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Patent Application Titled "Light-Sensitive Chimeric GPCR Protein" Published Online

July 8, 2014

By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Van Wyk, Michiel (Hinterkappelen, CH); Kleinlogel, Sonja (Hinterkappelen, CH), filed on June 22, 2012, was made available online on June 26, 2014 (see also Patents).

No assignee for this patent application has been made.

Reporters obtained the following quote from the background information supplied by the inventors: "Major causes of retinal photoreceptor degeneration include retinitis pigmentosa (RP), age-related macular degeneration (ARMD), diabetic retinopathy and other diseases. Approximately one in three thousand, or three million people worldwide, suffer from retinitis pigmentosa (RP), a genetic condition that leads to photoreceptor degeneration and eventually blindness. The rate and severity of photoreceptor degeneration is variable and highly dependant on the mutation itself. Over fifty genes may be affected (Hartong et al. Lancet 368:1795-1809; 2006). To date, little treatment is available for RP patients. Ongoing trials that focus on neuroprotective agents (e.g. ciliary neurotrophic factor) or gene addition therapy (introducing the 'non-mutated' gene), which aim to correct acquired or hereditary gene deficiencies to the natural functional gene, have so far shown only marginal success. Given that the adult retina has no ability to generate new photoreceptors after photoreceptor loss, gene addition therapy is only useful as long as photoreceptor loss is small and mainly slows down or stabilizes the early condition.

"An alternative approach employed in recent experimental studies is to render the remaining photoreceptors or surviving inner retinal neurons light-sensitive through transgenic expression of a light-sensitive protein.

"In US 2009/0088399 and US 2010/0015095 it is proposed to introduce the light-gated algal ion-channel channelrhodopsin-2 (ChR2) into the inner retina of patients suffering from photoreceptor cell degeneration This renders the naturally light-insensitive inner retinal cells, such as bipolar or amacrine cells, light-sensitive and capable of detecting visual information, which is subsequently relayed to the brain without receiving input from photoreceptors.

"Similarly, in US 2005/0208022 and US 2009/0208462 it is proposed to introduce a photoreceptive protein such as an opsin (including melanopsin) or cytochromes into the inner retinal neurons including amacrine, horizontal and bipolar cells of patients suffering from photoreceptor degeneration.

"The approach to express ChR2 in inner retinal neurons holds considerable promise and is currently tested in non-human primates (Fradot M et al. Human Gene Therapy 22(5), 587-593; 2011) and isolated human retinas (Ivanova E et al. Opthalmol Vis Sci 51(10), 5288-5296, 2010), raising hope for clinical trials in the near future.

"In recent years retinal gene-replacement therapy using recombinant Adeno-associated virus (rAAV) has been successful and has reached final clinical trials. In particular, Bainbridge and colleagues used rAAV to replace the defective retinal pigment epithelium-specific 65-kDa protein gene (RPE65). A deficiency in the RPE65 protein renders photoreceptors unable to respond to light, as it is required for the recycling of the chromophore, i.e. the conversion of all-trans retinal to 11-cis retinal (Bainbridge J W B et al., N Engl J Med 358(21), 2231-2239; 2008). Gene therapy is therefore a promising therapeutic approach to correct for visual deficiencies by the introduction of suitable genes into retinal neurons.

"The currently available light-activatable proteins that could be used in gene therapy to compensate for the loss of photoreceptor cells, however, still hold a number of substantial drawbacks: 1) Artificial expression of foreign, invertebrate or algal proteins, e.g. ChR2, could trigger unpredictable immune reactions in patients. 2) ChR2 has a relatively high permeability to calcium, which might be toxic over the long term. 3) The ChR2 response is inherently weak at natural light intensities as each captured photon can only activate a single protein. 4) Although, melanopsin is able to amplify light-signals by gating the activities of high-throughput enzymatic reactions, these enzymatic partners are not sufficiently available in inner retinal neurons. Therefore, the expression of melanopsin in ganglion cells and ON-bipolar cells does not elicit an amplification of the light signal sufficient to restore functional vision at natural light intensities. 5) Also, the regulatory mechanisms that naturally control protein activity through changes in turnover and modulation are absent when expressing foreign proteins.

"The object of the current invention is to provide a light-sensitive chimeric protein, which, when expressed in inner retinal neurons, overcomes these deficiencies. That is, it is an object of the invention to provide a superior light-sensitive protein for the improvement and restoration of vision, particularly in patients with retinal photoreceptor degeneration. This chimeric protein will improve or restore light-sensitivity to a higher extent compared to the light-sensitivity that is obtainable by proteins proposed in the state of the art. Further objects of the invention include the genetic information encoding the chimeric light-sensitive protein and methods of expressing this chimeric protein in living cells and organisms. Yet further objects of the invention include the expression of the genetic information encoding the chimeric light-sensitive protein in inner retinal cells in vivo for therapeutic treatment and biomedical products comprising the light-sensitive protein or genetic information encoding the chimeric protein."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "This technical problem is solved by a light-sensitive chimeric protein comprising domains from at least two members of the G-protein-coupled-receptor (GPCR) protein super family, which are fused to yield a light-sensitive GPCR chimera capable of coupling a light signal to the signaling cascade of the metabotropic glutamate receptor 6 (mGluR6).

"The G-protein-coupled-receptor (GPCR) protein super family members are transmembrane protein receptors transmitting signals from the cell surface to intracellular effectors. They have a structure, which typically comprises seven transmembrane domains (TM1 to TM7), three extracellular loops (EL1 to EL3), three intracellular loops (IL1 to IL3), an extracellular N-terminal domain (NT) and an intracellular C-terminal (CT) domain. The GPCR protein super family includes light-sensitive receptor proteins called photopigments such as opsins, for example rhodopsin and melanopsin. The GPCR super family also include ligand-gated metabotropic receptors, for example mGluR6. The metabotropic G-protein coupled receptors are indirectly linked to ion channels in the membrane via a signal transduction cascade mediated by specific G-proteins accomplishing an amplification of the signal. That is, activated G-proteins regulate the activity of enzymes, for example adenylate cyclase, which rapidly produce large quantities of product, for example cAMP, which may in turn activate large numbers of ion channels in the cell membrane. In contrast to such metabotropic GPCRs, ionotropic receptors are directly linked to ion channels in the membrane. Therefore, ionotropic receptors like channelrhodopsin are not capable of signal amplification like metabotropic receptors.

"One aspect of the invention concerns a chimeric GPCR protein, comprising domains which are derived from at least two GPCR family members:

"A first of the at least two GPCR family members contributes domains which mediate the light sensitivity to the chimeric light-sensitive GPCR protein. This first member belongs to the family of light-sensitive GPCR proteins also called photopigments, and in some embodiments this light-sensitive GPCR protein is melanopsin, in particular human melanopsin.

"A second of the at least two GPCR family members, namely mGluR6, contributes domains for coupling the light signal to the intracellular signalling cascade of mGluR6.

"mGluR6 is a native component of the cell membrane of ON-bipolar cells in the inner retina. For the therapeutic aspects of the current invention these ON-bipolar cells are the target cells in which the light-sensitive chimeric GPCR protein will be expressed. Physiologically, the native ON-bipolar cell mGluR6 activates its intracellular signal cascade upon extracellular binding of glutamate. Thus, the ON bipolar cells naturally contain the specific intracellular components mediating the mGluR6 signaling cascade.

"In the physiological light signal transduction pathway, light-activated healthy rod and cone photoreceptor cells respond to a decrease in light intensity with an increase in the level of glutamate released from their synaptic terminals, which then binds to mGluR6 on ON-bipolar cells, which in turn elicits an amplification of the light signal through the specific G-Protein coupled intracellular signaling cascade of mGluR6. In analogy to this natural pathway, the chimeric light-sensitive GPCR protein expressed in ON-bipolar cells of blind retinas transmits the light-signal to the still existing (Kriz{hacek over (a)}j D et al., Vision Res. 50:2460-65, 2010) intracellular signal cascade of the mGluR6 receptor upon light activation.

"Remarkably, the ON-bipolar cells, when complemented with the chimeric light-sensitive GPCR protein, directly perceive the light signal via the chimeric light-sensitive GPCR protein, bypassing the indirect glutamate signal that follows the light-stimulation of the photoreceptors. Thus, the chimeric light-sensitive GPCR protein is capable of directly coupling light activation to the mGluR6 signal cascade. In other words, light activation is independent of any functional rod or cone photoreceptor cells. Furthermore, the physiological amplification of the signal elicited by one photon is retained through the signalling cascade of the mGluR6.

"The term 'domain' in the context of this patent application refers to the intracellular and extracellular loops, the N- and C-termini and the transmembrane regions of a member of the GPCR protein family. The term 'domain derived from' such as domain derived from mGluR6 or a domain derived from an opsin includes any domain for which the physiologically relevant corresponding part has an identical amino acid sequence or a similar amino acid sequence to the sequence of such domain in the physiological counterpart of the GPCR family member. In general, similar amino acid sequences or similar domains exhibit at least a 60% homology, preferably at least a 80% homology and most preferably at least a 90% homology. Similar domains also particularly include domains comprising relevant conserved amino acids, independent of whether a part of the remaining sequence is deviating or missing from the native counterpart or whether additional sequences are present in the chimeric protein that are not present in the native GPCR family member.

"In some embodiments the chimeric protein comprises a light-activatable extracellular domain which is derived from a bi-stable photopigment, such as melanopsin but not rhodopsin for example. The advantage of bi-stable photopigments is that they are recycled after bleaching through recovery by light rather than by external cellular enzymes. The recovery rate is very fast and will sustain a high light-sensitivity even at high light intensities. With bi-stable photopigments, light bleaching and bleach recovery are increased equally at high light intensities, whereas rhodopsin, which is not bi-stable, looses its photosensitivity during illumination as more and more rhodopsins are bleached. Light bleaching in non-bi-stable photopigments such as rhodopsin can lead in the worst case to short-term blindness. The recovery rate could even be slower when a non-bi-stable photopigment such as rhodopsin is expressed in a foreign cell type, because the recovery enzymes are not necessarily available in proximity. In a healthy retina these enzymes are located in the retinal pigment epithelium.

"Accordingly the choice of the domains of the first member of the chimeric GPCR, to be derived from a bi-stable photopigment renders the recovery of the chimeric GPCR after light-bleaching independent of the availability of bleach-recovery enzymes. In some embodiments the light-activatable domain of a bi-stable photoreceptor protein is selected from the opsin family, and most preferably is melanopsin and, if used in human patients, it is human melanopsin to avoid an immune reaction.

"In some embodiments of the chimeric GPCR protein the first GPCR member contributes at least the domains containing the amino acid residues forming the Schiff base (linking the chromophore covalently to the GPCR), which are for melanopsin Tyrosine.sup.149 (Y149) in TM3 and Lysine.sup.321 (K321) in TM7, or all the domains derived from the domains which form the chromophore binding pocket in the physiological counterpart. The chromophore binding pocket refers to the binding site for the light pigment, which absorbs a photon such as for example 11-cis retinal in melanopsin (Hermann et al., Neuroscience letters, Vol. 376 p76-80, 2004.)

"In some other embodiments the chimeric GPCR protein comprises all of the extracellular domains of the first GPCR member, which are the N-terminus and the three extracellular loops (EL1, EL2, EL3) and additionally all of the seven transmembrane domains (TM1 to TM7) from the first GPCR member.

"In either of these embodiments, at least one of the intracellular domains of the chimeric GPCR protein, i.e. at least one of the intracellular loops IL1, IL2, IL3 and/or the C terminus is derived from the second GPCR, which is mGluR6. In some embodiments the at least one intracellular domain derived from mGluR6 is IL3 or is IL3 and additionally at least one of the other intracellular domains, e.g. IL3 and IL2 or IL 3 and IL 2 and the C-terminus or other combinations.

"Functional chimeric GPCR proteins according to the invention are light-sensitive and capable of coupling light activation to the mGluR6 signaling cascade. Depending on which photopigment is chosen as first GPCR member for the chimeric protein, either some or all transmembrane domains and extracellular domains of this photopigment are used. The domains required for forming a chromophore pocket are necessary to render the chimeric protein light activatable, which according to current knowledge are for example TM3 to TM 7 in melanopsin and TM2 to TM 7 in channelrhodopsin.

"The domains which are necessary for coupling light activation to the mGluR6 signaling cascade must be capable of binding to the G-Protein specific for the mGluR6 pathway, Galpha(o). IL3 appears to be particularly relevant for the specific binding to the G-protein of the GPCR signal cascade. Generally, the other intracellular loops and the C-terminus enhance the specificity of G-protein binding over embodiments in which some or all of IL1 and IL2 and the C-terminal domain are not derived from mGluR6.

"In some embodiments the chimeric GPCR protein comprises domains which are derived from another bi-stable GPCR protein (or opsin chimeras based on a bi-stable GPCR) which is not the first and not the second member.

"For minimizing potential immunogenic reactions and for optimizing the physiological coupling to the mGluR6 in some embodiments to be used for medical therapy in humans, the light-sensitive domains are derived from human GPCRs such as human melanopsin, human rhodopsin, human cone-opsin but also chimeric human opsins.

"The light-sensitive chimeric GPCR protein is constructed by fusing the genetic information encoding domains of the GPCR members with the desired functionalities of light-sensitivity and coupling of the light activation to the signaling cascade of mGluR6 according to techniques known in the art. Identification of the desired domains and determination of suitable cutting and ligation sites at the N- and C-terminal ends of any particular domain are primarily based on 1) alignment of gene sequences/conserved residues and 2) computer modeling of the secondary and tertiary structure of the light-sensitive GPCR family member and mGluR6, using standard software available in the art. This approach has an inherent variability in the exact definition of the length of the individual domains and such variability is included within the scope of this invention when speaking of domains. Furthermore, at individual fusion sites between domains, there are generally a number of possibilities of splicing the domains together to yield a functional protein. And, evidently, deletion of portions of an amino acid sequence not required for function, conservative amino acid substitutions, for example interchanging hydrophobic with hydrophobic or hydrophilic with hydrophilic amino acids, and nucleotide substitutions are also within the scope of the invention. Accordingly, a considerable number of sequence variants particularly in regions of the fusion sites between adjacent domains of the chimeric GPCR proteins fall within the scope of the invention, provided that they yield functional chimeric GPCR proteins. In embodiments in which all of the transmembrane and the extracellular domains are derived from the first GPCR member and at least one or all of the intracellular domains are replaced with corresponding domains derived from mGluR6, all feasible cutting and ligation sites for exchanging IL1, IL2, IL3 and the C-terminus are within the scope of the invention.

"Further aspects of the invention concern the genetic information of a light-activatable chimeric GPCR protein capable of coupling the light activation to the signaling cascade of mGluR6, vectors including viral vectors such as rAAVs comprising this genetic information, transgenic animals such as mice and zebra fish comprising this genetic information and cell culture cells comprising such genetic information or expressing light-activatable chimeric GPCR proteins capable of coupling the light activation to the signaling cascade of mGluR6, including in particular neuronal cell lines, inner retinal neuronal cell lines and bipolar cell lines in particular ON-bipolar cells.

"A further aspect of the invention concerns methods of introducing the genetic information for expression of a light-activatable chimeric GPCR protein capable of coupling the light activation to the signaling cascade of mGluR6 into the eye, preferably into ON-bipolar cells. Yet a further aspect of the invention concerns methods of introducing the genetic information for expression of a light-activatable chimeric GPCR protein capable of coupling the light activation to the signaling cascade of mGluR6 into cell culture cells, in particular into neural cell lines, including retinal cell lines, inner retinal cell lines and bipolar cell lines.

"A further aspect of the invention concerns gene therapeutic methods of introducing the light-sensitive chimeric GPCR protein capable of coupling light activation to the signaling cascade of mGluR6 into the eye, in particular into the vitreal or subretinal space to target retinal cells including ON-bipolar cells of both rod and cone photoreceptor cells, for improving vision in medical therapy. Such gene therapeutic methods include but are not limited to electroporation, viral transduction and chemical-based transfection. Such medical therapy in particular includes the treatment of partial or complete blindness, e.g. for the treatment of retinitis pigementosa (RP) and macular degeneration (ARMD) as well as other forms of photoreceptor degeneration.

"Yet a further aspect of the invention concerns the light-sensitive chimeric GPCR protein capable of coupling light-activation to the signaling cascade of mGluR6 or the genetic information encoding said chimeric protein and compositions comprising said protein or said genetic information as such or within vectors or cells for the purpose of medical therapy, in particular for improving vision, for the treatment of partial or complete blindness, for the treatment of retinitis pigmentosa (RP) and macular degeneration (ARMD) as well as other forms of photoreceptor degeneration.

"Physiologically, the metabotropic glutamate receptor of ON-bipolar cells in the inner nuclear layer of the retina is activated by the neurotransmitter glutamate in response to retinal photoreceptor cell activity. When the photoreceptors are stimulated by light, the concentration of glutamate released onto ON-bipolar cells changes. The light-sensitive chimeric GPCR protein is a variant of the native mGluR6 protein, which is activated by light directly whereas the native mGluR6 protein is activated indirectly via glutamate after stimulation of the photoreceptor cells by changes in light. Therefore, patients suffering from photoreceptor degeneration can be treated by expressing a chimeric light-activatable protein comprising intracellular domains of mGluR6 capable of coupling the light activation to the signaling cascade of the mGluR6 in their ON-bipolar cells.

"In some embodiments of the light-sensitive chimeric GPCR protein at least one or all of the intracellular components of melanopsin or another bi-stable photopigment are substituted with the intracellular components of mGluR6, resulting in a chimeric protein comprising the photoreceptor domains of melanopsin, which is able to drive existing intracellular mGluR6 signaling cascades in inner retinal neurons, in particular in ON-bipolar cells.

"Due to artificial expression of a chimeric light activatable mGluR6-melanopsin protein in ON-bipolar cells, weak light signals are amplified by steering the physiological pre-existing fast enzymatic reactions regulated by native mGluR6. Also, such chimeric proteins will escape immune reactions, when extracellular domains of native photoreceptor proteins such as human melanopsin are used, because the only part accessible to the immune system will be identical to that of native human melanopsin.

"An advantage of using mGluR6 as the first GPCR member is that mGluR6 is expressed only in ON-bipolar cells in the retina. Therefore, transgenically expressed chimeric mGluR6-melanopsin will efficiently couple to the mGluR6 signaling cascade in ON bipolar cells only. Moreover, the degradation and modulation of the chimeric protein (e.g. arrestin binding) will occur through pre-existing mGluR6 pathways, allowing full self-control of protein activity.

"There is yet another particular effect of the expression of the chimeric light-sensitive mGluR6-melanopsin protein in ON bipolar cells to restore vision, which differs from other vision recovery methods: Visual contrast will actually be inverted; dark will appear bright and bright will appear dark. That is, neural circuits naturally activated by an increase in light intensity will be activated by a decrease in light intensity and vice versa. This in fact might have a key advantage over the prior art as outlined below:

"Photoreceptors release relatively high levels of their neurotransmitter (glutamate) in the dark and less transmitter as the brightness increases. The ON-bipolar cells receive their input through mGluR6 receptors, which hyperpolarize the bipolar cells when activated (in the dark) and vice versa. If there are no photoreceptors, there is no glutamate, the ON-bipolar cells are depolarized and the surviving inner retina is effectively in an 'extremely bright light' adaptive mode. In fact, the very slow degeneration of ON bipolar cells may be due to this sustained depolarization. Retinitis pigmentosa patients are not aware of the light adaptation of their retina, because the retinal output only signals spatial and temporal changes in light intensity. That is, if changes in intensity are not detected, the retina will effectively send no signal to the brain, although the retina is in the fully light adapted state.

"For improving vision in patients with partial or total loss of photoreceptor cells, it is important to take into consideration that the retina is in a fully light-adapted state. This implies that the ON-bipolar cells are permanently relatively depolarized. Channelrhodopsin-2 expressed in ON-bipolar cells will only depolarize these cells further and thus the signal difference between the light-ON and the light-OFF state is relatively small. In contrast, the ON-bipolar cells expressing the chimeric light-sensitive mGluR6-GPCR protein according to the invention are hyperpolarized by light. Evidently, this increases signal difference and thus enhances output and accordingly light sensitivity.


"FIG. 1: Schematic drawing showing the domains and orientation across the cell membrane of an embodiment of the light-sensitive chimeric GPRC protein with the N-terminus (NT), transmembrane domains (TM1-TM7) and extracellular loops 1-3 (EL1-EL3) from melanopsin and the intracellular loops 1-3 (IL1-IL3) and the C-terminus (CT) from mGluR6.

"FIG. 2: Example 1 Whole cell current responses of HEK293(GIRK) cells transfected with mouse mGluR6-melanopsin (IL2(DRIY), IL3(I) and CT from mGluR6, exemplary embodiment D with Seq. No. 7/8)--presently preferred sequence with biggest currents measured in HEK293 (GIRK) cells

"FIG. 3: Example 1: Outward K.sup.+ currents

"FIG. 4: Example 2: Successful and specific mGluR6-melanopsin transduction of mouse ON-bipolar cells using a rAAV2 capsid mutant vector

"FIG. 5: Light responses recorded from retinal ganglion cells in eight week old rd1 mouse retina (retina without photoreceptor cells), one month after introducing mGluR6-melanopsin into the retinal ON bipolar cells using a rAAV2 vector

"FIG. 6: Immunolabelling with the rabbit anti-Rab1A antibody shows that the dark-adapted retina of a blind rd1 mouse is in a light-adapted, depolarized state."

For more information, see this patent application: Van Wyk, Michiel; Kleinlogel, Sonja. Light-Sensitive Chimeric GPCR Protein. Filed June 22, 2012 and posted June 26, 2014. Patent URL:

Keywords for this news article include: Patents, Therapy, Peptides, Glutamates, Amino Acids, Ion Channels, Cell Membrane, Glutamic Acid, Retinal Neurons, Retinal Pigments, Biological Pigments, Cellular Structures, Photoreceptor Cells, Enzymes and Coenzymes, Information Technology, Membrane Glycoproteins, Sensory Receptor Cells, Membrane Transport Proteins.

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Source: Life Science Weekly

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