity Hub, channelrhodopsin-2 is the same "magical switch" protein already famous for its ability to "turn a gentle mouse aggressive, shut down obsessive grooming behavior, and implant false memories in unsuspecting mice."
The protein is placed directly into the retina using gene therapy, allowing the rod-and-cone system to be bypassed entirely and giving the eye's ganglion cells the ability to sense light on their own. Though some levels of colorblindness may persist even with successful implementation of the forthcoming human trials, some researchers speculate that the human brain could potentially make adjustments in order to counteract the color loss.
Keep up the good work, science. We're all counting on you.
US2015044181
IDENTIFICATION OF CHANNELRHODOPSIN-2 (CHOP2) MUTATIONS AND METHODS OF USE.
The invention provides compositions and kits including at least one nucleic acid or polypeptide molecule encoding for a mutant ChR2 protein. Methods of the invention include administering a composition comprising a mutant ChR2 to a subject to preserve, improve, or restore phototransduction. Preferably, the compositions and methods of the invention are provided to a subject having impaired vision, thereby restoring vision to normal levels.
FIELD OF THE INVENTION
[0004] This invention relates generally to the field of molecular biology. Mutations in the Channelopsin-2 (Chop2) gene are identified. Compositions comprising a mutant Chop2 gene are used in therapeutic methods to improve and restore vision loss.
BACKGROUND OF THE INVENTION
[0005] The retina is composed of photoreceptors (or photoreceptor cells, rods and cones). Photoreceptors are highly specialized neurons that are responsible for phototransduction, or the conversion of light (in the form of electromagnetic radiation) into electrical and chemical signals that propagate a cascade of events within the visual system, ultimately generating a representation of our world.
[0006] Photoreceptor loss or degeneration severely compromises, if not completely inhibits, phototransduction of visual information within the retina. Loss of photoreceptor cells and/or loss of a photoreceptor cell function are the primary causes of diminished visual acuity, diminished light sensitivity, and blindness. There is a long-felt need in the art for compositions and method that restore photosensitivity of the retina of a subject experiencing vision loss.
SUMMARY OF THE INVENTION
[0007] The invention provides a solution for the long-felt need for a method of restoring and/or increasing the light sensitivity of photoreceptor cells by expression of advantageous mutations, and/or combinations thereof, of the Channelopsin-2 (Chop2) gene, and subsequently providing methods for Channelopsin-2 (Chop2)-based gene therapy.
[0008] Channelopsin-2 (Chop2)-based gene therapy offers a superior strategy for restoring retinal photosensitivity after photoreceptor degeneration. The protein product of the Chop2 gene, when bound to the light-isomerizable chromophore all-trans-retinal, forms a functional light-gated channel, called channelrhodopsin-2 (ChR2). Native ChR2 shows low light sensitivity. Recently, two mutant ChR2s, L132C and T159C, were reported to markedly increase their light sensitivity (Kleinlogel et al. (2011) Nat. Neurosci. 14:513-8; Berndt et al. (2011) Proc Natl Acad Sci USA. 108:7595-600; Prigge et al. (2012) J Biol. Chem. 287(38)3104:12; the contents of each of which are incorporated herein in their entireties). The properties of these two ChR2 mutants (i.e., L132C and T159C) were examined and compared with a number of double mutants at these two sites to identify suitable candidates for therapeutic methods. Compositions comprising one or more of these mutations are provided to a subject in need thereof for the purpose of restoring vision. Specifically, desired mutations in the Chop2 gene are introduced to a cell and/or integrated into the genomic DNA of a cell to improve or restore vision. Desired mutations in the Chop2 gene that are introduced to a cell to improve or restore vision may also remain episomal, not having integrated into the genomic DNA.
[0009] Mutations at the L132 or T159 amino acid positions of Chop2 (and therefore, the resulting ChR2) markedly lower the threshold light intensity that is required to elicit the ChR2-mediated photocurrent. Double mutants at the amino acid positions L132 and T159 further increase the photocurrent at low light intensities, exceeding that of either of the corresponding single mutations. Retinal ganglion cells expressing the double mutants at the L132 and T159 positions can respond to light intensities that fall within the range of normal outdoor lighting conditions but should still maintain adequate, and high temporal resolution that are suitable for restoring useful vision. Thus, mutant Chop2 protein of the present invention that form mutant ChR2s having improved light sensitivity are used alone or in combination to restore or improve vision.
[0010] Specifically, the invention provides an isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L). In certain embodiments of the isolated polypeptide molecule, the amino acid at position 132 is cysteine (C) or alanine (A). When the amino acid at position 132 is cysteine (C), the polypeptide molecule may comprise or consist of SEQ ID NO: 13. When the amino acid at position 132 is alanine (A), the polypeptide molecule may comprise or consist of SEQ ID NO: 20.
[0011] The invention provides an isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 159 of SEQ ID NO: 26 is not a threonine (T). In certain embodiments of the isolated polypeptide molecule, the amino acid at position 159 is cysteine (C), serine (S), or alanine (A). When the amino acid at position 159 is cysteine (C), the polypeptide molecule may comprise or consist of SEQ ID NO: 14. When the amino acid at position 159 is serine (S), the polypeptide molecule may comprise or consist of SEQ ID NO: 17. When the amino acid at position 159 is alanine (A), the polypeptide molecule may comprise or consist of SEQ ID NO: 23.
[0012] The invention provides isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T). In certain embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), the amino acid at position 132 is cysteine (C), and the amino acid at position 159 is cysteine (C). In a preferred embodiment of this isolated polypeptide molecule, the polypeptide molecule comprises or consists of SEQ ID NO: 16. The invention provides an isolated nucleic acid molecule that encodes for the isolated polypeptide comprising or consisting of SEQ ID NO: 16. Preferably, the isolated nucleic acid molecule that encodes for the isolated polypeptide comprising or consisting of SEQ ID NO: 16, is a nucleic acid molecule that comprises or consists of SEQ ID NO: 15.
[0013] In certain embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is serine(S). The isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), may comprise or consist of SEQ ID NO: 19. Alternatively, or in addition, the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), wherein the amino acid at position 132 is cysteine (C) and wherein the amino acid at position 159 is serine(S) may comprise or consist of SEQ ID NO: 19. The invention provides an isolated nucleic acid molecule that encodes for the isolated polypeptide that comprises or consists of SEQ ID NO: 19. Preferably, the nucleic acid molecule comprises or consists of SEQ ID NO: 18.
[0014] In certain embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), the amino acid at position 132 is alanine (A) and the amino acid at position 159 is cysteine (C). The isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T) may comprise or consist of SEQ ID NO: 22. Alternatively, or in addition, the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), wherein the amino acid at position 132 is alanine (A) and wherein the amino acid at position 159 is cysteine (C) may comprise or consist of SEQ ID NO: 22. The invention provides an isolated nucleic acid molecule that encodes for the isolated polypeptide that comprises or consists of SEQ ID NO: 22. Preferably, this nucleic acid molecule comprises or consists of SEQ ID NO: 21.
[0015] In certain embodiments of the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), the amino acid at position 132 is cysteine (C) and the amino acid at position 159 is alanine (A). The isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T) may comprise or consist of SEQ ID NO: 25. Alternatively, or in addition, the isolated polypeptide molecule comprising or consisting of SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino acid at position 159 is not threonine (T), wherein the amino acid at position 132 is cysteine (C) and wherein the amino acid at position 159 is alanine (A) may comprise or consist of SEQ ID NO: 25. The invention provides an isolated nucleic acid molecule that encodes for the isolated polypeptide that comprises or consists of SEQ ID NO: 25. Preferably, this nucleic acid molecule comprises or consists of SEQ ID NO: 24.
[0016] The invention provides any one of the isolated polypeptide molecules described herein, wherein the polypeptide molecule encodes for a mutant Chop2 protein that forms a mutant ChR2, which elicits a current in response to a threshold intensity of light that is lower than the threshold of a wild type ChR2 protein. Moreover, the current conducts cations. Exemplary cations include, but are not limited to, H<+>, Na<+>, K<+>, and Ca<2+> ions. The ChR2 wild type and mutant proteins described herein non-specifically conduct cations. Consequently, the current conducts one or more of the following: H<+>, Na<+>, K<+>, and Ca<2+> ions.
[0017] The invention provides any one of the isolated polypeptide molecules described herein further comprising a pharmaceutically acceptable carrier. The invention also provides a composition comprising at least one isolated polynucleotide molecule described herein. The composition may further comprise a pharmaceutically-acceptable carrier.
[0018] The invention provides an isolated nucleic acid molecule that encodes for any of the isolated polypeptides described herein. Moreover, the isolated nucleic acid molecule may further include a pharmaceutically acceptable carrier. The invention also provides a composition comprising at least one isolated nucleic acid molecule described herein. The composition may further comprise a pharmaceutically-acceptable carrier.
[0019] The invention provides a cell, wherein the cell has been contacted with or comprises an isolated polypeptide molecule of the invention. Moreover, the invention provides a cell, wherein the cell has been contacted with or comprises an isolated nucleic acid molecule that encodes for an isolated polypeptide molecule of the invention. The invention provides, a composition comprising, consisting essentially of, or consisting of a cell that comprises an isolated polypeptide molecule of the invention or a nucleic acid molecule that encodes for an isolated polypeptide molecule of the invention. Cells of the invention may be contacted with the isolated polypeptide or an isolated nucleic acid encoding the polypeptide in vitro, ex vivo, in vivo, or in situ. In certain embodiments of the invention, the cell is a photoreceptor; a horizontal cell; a bipolar cell; an amacrine cell, and, especially, an AII amacrine cell; or a retinal ganglion cell, including a photosensitive retinal ganglion cell. Preferably, the cell is a retinal ganglion cell, a photosensitive retinal ganglion cell, a bipolar cell, an ON-type bipolar cell, a rod bipolar cell, or an AII amacrine cell. In certain aspects of the invention, the cell is a photoreceptor, a bipolar cell, a rod bipolar cell, an ON-type cone bipolar cell, a retinal ganglion cell, a photosensitive retinal ganglion cell, a horizontal cell, an amacrine cell, or an AII amacrine cell.
[0020] The invention provides a method of improving or restoring vision, comprising administering to a subject any one of the compositions described herein. The invention further provides a prophylactic method of preserving vision, comprising administering to a subject any one of the compositions described herein.
[0021] The methods described herein may also be applied to those subjects who are healthy, blind (in part or in total), and/or those subjects with retinal degeneration (characterized by a loss of rod and/or cone photoreceptor cells), but may be dependent upon the activity of photosensitive retinal ganglion cells for a determination of ambient light levels. For example, the methods described herein can be used to preserve, improve, or restore the activity of a photosensitive retinal ganglion cell that mediates the transduction of light information for synchronizing circadian rhythms to the 24-hour light/dark cycle, pupillary control and reflexes, and photic regulation of melatonin release.
[0022] In certain embodiments of the methods of the invention, the subject may have normal vision or impaired vision. Alternatively, or in addition, the subject may be at risk for developing an ocular disease that leads to impairment of vision. For example, the subject may have a family history of, ocular disease, including, macular degeneration and retinitis pigmentosa. The subject may be at risk for incurring an eye injury that causes damage to photosensitive cells in the retina. The subject may have a genetic marker or genetic/congenital condition that results in impaired vision, low vision, legal blindness, partial blindness, or complete blindness. Subjects may have a refractive defect that results in myopia (near-sightedness) or hyperopia (far-sightedness).
US8470790
Restoration of Visual Responses by In Vivo Delivery of Rhodopsin Nucleic Acids
Nucleic acid vectors encoding light-gated cation-selective membrane channels, in particular channelrhodopsin-2 (Chop2), converted inner retinal neurons to photosensitive cells in photoreceptor-degenerated retina in an animal model. Such treatment restored visual perception and various aspects of vision. A method of restoring light sensitivity to a retina of a subject suffering from vision loss due to photoreceptor degeneration, as in retinitis pigmentosa or macular degeneration, is provided. The method comprises delivering to the subject by intravitreal or subretinal injection, the above nucleic acid vector which comprises an open reading frame encoding a rhodopsin, to which is operatively linked a promoter and transcriptional regulatory sequences, so that the nucleic acid is expressed in inner retinal neurons. These cells, normally light-insensitive, are converted to a light-sensitive state and transmit visual information to the brain, compensating for the loss, and leading to restoration of various visual capabilities.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention in the field of molecular biology and medicine relates to the use of microbial-type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2) to convert inner retinal neurons to photosensitive cells in photoreceptor-degenerated retina, thereby restoring visual perception and various aspects of vision.
US2013259833
AAV-Mediated Subcellular Targeting of Heterologous Rhodopsins in Retinal Ganglion Cells
Microbial type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2/ChR2) or the ion pump halorhodopsin (HaloR) are expressed in retinal ganglion cells upon transduction using recombinant AAV vectors. Selective targeting of these transgenes for expression in discrete subcellular regions or sites is achieved by including a sorting motif in the vector that can target either the central area or surround (off-center) area of these cells. Nucleic acid molecules comprising nucleotide sequences encoding such rhodopsins and sorting motifs and their use in methods of differential expression of the transgene are disclosed. These compositions and methods provide significant improvements for restoring visual perception and various aspects of vision, particular in patients with retinal disease.