G-PROTEIN-GATED-K+ CHANNEL-MEDIATED ENHANCEMENTS IN LIGHT SENSITIVITY IN ROD-CONE DYSTROPHY (RCD)

Abstract

The present invention concerns a new gene therapy approach to increase light-sensitivity in degenerating cones in advanced stages of rod-cone dystrophy (RCD) mediated by G-protein-gated-K+ channel (GIRK), in particular GIRK2, activated by G proteins recruited by cone opsin expressed in degenerating cones.

Claims

1. A vector comprising a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a functional derivative thereof.

2. The vector according to claim 1, wherein the nucleotide sequence encoding GIRK2 or a derivative thereof comprises the sequence SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

3. The vector according to claim 1, wherein the vector is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, and an SV40 viral vector.

4. The vector according to claim 1, wherein the vector is an AAV2 or AVV9 virus comprising a 7 to 11 amino acid long insertion peptide in the GH loop of the VP1 capsid protein, wherein the insertion peptide comprises the amino acid sequence LGETTRP (SEQ ID NO: 7).

5. The vector according to claim 1, wherein the vector is a recombinant AAV9 vector comprising: a VP1 capsid protein wherein in a 7 to 11 amino acid long insertion peptide is inserted in the GH loop of said VP1 capsid protein relative to wild-type AAV9 VP1 capsid protein, at a position localized between amino acids 588 and 589 of wild-type AAV9 VP1 capsid protein, wherein said peptide comprises the amino acid sequence LGETTRP (SEQ ID NO: 7); and the nucleotide sequence encoding GIRK2 or a functional derivative thereof under the control of a pR1.7 promoter.

6. The vector according to claim 4, wherein said insertion peptide comprises or consists of the amino acid sequence AALGETTRPA (SEQ ID NO: 10), LALGETTRPA (SEQ ID NO: 11), or GLGETTRPA (SEQ ID NO: 12).

7. The vector according to claim 1, further comprising a nucleotide sequence encoding a mammalian cone opsin.

8. A carrier including the vector of claim 1.

9. The carrier of claim 8, further comprising a vector comprising a nucleotide sequence encoding a mammalian cone opsin.

10. The carrier according to claim 9, wherein the vector comprising a nucleotide sequence encoding a mammalian cone opsin: a) is selected from the group consisting of an adeno-associated virus (AAV), an adenovirus, a lentivirus, and SV40 viral vector; or b) is an AAV2 or AVV9 virus comprising a 7 to 11 amino acid long insertion peptide in the GH loop of the VP1 capsid protein, wherein the insertion peptide comprises the amino acid sequence LGETTRP (SEQ ID NO: 7); or c) is a recombinant AAV9 vector comprising: a VP1 capsid protein in a 7 to 11 amino acid long insertion peptide is inserted in the GH loop of said VP1 capsid protein relative to wild-type AAV9 VP1 capsid protein, at a position localized between amino acids 588 and 589 of wild-type AAV9 VP1 capsid protein, wherein said peptide comprises the amino acid sequence LGETTRP (SEQ ID NO: 7); and the nucleotide sequence encoding the mammalian cone opsin is under the control of a pR1.7 promoter.

11. The carrier according to claim 8, wherein the carrier is selected from the group consisting of solid-lipid nanoparticles, chitosan nanoparticles, liposomes, lipoplexes and cationic polymers.

12. The vector according to claim 7 or a carrier comprising the vector, wherein the mammalian cone opsin is a short wavelength cone opsin (SWO).

13. A pharmaceutical composition comprising the vector according to claim 1, or a carrier comprising the vector, and a pharmaceutically acceptable carrier, diluent or excipient.

14. A method of treating rod-cone dystrophy (RCD) in a subject in need thereof, comprising, administering to the subject a therapeutically effective amount of i) a nucleic acid comprising a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a functional derivative thereof, ii) a vector according to encoding the nucleic acid, iii) a carrier comprising the vector iv) a pharmaceutical composition comprising the nucleic acid, the vector or the carrier, or v) a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof.

15. The method according to claim 14, wherein the nucleic acid, vector, carrier or pharmaceutical composition is administered by subretinal injection at a distance of from the fovea.

16. The method according to claim 14, wherein the nucleic acid, vector, carrier or pharmaceutical composition is administered by subretinal injection a) in a region adjacent to a superior or inferior temporal branch of a retinal artery; b) at a distance of 2-3 optic disk diameters away from the center of the fovea; and c) at a position localized in a geometric shape delineated by the branches of a temporal retinal artery and a temporal retinal vein.

17. (canceled)

18. (canceled)

19. The method according to claim 14, wherein the nucleotide sequence of the nucleic acid comprises the sequence SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

20. (canceled)

21. The method according to claim 14, wherein the cone precursor cell is obtained from the RCD subject to be treated.

22. The method according to claim 14, wherein the cone precursor cell is obtained by differentiation induced pluripotent stem cells obtained from somatic cells of the RCD subject to be treated.

23. The according to claim 14, wherein the cone precursor cell is administered by subretinal space injection.

24. A method of preparing a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof, said method comprising infecting a cone precursor cell with a viral vector according to claim 1, or a carrier comprising the vector.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] FIG. 1 represents phototransduction cascade (A) normal phototransduction cascade (B) short phototransduction cascade with an animal opsin and GIRK2 channel. PDE: phosphodiesterase. CNG: cyclic-nucleotic gated channels. cGMP: cyclic guanosine monophosphate.

[0069] FIG. 2 represents alignments of GIRK2 (A) rat truncated GIRK2 vs mouse GIRK2 (B) mouse GIRK2 vs human GIRK2.

[0070] FIG. 3 represents plasmids (A) CMV-GIRK2-GFP and (B) CMV-SWO-mCherry.

[0071] FIG. 4 represents what remained in the phototransduction cascade in rd10 mice using immunohistochemistry (A-D) retinal cross-section of a control WT mouse stained with (A) opsin, (B) transducin, (C) PDE and (D) cone arrestin. (E-H) retinal cross-section of a rd10 mouse at P14 stained with (E) opsin, (F) transducin, (G) PDE and (H) cone arrestin. (I-L) Retinal cross-section of a rd10 mouse at P150 stained with (1) opsin, (J) transducin, (K) PDE and (L) cone arrestin. ONL: outer nuclear layer. INL: inner nuclear layer. GC: ganglion cells. Scale bar is 50 μm. Inset scale bar is 25 μm.

[0072] FIG. 5 represents preliminary data. (A) Eye fundus of GIRK2-GFP expression in rd10 mouse one week post-injection (*site ofinjection) (B) Photopic ERG amplitude in rd10 mice at P33, injected with AAV-SWO-tdTomato and AAV-GIRK2-GFP. Control mice are injected with AAV-GFP (n=12). P=0,0002. (C) Representative flickers ERG at P33. (D) Measure of the visual acuity by optokinetic test in rd10 mice, injected with AAV-SWO-tdTomato and AAV-GIRK2-GFP. Control mice are injected with AAV-GFP. Control mice were injected with AAV-GFP (n=8).

[0073] FIG. 6 represents GIRK2-mediated vision. (A) Photopic ERG amplitude in rd10 mice at P41, injected with AAV-SWO-tdTomato and/or AAV-GIRK2-GFP. Control mice are injected with AAV-GFP (n=12). P.sub.SWO+GIRK2=0.0381 and P.sub.GIRK2=0.0021. (B) Measure of the visual acuity by optokinetic test in rd10 mice, injected with AAV-SWO-tdTomato and/or AAV-GIRK2-GFP. Control mice are injected with AAV-GFP. Control mice were injected with AAV-GFP (n=7). (C) Representative flickers ERG at P41.

[0074] FIG. 7 represents long term efficiency. (A) Photopic ERG amplitude in rd10 mice, injected with AAV-GIRK2-GFP. Control mice are injected with PBS (n=6). (B) Measure of the visual acuity by optokinetic test in rd10 mice, injected with AAV-GIRK2-GFP. Control mice are injected with PBS (n=6). (C) Number of cones of wild-type mice and non-injected rd0 mice over time (n=6). Pvalue.sub.(P50-P365)=0.0022. (D) Linear regression correlation between the ERG amplitudes and the number of cones in rd10 mice (n=6). Pvalue.sub.non-injected=0.0482. Pvalue.sub.AAV-GIRK2-GFP=0,0007. Pvalue.sub.PBS=0.0104.

[0075] FIG. 8 represents what remained in the phototransduction cascade in huP347S.sup.+/− mice using immunohistochemistry. (A-D) Retinal cross-section of a control WT mouse stained with (A) opsin, (B) transducin, (C) PDE and (D) cone arrestin. (E-H) retinal cross-section of a huP347S.sup.+/− mouse at P14 stained with (E) opsin, (F) transducin, (G) PDE and (H) cone arrestin. (I-L) retinal cross-section of a huP347S.sup.+/− mouse at P150 stained with (1) opsin, (J) transducin, (K) PDE and (L) cone arrestin. ONL: outer nuclear layer. INL: inner nuclear layer. GC: ganglion cells. Scale bar is 50 μm. Inset scale bar is 25 μm.

[0076] FIG. 9 represents universality of the approach. (A) Photopic ERG amplitude in huP347S.sup.+/− mice, injected with AAV-GIRK2-GFP. Control mice are injected with PBS (n=6). (B) Measure of the visual acuity by optokinetic test in huP347S.sup.+/− mice, injected with AAV-GIRK2-GFP. Control mice are injected with PBS (n=6). (C) Number of cones of wild-type mice and non-injected huP347S.sup.+/− mice over time (n=6). Pvalue.sub.(P50-P365)=0.0022. (D) Linear regression correlation between the ERG amplitudes and the number of cones in huP347S.sup.+/− mice (n=5). Pvalue.sub.non-injected=0.0313. Pvalue.sub.AAV-GIRK2-GFP=0, 0146. Pvalue.sub.PBS=0.0497.

[0077] FIG. 10 represents the efficiency of the mouse GIRK2 in HEK cells transfected with two plasmids: CMV-SWO-mCherry and CMV-GIRK2-GFP.

[0078] FIG. 11 represents phenotyping of a normal volunteer and retinitis pigmentosa patients for eligible patient population. Upper panel (A) shows the fundus and OCT images of the back of the eye in a normal individual along with adaptive optics images of cone dominated regions of the retina. Middle panel (B) shows a pie-chart distribution of advanced RCD patients. Lower panel (C) represent OCT and AOSLO images of different patients. AOSLO confocal (up) and AOSLO split detection (low) in vivo retinal images of a patient with retinitis pigmentosa (age 77, male). Acquired fields are located at the transition between regions of presumed dormant cones (i.e. morphologically intact—as indicated by the IS/OS line visible in OCT, clear inner segment mosaic visible in AOSLO split detection, and clear cone mosaic indicating intact inner and outer segments in AOSLO confocal—though with reduced function according to the patient's visual acuity; yellow bars) and damaged or absent cones (indicated by the absent IS/OS line in OCT, and the blurred inner segment and cone mosaics in AOSLO split detection and confocal respectively; red bars). Arrows indicate cones that appear to be degenerating, with absent OS in confocal but present IS in split detection. Scale bars, 200 μm.

[0079] FIG. 12 represents immunohistochemistry labeling cone phototransduction cascade proteins in normal and RP human retina. (A) Retinal cross-section of a 86 years old control human retina (20×). (B) Retinal cross-section of a 75 years old human retina affected by retinitis pigmentosa (RP) and having night blindness and loss of peripheral vision (40×). (A-B) stained with Opn1mw, (bright grey) and nuclear stain DAPI (dark grey). ONL: outer nuclear layer. INL: inner nuclear layer. GC: ganglion cells. Scale bar is 50 μm. Inset scale bar is 25 μm.

[0080] FIG. 13: Localization of subretinal injection sites to deliver the AAV solution under the retina, close to the fovea but without foveal detachment.

EXAMPLES

Example 1: Material and Methods

[0081] 1. Animals

[0082] C57BL/6j.sup.rd10/rd10 (rd10) mice were used in these experiments. They have a mutation on the rod PDE gene leading to a dysfunctional phototransduction cascade and a rod-cone dystrophy. The second model used is the huRhoP347S.sup.+/− mouse. The homozygous strand of this mouse present a KO of mouse rhodopsin (mRho) gene and a KI of human rhodopsin (huRho) with a mutation (P347S) (Millington-Ward et al., 2011) [30]. The homozygous males were crossed C57BL/6j (wild-type) females to obtain heterozygous mice. These mice have a similar phenotype as the rd10 mice but the degeneration rate is lower.

[0083] 2. AAV Injections

[0084] Mice were first anesthetised with intraperitoneal injections of 0.2 ml/20 g ketamine (Ketamine 500, Vibrac France) and xylazine (Xylazine 2%, Rompun) diluted in 0.9% NaCl. Eyes were dilated with 8% Neosynephrine (Neosynephrine Faure 10%, Europhta) and 42% Mydriaticum (Mydriaticum 0.5%, Thea) diluted in 0.9% NaCl.

[0085] A total volume of 1 μl of vector solution was injected subretinally. Fradexam, an ophthalmic ointment, was applied after injection. The list of injected viral vectors is presented below:

TABLE-US-00005 Injection Table Mice Eyes viral vector injected viral vector titration volume injected rd10 both AAV8-mCAR-GFP 10.sup.11 rAAV 1 μl for all particles conditions rd10 both AAV8-mCAR-GIRK2-GFP + AAV8-mCAR-SWO-tdTomato rd10 both AAV8-mCAR-GIRK2-GFP rd10 right PBS rd10 left AAV8-PR1.7-GIRK2-GFP 5.10.sup.8 rAAV particles huRhoP347S.sup.+/− right PBS huRhoP347S.sup.+/− left AAV8-PR1.7-GIRK2-GFP 5.10.sup.8 rAAV particles

[0086] 3. Eye Fundus Examination

[0087] One week after subretinal injection, mice were anesthetised by isofluorane inhalation. Eyes were dilated and then protected with Lubrithal eye gel (VetXX). Fundus imaging was performed with a fundus camera (Micron III; Phoenix research Lab) equipped with specific filters to monitor GFP or tdTomato expression in live anesthetised mice.

[0088] 4. Electroretinography (ERG) Recordings

[0089] To evaluate retinal function, electroretinography recordings (ERG) were recorded (espion E2 ERG system; Diagnosys). Several tests were performed at different time points after injections of the viral vectors. Mice were anesthetised with intraperitoneal injections of 0.2 ml/20 g ketamine (Ketamine 500, Vibrac France) and xylazine (Xylasine 2%, Rompun) diluted in 0.9% NaCl. Mice were then placed on a heated pad at 37° C. Eyes were dilated with Neosyhephrine (Neosynephrine Faure 10%, Europhta) and Mydriaticum (Mydriaticum 0.5%, Thea) diluted in 0.9% NaCl. Eyes were protected with Lubrithal eye gel before putting electrodes on the corneal surface of each eye. The reference electrode was inserted under the skin into the forehead and a ground electrode under the skin in the back.

[0090] ERG recordings were done under two conditions: (i) photopic condition, which reflects con-driven light responses—6 ms light flashes were applied every second during 60 seconds at increasing light intensities (0.1/1/10/50cd s/m) after an adaptation of 5 minutes at 20cd s/m—and (ii) flicker condition, which are rapid frequency light stimuli that reflect cone function (70 flashes at 10 Hz et 1 cd s/m).

[0091] Graph and statistical analysis were performed using GraphPad.

[0092] 5. Optokinetic Test

[0093] Visual acuity was measured using an optokinetic test scoring the head turning movement of a mouse placed in front of moving bars. Testing was performed using a computer-based machine consisting of four computer monitors arranged in a square to form an optokinetic chamber. A computer program was designated to generate the optokinetic stimuli, consisting of moving alternate black and white stripes. The spatial frequency is ranging from 0.03 to 0.6 cyc/deg. The program enabled modulation of stripe width and direction of bar movement.

[0094] 6. Immunohistochemistry and Confocal Imaging

[0095] Animals were sacrificed by CO.sub.2 inhalation, and the eyes were enucleated and fixed in 4% paraformaldehyde-PBS for 1 h at room temperature. The eyes were dissected either as eyecups for immunohistochemistry or prepared as flat mounts for cell counting. The eyecups were then cryoprotected with a gradient of PBS-Sucrose 10% for 1 h and then in PBS-Sucrose 30% overnight. The eyecups were embedded in OCT and 12 μm thick cryostat sections (ThermoFisher) were cut and mounted on glass slides. The sections were washed in PBS (3×5 mins) and stained against different antibodies (see table below) and DAPI (1:2000). The sections were finally washed in PBS, mounted in Fluoromount Vactashield (Vector Laboratories) and coverslipped for imaging using laser-confocal microscopy (Olympus IX81). For flat-mount retina stainings, the protocol is the same except that the tissue was not cryoprotected. Images were analysed using FIJI software.

TABLE-US-00006 Antibody table Target Host Clonality/Conjugated PRIMARY ANTIBODIES Red/Green opsin (M/L opsin) Rabbit Polyclonal Mouse Cone arrestin Rabbit Polyclonal PDE6C Rabbit Polyclonal GNAT2 Rabbit Polyclonal PNA-Lectin Conjugated with FITC SECONDARY ANTIBODIES Anti-rabbit Donkey AF 546

[0096] 7. Cell Counts

[0097] Flat mount retinas of rd10 and huRhoP347S.sup.+/− mice were stained using antibodies against mouse cone arrestin—mCAR (1:10000) and DAPI (1:2000). The double stained cells counted at different ages. Retinas from 5 animals (n=10) were used for each age and were oriented dorso-ventrally and naso-temporally. Serial optical sections were obtained to cover the thickness of the entire outer nuclear layer (ONL). Two scanning areas of 211.97×211.97 μm were made in each of the four regions in all retinas. Counts of cone cells were performed manually using the FIJI software by the reconstruction of the images (z stack) covering the entire thickness of the ONL. Average density values of each retina were calculated to obtain the number of cone cells per mm.sup.2 at different ages.

[0098] 8. In Vitro Test of the Efficiency of Mouse GIRK2

[0099] HEK cells were transfected with two plasmids: CMV-SWO-mCherry and CMV-GIRK2-GFP (FIG. 3) according to a well-known procedure in the art. HEK293 cells were cultured and recorded in dark room conditions after transfection. Cells were placed in the recording chamber of a microscope equipped with a 25× water immersion objective (XLPlanN-25×-W-MP/NA1.05, Olympus) at 36° C. in oxygenated (95% O2/5% CO2) Ames medium (Sigma-Aldrich) enriched with an addition of 1 mM9-cis-retinal. KGluconate was added to the external solution in order to get a high extracellular potassium concentration leading to a cell potassium reversal potential of −40 mV.

[0100] For Whole-cell recordings, the Axon Multiclamp 700B amplifier (Molecular Device Cellular Neurosciences) was used, GIRK-mediated K+-currents were recorded in voltage-clamp configuration at −80 mV, using borosilicate glass pipettes (BF100-50-10, Sutter Instrument) pulled to 5MΩ and filled with 115 mMK Gluconate, 10 mM KCl, 1 mM MgCIl, 0.5 mM CaCl2, 1.5 mM EGTA, 10 mM HEPES, and 4 mM ATP-Na2 (pH 7.2).

[0101] During experiments, a CCD camera (Hamamatsu Corp.) was used to visualize cells using a trans-illuminated infrared-light. A monochromatic light source (Polychrome V, TILL photonics) was used to stimulate cells during electrophysiological experiments with light flashes at 400 nm.

[0102] 9. Patient Eye Fundus Imaging

[0103] Adaptive optics scanning laser ophthalmoscopy (AOSLO) (Roorda et al., 2002) [60] was used to image cone photoreceptor mosaic at cell resolution. The AOSLO device used (MAORI, PSI, Andover, Mass., USA) allows simultaneous imaging over a 2-degree field of view of intact cones with both inner and outer segments (IS, OS) from light scattered along the optical axis (confocal mode) and inner segments (IS) from multiply scattered light scattered off axis (split detection mode). This allows us to evaluate cone presence and health, with differential imaging of IS versus IS+OS for each cone.

Example 2: Results

1. The Changes in the Phototransduction Cascade in Degenerating Cones

[0104] The phototransduction cascade was first analysed in the rd10 mouse model by studying its components using immunohistochemistry, at different time points during retinal degeneration. Immunofluorescence staining was performed against cone opsin, transducing, phosphodiesterase and cone arrestin proteins of the phototransduction cascade that interact directly with cone opsin.

[0105] FIG. 4 shows that only the cone opsin and arrestin were still expressed and localized around the cone cell body at late stage of the disease.

2. Cone Opsin and GIRK2-Mediated Vision Restoration

[0106] Based on immunohistochemistry and previous findings with cone opsins expressed in neurons, it was first studied why delivering a mouse short wavelength cone opsin (SWO) fused with tdTomato and GIRK2 fused with GFP using two AAV vectors mixed in equimolar ratios would enhance cone cell's response to light. Thus two AAVs were injected subretinally to degenerating rd10 mouse retinas at p15 (FIG. 5A). This led to a significant increase in phototpic ERG amplitudes in treated eyes compared to controls (FIG. 5B). Flicker ERGs confirmed that the recovery mechanism was still active in these cone cells expressing GIRK2 allowing them to follow a fast stimulus (FIG. 5C). The rd10 animals treated with GIRK2 showed also an improved optokinetic reflex compared to controls (FIG. 5D).

[0107] Next it was studied if the endogenous cone opsin, still present in degenerating cones, was functional and sufficient to activate GIRK2 channel in this mouse model. For this, a single AAV8 vector encoding GIRK2 in fusion with GFP was delivered. This led to similar increases in photopic ERG amplitudes and optokinetic reflex in treated eyes compared to controls confirming that GIRK2 alone was sufficient to increase light sensitivity via G protein coupled signalling involving cone opsin (FIG. 6A-B). Flicker ERGs were also robustly amplified with this approach (FIG. 6C).

3. GIRK2-Mediated Vision Restoration: Long-Term Efficacy

[0108] Photopic ERG recordings were performed to monitor the cone response to light stimuli at different time points after treatment with GIRK2 and in absence of treatment. These ERGs were done under two conditions: (i) photopic with light flashes applied every second during 60 seconds at increasing light intensities and (ii) flicker stimulation with repetitive flashes during 60 seconds. Data were collected on a weekly basis until p50 and then every 10 to 13 days until 11 weeks of age and showed a gradual decline in ERG amplitudes for both controls and treated eyes (FIG. 7A). Moreover, these results are consistent with the optokinetic test, both controls and treated eyes with GIRK2 show a decreased optokinetic reflex over time (FIG. 7B). This decline was to be expected as cone numbers also decreased over time in the rd10 mice (FIG. 7C). The number of cone photoreceptors remaining in rd10 retinas were counted to correlate decreases in cone numbers with decrease in ERG amplitudes. Indeed, the decrease in light responses was proportional to number of remaining photoreceptors (FIG. 7D). It was thus concluded that GIRK2 increased light responses in remaining cones so long as cones remained alive but, as expected, it did not slow down the loss of cone cells.

4. GIRK2-Mediated Vision Restoration in an RCD Model Caused by Mutant Rhodopsin

[0109] Having in mind the goal of creating a mutation-independent therapy, the approach was tested in another mouse model—with a different causal mutation. To this aim experiments were done in a heterozygous mouse model called mRho.sup.−/−-huRhoP347S.sup.+/− carrying a knock in for P347S mutant human rhodopsin. Mutant human rhodopsin and absence of mouse rhodopsin led to a rod-cone dystrophy in this complementary model. Here, the same set of experiments was repeated as that was done in the rd10 mouse model. First, the phototransduction cascade proteins interacting with cone opsin at different time points was analysed (FIG. 8). It was noticed that: (i) the degeneration rate was slower compared to rd10 and (ii) similar to the rd10 model only the opsin and the arrestin persisted in cone cell bodies at P150.

[0110] Next, mice were injected at P15 with the same AAV vectors encoding for GIRK2 fused with GFP and recorded ERGs to monitor cone response to light stimuli at various time points (FIG. 9A). The response amplitudes of treated eyes were significantly higher than that of control eyes until P100. Moreover, flicker ERG responses were also similarly improved in this mouse model. Similarly to rd10 mice, this mouse model also shows an improved optokinetic reflex that decreases over time in both control and treated conditions (FIG. 9B). This decline is to be expected as cone numbers also decreases over time in this RCD mouse model (FIG. 9C). The decrease in time in ERG amplitudes also correlated with a decrease in cone numbers in this model (FIG. 9D). This was again consistent with the fact that the approach did not stop the degeneration but allowed for enhanced light sensitivity via GIRK2.

5. Efficiency of the Mouse GIRK2 in an In Vitro Test

[0111] Light stimulations (400 nm, 5 seconds, fullfield) activated GIRK currents in HEK cells expressing both GIRK and SWO (Short Wavelength Opsin) (FIG. 10). GIRK channels are modulated in a membrane-delimited, fast manner via the Gi/o pathway and the expression of the mouse GIRK channel was membrane bound. The amplitudes and kinetics of light-induced activation and deactivation of GIRK channels with SWO, induce large GIRK current amplitudes during a 5 s light pulse.

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