Patent Publication Number: US-2023159609-A1

Title: G-protein-gated-k+ channel-mediated enhancements in light sensitivity in rod-cone dystrophy (rcd)

Description:
TECHNICAL FIELD OF THE INVENTION 
     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. 
     In the description below, references in square brackets ([ ]) refer to the list of references at the end of the text. 
     STATE OF THE ART 
     Retina is the light sensitive tissue of the eye composed of three layers of neurons interconnected by synapses. The primary neurons of the retina are the light-sensing photoreceptors (PR), which are of two types: the rods for night vision and the cones for daylight vision. Cone-mediated vision is mostly supported by the fovea and is responsible for high acuity central vision most valuable to our daily visual tasks (Sinha et al., 2017) [1]. The light sensitive G protein coupled receptors that link photon capture to intracellular signaling leading to membrane hyperpolarization in photoreceptors are called opsins (Yau and Hardie, 2009) [2]. There is one type of rod opsin found in rods and three types of cone opsins—responsible for trichromatic vision—in the primate retina. The structural properties and phototransduction cascades are similar between these opsins. 
     The phototransduction cascade is composed of several proteins that are concentrated in the photoreceptor outer-segments in normal retinas ( FIG.  1 A ). The role of the photoreceptor is to sense light via this phototransduction cascade and induce an electrical signal that is then processed and transmitted towards downstream neurons (Ebrey and Koutalos, 2001) [3]. 
     The absorption of a photon activates the opsin composed of two parts: the protein part, and the light absorbing part, which is the retinal—a derivative of vitamin A. The latter isomerizes from 11-cis-retinal (dark adapted state) into all-trans-retinal configuration (light adapted state). As a result, the opsin becomes catalytically active recruiting the G protein transducin. The α-subunit of transducin is activated by the replacement of GDP by GTP. After, the α-subunit dissociates from the βγ-subunits to activate the membrane-associated phosphodiesterase 6 (PDE) by binding its two inhibitory γ subunits. The activated PDE hydrolyses cGMP into GMP. The reduction of cGMP clones the nucleotide-gated channels (CNG) and this stops cation entry, resulting in PR hyperpolarization and reduction in glutamate release by the photoreceptor (Larhammar et al., 2009) [4]. 
     In order to respond to another photon, this phototransduction cascade is deactivated by two mechanisms: (i) the transducin inactivates itself by hydrolyzing the bound GTP and (ii) the rhodopsin kinase (GRK) phosphorylates the opsin that interacts with the regulatory protein arrestin, leading to opsin inactivation. Retinal is then recycled by the retinal pigment epithelium (RPE) and Müller glial cells. Each and every protein of this cascade plays an important role in converting the light signal into an electrical signal conveyed to the second and third order neurons (Maeda et al., 2003) [5]. 
     Inherited retinal degenerations are mostly due to mutations in photoreceptor or RPE cells leading to the degeneration of the rods, followed by cone outer segment degeneration eventually leading to blindness (Buch et al., 2004) [6]. Among them, rod-cone dystrophy (RCD) represents the largest category where genetic causes are highly heterogeneous. More than 60 different genes expressed in rod photoreceptors or the retinal pigment epithelium are involved (Wright et al., 2010) [7]. The first gene that has been linked to RCD is the rhodopsin gene RHO that counts for 25% of RCD autosomal dominant cases. Many other causative genes have also been identified: the cGMP-PDE subunit gene and the cyclic GMP gated channel protein a subunit gene. Although there are many causative genes, the resulting RCD phenotype is the same across different mutations (Ferrari et al., 2011) [8]. The common RCD phenotype is characterized by the progressive rod degeneration, causing night blindness, and followed by progressive peripheral cone degeneration, causing “tunnel vision”, mediated entirely by the remaining foveal cones then eventually resulting in complete blindness in the latest stages of disease. Usually, when patients are diagnosed with RCD, they already show night blindness, meaning their rods have degenerated. However, the cones remain until the late stages of the disease; particularly in the foveal region responsible for high acuity leading to tunnel vision in early stages (Li et al., 1995) [9]. In later stages of the disease, these cones lose their outer segment structures leading to complete blindness before the complete loss of the cone soma and pedicle (Li et al., 1995) [9]. 
     In order to preserve the vision in these patients presenting light sensitive cone cell bodies, one innovative strategy is retinal gene therapy, which broadly refers to the transfer of a therapeutic gene into retinal cells to mediate a therapeutic effect (Bennet, 2017) [10]. Although the first successful clinical trials of gene therapy have focused on gene replacement, where a gene carrying a recessive mutation is replaced by a functional cDNA copy, this strategy is limited because it cannot be used for the majority of retinal degenerations (Bennet, 2017) [10]. For example, in RCD, the huge variability of mutations makes it difficult to apply to each specific mutation. Moreover, dominant mutations cannot be treated using this approach. Furthermore, in 50% of cases, the causative mutation is not elucidated or the rod photoreceptors bearing the most frequent mutations are already lost (Dalkara et al., 2015) [11]. For these reasons, mutation-independent gene therapies that can be applied beyond the loss of rods must be developed to treat a large number of patients without knowledge of the mutant gene. 
     Taking this goal into account, previous studies used the ectopic expression of microbial opsins, like channelrhodopsin in bipolar cells or halorhodopsin in cones PRs, to modulate membrane potential and induce a depolarization or hyperpolarization respectively (Busskamp et al., 2012; Dalkara and Sahel, 2014; Scholl et al., 2016) [12-14]. Thus, in RCD, optogenetics can be used as a therapeutic strategy to restore vision in blind retinas (Baker and Flannery, 2008) [15]. There are many parameters to consider: (i) the choice of cell target to make the most out of the retinal circuitry sending the most interpretable signal to the brain (ii) the choice of the appropriate optogenetic tool to obtain a close to natural electrophysiological response in these target cells. Concerning the second point, the relatively weak capacity of microbial opsins has been a major limitation: potential immunogenicity of using an opsin from prokaryotic species, high intensities required due to the lack of signal amplification by these directly light gated channels and pumps originating from single cell microorganisms, as in the case of halorhodopsin (Baker and Flannery, 2008) [15], (Cehajic-Kapetanovic et al., 2015; Gaub et al., 2015; Van Gelder and Kaur, 2015; van Wyk et al., 2015) [16-19]. Unlike rhodopsins or cone opsins, microbial opsins are not able to activate G protein coupled cascades such as the phototransduction cascade present in healthy retinas. One of the possible ways to go beyond the limits of microbial opsins is the use of animal opsins, which are all G protein coupled receptors. However all work in this field has so far been focused on inner retinal neurons (Berry et al., 2019; Cehajic-Kapetanovic et al., 2015; De Silva et al., 2017; Gaub et al., 2015; Lin et al., 2008; van Wyk et al., 2015) [20, 16, 21, 17, 22, 19]. 
     In a previous study, it has been shown that light-activation of two animal cone opsins can stimulate the G i/o  signalling pathways in human kidney cells and in neuronal cells in vitro and in vivo (Masseck et al., 2014) [23]. This pathway is involved in fast dampening of neuronal activity and inhibition of intrinsic ion channels. However, it has also been shown that animal cone opsins can directly modulate the G protein-gated inwardly rectifying potassium channels (GIRK) upon co-expression in any given cell (Berry et al., 2019; Masseck et al., 2014) [20, 23]. GIRK channels are composed of two subunits. There are four types of subunits: GIRK1 to 4. GIRK1 and 3 cannot form homotetramers; they have to be associated with GIRK2 to be functional (Mark and Herlitze, 2000) [24]. Conversely, GIRK2 alone can form homotetramers. The GIRK channel is predominantly closed at resting membrane potentials. After its activation by the βγ subunit of a G i/o  protein, potassium ions flow out of the cell, thus, hyperpolarizing the neuron ( FIG.  1 B ). It has therefore been possible to use vertebrate cone opsins SWO (for short wavelength opsin) and LWO (for long wavelength opsin) for repetitive G i/o  activation of a specific wavelength in vivo in the anxiety circuitry, and the combination of cone opsins with GIRK has proven more efficient that microbial opsins at low light intensities (Masseck et al., 2014) [23]. 
     DESCRIPTION OF THE INVENTION 
     The Inventors have thus investigated if it was possible to implement this cone opsin based system in a vision restoration setting and investigated patient retinas in preparation for clinical candidate selection. Therefore in order to develop a light-sensitive cone reactivation strategy, the expression of the phototransduction cascade elements in cones during degeneration was first examined in two RCD mouse models. After exploring the phosphotransduction cascade in two RCD mouse models, a target molecule approach acting via G i/o  proteins recruited by the activation of remaining cone opsin was proposed for the first time. It has thereby created a new “short phototransduction cascade” that is independent from the expression of PDE and transducin. 
     First, the state of the endogeneous phototransduction cascade in degenerating cones through the progression of disease was investigated. In both mouse models, only opsin and arrestin were found to migrate to the cone cell bodies after outer segment degeneration. Thus it was hypothesized that cone reactivation based on cone opsin signaling may be feasible, which in turn will allow to recover high sensitivity vision. It was found that endogenous cone opsins were still expressed at the level of the cone cell body in rd10 mouse model ( FIG.  4   ) suggesting the possibility of linking their activity to GIRK channels, in particular GIRK2 channel, even in absence of transducin and phosphodiesterase (Figure is 1B). In this configuration, the opening of GIRK2 channel will allow the exit of potassium ions due to the resting membrane potential of dormant cones (Busskamp, 2010) [25]. K +  efflux via the GIRK2 channel will hyperpolarize the cones in response to light as it was seen in the two mouse models of RCD. 
     Next, the target GIRK2 channel activated by G proteins recruited by cone opsin was expressed in degenerating cones. Moreover, since the remaining opsin in the cone cell bodies is still functional and sufficient to induce a light response in the degenerated cones, the insertion of GIRK2 in all cones leads to light responses following the spectral properties of each of the opsins preserving color vision. The results pointed towards enhanced light-sensitivity in cones of RCD retinas during and after degeneration of cone outer-segments. The results thus demonstrated that vertebrate light sensitive proteins combined with GIRK channel, in particular GIRK2 channel, activated by an endogenous G protein, could improve the visual function in the two mouse models, as demonstrated by electroretinography and behaviour. This is the first time that a light insensitive mammalian ion channel has been linked to intrinsic opsins providing new avenues in vision restoration that can be implemented to increase light sensitivity even before complete outer segment degeneration. Since this new system makes use of intrinsic opsins expressed in degenerating cones, it also enables for the first time, color vision restoration/maintenance. 
     Similarly to the RCD mouse models, the cone opsin and the cone arrestin remain in the cone cell bodies of RP human patients ( FIG.  13   ). This result demonstrates the feasibility of reactivating the cone function in the foveal region of RCD human patients with the short GIRK2/opsin phototransduction cascade. The activation of the remaining cone opsin by a light stimulus will trigger the short phototransduction cascade and lead to vision restoration in RCD patients, even at an intermediate or advanced stage of the disease. 
     This new approach has thus the potential to maintain and/or restore, high acuity and color vision requiring only low light intensities in human patients. 
     A clear advantage of microbial opsins is their robustness and millisecond scale kinetics (Packer et al., 2013) [26]. For systems using other opsins, it should be considered that in order to respond to another light stimulus, the cascade has to be deactivated to recover light sensitivity. In absence of this, cones may stay hyperpolarized after GIRK2 channel activation limiting their ability to modulate synaptic transmission at a movie rate compatible with motion vision. In the present approach, depolarization of the cones was made possible thanks to the arrestin that is still maintained at very late stages of the disease in both RCD models—essential point which was not known in the art before the present invention. This was noticeable in the flicker ERG traces showing responses of the retina during repetitive light stimuli and also by the improved optokinetic reflex of treated mice. 
     Lastly, the fact that incorporation of GIRK2 enhances existing light responses in cones even prior to complete outer segment loss offers the possibility of implementing this gene therapy in mid stages of the disease. Retinal degeneration in mice is much faster than in humans, thus a few days of therapeutic efficiency in mice is equal to several years in humans. Nonetheless, even when endogenous light responses disappear, retinas expressing GIRK2 are still able to respond to light, generating response amplitudes that correspond to remaining cone numbers. This suggests that patients with remaining foveal cones can benefit from this treatment even if they have no detectable light perception at the beginning of treatment ( FIG.  11   ). Thus this approach can be used as long as cone cells remain. Indeed a decrease in the response of treated cones to light stimuli was recorded, which was consistent with decrease in cone numbers and the fact that we did not transduce all cones due to subretinal injection further limited the beneficial effect. AAV vectors showing better lateral spread can be used to increase transduced cone numbers beyond the bleb (Khabou et al., 2018; International application WO 2018134168) [27, 28]. In order to increase the therapeutic window, neurotrophic factors can be implemented alongside the approach of the present invention. Indeed, AAV-mediated secretion of neurotrophic factors such as the rod-derived cone viability factor (RdCVF) have been shown to delay cone cell death and may be combined with GIRK2 mediated sensitization (Byrne et al., 2015) [29]. 
     An object of the present invention is therefore a vector comprising a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a functional derivative thereof. The vector of the present invention can further comprise a nucleotide sequence encoding a mammalian cone opsin. For example, the mammalian cone opsin is a short wavelength cone opsin (SWO), e.g. from  Mus musculus  or human cone opsin. Where present in the same vector, the nucleotide sequence encoding GIRK2 or a functional derivative thereof, and the nucleotide sequence encoding a mammalian cone opsin are preferably under the control of a same promoter, in particular a cone-specific promoter such as pR1.7 or a functional variant thereof, or minimal M-opsin promoter, in particular in a pMNTC expression cassette. 
     For the purposes of the present invention, “a [GIRK2] functional derivative thereof” means a nucleotide sequence encoding an isoform or variant of GIRK2 which differs by only a few nucleotides compared to the WT form (e.g. mouse) or a nucleotide sequence encoding a truncated GIRK2 ( FIG.  2   ), but all of which retain the ability to respond to light when co-expressed with an opsin. For example, a nucleotide sequence encoding GIRK2 or a derivative thereof comprises or consists of the nucleotide sequence SEQ ID NOs: 1, 3 or 5. SEQ ID NO: 4, the polypeptide encoded by SEQ ID NO: 3, comprises a mutation VL to AA at positions 13-14 of the polypeptide sequence which leads to increased cell surface expression of the GIRK2 variant compared to wild-type GIRK2 (Ma et al., 2002) [31]. 
     
       
         
           
               
             
               
                   
               
             
            
               
                 Human GIRK2 
               
            
           
           
               
               
               
            
               
                   
                 Nucleotide 
                 ATGGCCAAGCTGACAGAATCCATGA 
               
               
                   
                 sequence 
                 CTAACGTCCTGGAGGGCGACTCCAT 
               
               
                   
                   
                 GGATCAGGACGTCGAAAGCCCAGTG 
               
               
                   
                   
                 GCCATTCACCAGCCAAAGTTGCCTA 
               
               
                   
                   
                 AGCAGGCCAGGGATGACCTGCCAAG 
               
               
                   
                   
                 ACACATCAGCCGAGATCGGACCAAA 
               
               
                   
                   
                 AGGAAAATCCAGAGGTACGTGAGGA 
               
               
                   
                   
                 AAGACGGAAAGTGCAATGTTCATCA 
               
               
                   
                   
                 CGGCAACGTGAGGGAGACCTATCGC 
               
               
                   
                   
                 TACCTGACCGATATCTTCACCACAT 
               
               
                   
                   
                 TAGTGGACCTGAAGTGGAGATTCAA 
               
               
                   
                   
                 CCTATTGATTTTTGTCATGGTTTAC 
               
               
                   
                   
                 ACAGTGACCTGGCTCTTTTTTGGAA 
               
               
                   
                   
                 TGATCTGGTGGTTGATCGCATACAT 
               
               
                   
                   
                 ACGGGGAGACATGGACCACATAGAG 
               
               
                   
                   
                 GACCCCTCCTGGACTCCTTGTGTTA 
               
               
                   
                   
                 CCAACCTCAACGGGTTCGTCTCTGC 
               
               
                   
                   
                 TTTTTTATTCTCAATAGAGACAGAA 
               
               
                   
                   
                 ACCACCATTGGTTATGGCTACCGGG 
               
               
                   
                   
                 TCATCACAGATAAATGCCCAGAGGG 
               
               
                   
                   
                 AATTATTCTTCTCTTAATCCAATCT 
               
               
                   
                   
                 GTGTTGGGGTCCATTGTCAATGCAT 
               
               
                   
                   
                 TCATGGTGGGATGCATGTTTGTAAA 
               
               
                   
                   
                 AATCTCTCAACCCAAGAAGAGGGCA 
               
               
                   
                   
                 GAGACCCTGGTCTTTTCCACCCATG 
               
               
                   
                   
                 CAGTGATCTCCATGCGGGATGGGAA 
               
               
                   
                   
                 ACTGTGCCTGATGTTCCGGGTAGGG 
               
               
                   
                   
                 GACCTTAGGAATTCCCACATTGTGG 
               
               
                   
                   
                 AGGCTTCCATCAGAGCCAAGTTGAT 
               
               
                   
                   
                 CAAATCCAAACAGACCTCGGAGGGG 
               
               
                   
                   
                 GAGTTCATCCCGTTGAACCAGACGG 
               
               
                   
                   
                 ATATCAACGTAGGGTATTACACGGG 
               
               
                   
                   
                 GGATGACCGTCTGTTTCTGGTGTCA 
               
               
                   
                   
                 CCGCTGATCATTAGCCATGAAATTA 
               
               
                   
                   
                 ACCAACAGAGTCCTTTCTGGGAGAT 
               
               
                   
                   
                 CTCCAAAGCCCAGCTGCCCAAAGAG 
               
               
                   
                   
                 GAACTGGAAATTGTGGTCATCCTAG 
               
               
                   
                   
                 AAGGAATGGTGGAAGCCACAGGGAT 
               
               
                   
                   
                 GACATGCCAAGCTCGAAGCTCCTAC 
               
               
                   
                   
                 ATCACCAGTGAGATCCTGTGGGGTT 
               
               
                   
                   
                 ACCGGTTCACACCTGTCCTGACCCT 
               
               
                   
                   
                 GGAGGACGGGTTCTACGAAGTTGAC 
               
               
                   
                   
                 TACAACAGCTTCCATGAGACCTATG 
               
               
                   
                   
                 AGACCAGCACCCCATCCCTTAGTGC 
               
               
                   
                   
                 CAAAGAGCTGGCCGAGTTAGCCAGC 
               
               
                   
                   
                 AGGGCAGAGCTGCCCCTGAGTTGGT 
               
               
                   
                   
                 CTGTATCCAGCAAACTCAACCAACA 
               
               
                   
                   
                 TGCAGAACTGGAGACTGAAGAGGAA 
               
               
                   
                   
                 GAAAAGAACCTCGAAGAGCAAACAG 
               
               
                   
                   
                 AAAGAAATGGTGATGTGGCAAACCT 
               
               
                   
                   
                 GGAGAATGAATCCAAAGTT 
               
               
                   
                   
                 (SEQ ID NO: 1) 
               
               
                   
                   
               
               
                   
                 Amino acid 
                 MAKLTESMTNVLEGDSMDQDVESPV 
               
               
                   
                 sequence 
                 AIHQPKLPKQARDDLPRHISRDRTK 
               
               
                   
                 (423Aa) 
                 RKIQRYVRKDGKCNVHHGNVRETYR 
               
               
                   
                   
                 YLTDIFTTLVDLKWRFNLLIFVMVY 
               
               
                   
                   
                 TVTWLFFGMIWWLIAYIRGDMDHIE 
               
               
                   
                   
                 DPSWTPCVTNLNGFVSAFLFSIETE 
               
               
                   
                   
                 TTIGYGYRVITDKCPEGIILLLIQS 
               
               
                   
                   
                 VLGSIVNAFMVGCMFVKISQPKKRA 
               
               
                   
                   
                 ETLVFSTHAVISMRDGKLCLMFRVG 
               
               
                   
                   
                 DLRNSHIVEASIRAKLIKSKQTSEG 
               
               
                   
                   
                 EFIPLNQTDINVGYYTGDDRLFLVS 
               
               
                   
                   
                 PLIISHEINQQSPFWEISKAQLPKE 
               
               
                   
                   
                 ELEIVVILEGMVEATGMTCQARSSY 
               
               
                   
                   
                 ITSEILWGYRFTPVLTLEDGFYEVD 
               
               
                   
                   
                 YNSFHETYETSTPSLSAKELAELAS 
               
               
                   
                   
                 RAELPLSWSVSSKLNQHAELETEEE 
               
               
                   
                   
                 EKNLEEQTERNGDVANLENESKV 
               
               
                   
                   
                 (SEQ ID NO: 2) 
               
               
                   
                   
               
            
           
           
               
            
               
                 Mouse GIRK2 
               
            
           
           
               
               
               
            
               
                   
                 Nucleotide 
                 ATGACAATGGCCAAGTTAACTGAAT 
               
               
                   
                 sequence 
                 CCATGACTAACGCCGCCGAAGGCGA 
               
               
                   
                   
                 TTCCATGGACCAGGATGTGGAAAGC 
               
               
                   
                   
                 CCAGTGGCCATTCACCAGCCAAAGT 
               
               
                   
                   
                 TGCCTAAGCAGGCCAGGGACGACCT 
               
               
                   
                   
                 GCCGAGACACATCAGCCGAGACAGG 
               
               
                   
                   
                 ACCAAAAGGAAAATCCAGAGGTACG 
               
               
                   
                   
                 TGAGGAAGGATGGGAAGTGCAACGT 
               
               
                   
                   
                 TCACCACGGCAATGTGCGGGAGACG 
               
               
                   
                   
                 TACCGATACCTGACGGACATCTTCA 
               
               
                   
                   
                 CCACCCTGGTGGACCTGAAGTGGAG 
               
               
                   
                   
                 ATTCAACCTGTTGATCTTTGTCATG 
               
               
                   
                   
                 GTCTACACAGTGACGTGGCTTTTCT 
               
               
                   
                   
                 TTGGGATGATCTGGTGGCTGATTGC 
               
               
                   
                   
                 GTACATCCGGGGAGATATGGACCAC 
               
               
                   
                   
                 ATAGAGGACCCCTCGTGGACTCCTT 
               
               
                   
                   
                 GTGTCACCAACCTCAACGGGTTTGT 
               
               
                   
                   
                 CTCTGCTTTTTTATTCTCCATAGAG 
               
               
                   
                   
                 ACAGAAACCACCATCGGTTATGGCT 
               
               
                   
                   
                 ACCGGGTCATCACGGACAAGTGCCC 
               
               
                   
                   
                 TGAGGGGATTATTCTCCTCTTAATC 
               
               
                   
                   
                 CAGTCCGTGTTGGGGTCCATTGTCA 
               
               
                   
                   
                 ACGCCTTCATGGTAGGATGTATGTT 
               
               
                   
                   
                 TGTGAAAATATCCCAACCCAAGAAG 
               
               
                   
                   
                 AGGGCAGAGACCCTGGTCTTTTCCA 
               
               
                   
                   
                 CCCACGCGGTGATCTCCATGCGGGA 
               
               
                   
                   
                 TGGGAAACTGTGCTTGATGTTCCGG 
               
               
                   
                   
                 GTGGGGGACTTGAGGAATTCTCACA 
               
               
                   
                   
                 TTGTGGAGGCATCCATCAGAGCCAA 
               
               
                   
                   
                 GTTGATCAAGTCCAAACAGACTTCA 
               
               
                   
                   
                 GAGGGGGAGTTTATTCCCCTCAACC 
               
               
                   
                   
                 AGACTGATATCAACGTGGGGTACTA 
               
               
                   
                   
                 CACAGGGGACGACCGGCTCTTTCTG 
               
               
                   
                   
                 GTGTCACCATTGATTATTAGCCATG 
               
               
                   
                   
                 AAATTAACCAACAGAGTCCCTTCTG 
               
               
                   
                   
                 GGAGATCTCCAAAGCGCAGCTGCCT 
               
               
                   
                   
                 AAAGAGGAACTGGAGATTGTGGTCA 
               
               
                   
                   
                 TCCTGGAGGGAATGGTGGAAGCCAC 
               
               
                   
                   
                 AGGAATGACGTGCCAAGCCCGAAGC 
               
               
                   
                   
                 TCCTACATCACCAGTGAGATCTTGT 
               
               
                   
                   
                 GGGGTTACCGGTTCACACCTGTCCT 
               
               
                   
                   
                 AACGCTGGAAGACGGGTTCTACGAA 
               
               
                   
                   
                 GTTGACTACAACAGCTTCCATGAGA 
               
               
                   
                   
                 CCTATGAGACCAGCACCCCGTCCCT 
               
               
                   
                   
                 TAGTGCCAAAGAGCTAGCGGAGCTG 
               
               
                   
                   
                 GCTAACCGGGCAGAGCTGCCTCTGA 
               
               
                   
                   
                 GTTGGTCTGTGTCCAGCAAACTGAA 
               
               
                   
                   
                 CCAACATGCAGAATTGGAGACAGAA 
               
               
                   
                   
                 GAGGAAGAGAAGAACCCGGAAGAAC 
               
               
                   
                   
                 TGACGGAGAGGAATGGTGACGTGGC 
               
               
                   
                   
                 AAACCTAGAGAATGAATCCAAAGTT 
               
               
                   
                   
                 (SEQ ID NO: 3) 
               
               
                   
                   
               
               
                   
                 Amino acid 
                 [two AA difference V 13 L 14   
               
               
                   
                 sequence 
                 to A 13 A 14  compared to WT 
               
               
                   
                 (425Aa) 
                 form (NCBI NP_034736.2)] 
               
               
                   
                   
                 MTMAKLTESMTNAAEGDSMDQDVES 
               
               
                   
                   
                 PVAIHQPKLPKQARDDLPRHISRDR 
               
               
                   
                   
                 TKRKIQRYVRKDGKCNVHHGNVRET 
               
               
                   
                   
                 YRYLTDIFTTLVDLKWRFNLLIFVM 
               
               
                   
                   
                 VYTVTWLFFGMIWWLIAYIRGDMDH 
               
               
                   
                   
                 IEDPSWTPCVTNLNGFVSAFLFSIE 
               
               
                   
                   
                 TETTIGYGYRVITDKCPEGIILLLI 
               
               
                   
                   
                 QSVLGSIVNAFMVGCMFVKISQPKK 
               
               
                   
                   
                 RAETLVFSTHAVISMRDGKLCLMFR 
               
               
                   
                   
                 VGDLRNSHIVEASIRAKLIKSKQTS 
               
               
                   
                   
                 EGEFIPLNQTDINVGYYTGDDRLFL 
               
               
                   
                   
                 VSPLIISHEINQQSPFWEISKAQLP 
               
               
                   
                   
                 KEELEIVVILEGMVEATGMTCQARS 
               
               
                   
                   
                 SYITSEILWGYRFTPVLTLEDGFYE 
               
               
                   
                   
                 VDYNSFHETYETSTPSLSAKELAEL 
               
               
                   
                   
                 ANRAELPLSWSVSSKLNQHAELETE 
               
               
                   
                   
                 EEEKNPEELTERNGDVANLENESKV 
               
               
                   
                   
                 (SEQ ID NO: 4) 
               
               
                   
                   
               
            
           
           
               
            
               
                 Rat truncated GIRK2 
               
            
           
           
               
               
               
            
               
                   
                 Nucleotide 
                 ATGGACCAAGACGTGGAAAGCCCAG 
               
               
                   
                 sequence 
                 TGGCCATTCACCAGCCAAAGTTGCC 
               
               
                   
                   
                 TAAGCAGGCCAGGGATGACCTGCCA 
               
               
                   
                   
                 AGACACATCAGCCGAGACAGGACCA 
               
               
                   
                   
                 AAAGGAGAATCCAGAGGTACGTGAG 
               
               
                   
                   
                 GAAGGATGGGAAGTGTAACGTCCAC 
               
               
                   
                   
                 CACGGCAACGTGCGGGAGACGTACC 
               
               
                   
                   
                 GATACCTGACGGACATCTTCACCAC 
               
               
                   
                   
                 CCTGGTGGACCTAAAGTGGAGATTC 
               
               
                   
                   
                 AACCTATTGATCTTTGTCATGGTCT 
               
               
                   
                   
                 ACACAGTGACGTGGCTTTTCTTTGG 
               
               
                   
                   
                 GATGATCTGGTGGCTAATTGCATAC 
               
               
                   
                   
                 ATCCGGGGAGATATGGACCACATAG 
               
               
                   
                   
                 AGGACCCCTCGTGGACTCCCTGTGT 
               
               
                   
                   
                 TACCAACCTCAACGGGTTTGTCTCC 
               
               
                   
                   
                 GCTTTTTTATTCTCAATAGAGACAG 
               
               
                   
                   
                 AAACCACCATTGGTTATGGCTACAG 
               
               
                   
                   
                 GGTCATCACGGACAAGTGCCCAGAA 
               
               
                   
                   
                 GGAATTATTCTCCTCTTAATCCAGT 
               
               
                   
                   
                 CCGTGTTGGGGTCCATTGTCAACGC 
               
               
                   
                   
                 CTTCATGGTAGGATGTATGTTTGTG 
               
               
                   
                   
                 AAAATATCCCAACCCAAGAAGAGGG 
               
               
                   
                   
                 CAGAGACCCTGGTCTTTTCCACCCA 
               
               
                   
                   
                 TGCGGTAATCTCCATGCGGGATGGG 
               
               
                   
                   
                 AAACTATGCCTGATGTTCCGGGTAG 
               
               
                   
                   
                 GGGACTTGAGGAATTCCCACATTGT 
               
               
                   
                   
                 GGAGGCCTCCATCAGAGCCAAGTTG 
               
               
                   
                   
                 ATCAAGTCCAAACAGACTTCAGAGG 
               
               
                   
                   
                 GGGAGTTCATTCCCCTCAACCAGAC 
               
               
                   
                   
                 GGATATCAACGTAGGGTACTACACC 
               
               
                   
                   
                 GGGGATGACCGACTCTTTCTCGTGT 
               
               
                   
                   
                 CACCGCTGATTATTAGCCATGAAAT 
               
               
                   
                   
                 TAACCAACAGAGTCCCTTCTGGGAG 
               
               
                   
                   
                 ATCTCCAAAGCCCAGCTGCCTAAAG 
               
               
                   
                   
                 AGGAACTGGAGATTGTGGTCATCCT 
               
               
                   
                   
                 GGAGGGAATGGTGGAAGCCACAGGA 
               
               
                   
                   
                 ATGACGTGCCAAGCTCGAAGCTCCT 
               
               
                   
                   
                 ACGTCACCAGTGAGATCCTGTGGGG 
               
               
                   
                   
                 TTACCGGTTCACACCAGTCCTGACA 
               
               
                   
                   
                 CTGGAGGACGGGTTCTATGAAGTTG 
               
               
                   
                   
                 ACTACAACAGCTTCCATGAGACCCA 
               
               
                   
                   
                 TGAGACCAGCACCCCGTCCCTTAGC 
               
               
                   
                   
                 GCCAAAGAGCTAGCCGAGCTGGCTA 
               
               
                   
                   
                 ACCGGGCAGAGCTGCCCCTGAGCTG 
               
               
                   
                   
                 GTCTGTGTCCAGCAAACTGAACCAA 
               
               
                   
                   
                 CATGCAGAACTGGAGACGGAAGAGG 
               
               
                   
                   
                 AAGAGAAGAACCCGGAAGAACTGAC 
               
               
                   
                   
                 AGAGAGGAATGGTGATGTGGCAAAC 
               
               
                   
                   
                 CTAGAGAATGAGTCCAAAGTG 
               
               
                   
                   
                 (SEQ ID NO: 5) 
               
               
                   
                   
               
               
                   
                 Amino acid 
                 [lacks the first 18AA at 
               
               
                   
                 sequence 
                 N-terminalcompared to WT 
               
               
                   
                 (407Aa) 
                 form (NCBI P48550.1)] 
               
               
                   
                   
                 MDQDVESPVAIHQPKLPKQARDDLP 
               
               
                   
                   
                 RHISRDRTKRRIQRYVRKDGKCNVH 
               
               
                   
                   
                 HGNVRETYRYLTDIFTTLVDLKWRF 
               
               
                   
                   
                 NLLIFVMVYTVTWLFFGMIWWLIAY 
               
               
                   
                   
                 IRGDMDHIEDPSWTPCVTNLNGFVS 
               
               
                   
                   
                 AFLFSIETETTIGYGYRVITDKCPE 
               
               
                   
                   
                 GIILLLIQSVLGSIVNAFMVGCMFV 
               
               
                   
                   
                 KISQPKKRAETLVFSTHAVISMRDG 
               
               
                   
                   
                 KLCLMFRVGDLRNSHIVEASIRAKL 
               
               
                   
                   
                 IKSKQTSEGEFIPLNQTDINVGYYT 
               
               
                   
                   
                 GDDRLFLVSPLIISHEINQQSPFWE 
               
               
                   
                   
                 ISKAQLPKEELEIWILEGMVEATGM 
               
               
                   
                   
                 TCQARSSYVTSEILWGYRFTPVLTL 
               
               
                   
                   
                 EDGFYEVDYNSFHETHETSTPSLSA 
               
               
                   
                   
                 KELAELANRAELPLSWSVSSKLNQH 
               
               
                   
                   
                 AELETEEEEKNPEELTERNGDVANL 
               
               
                   
                   
                 ENESKV  
               
               
                   
                   
                 (SEQ ID NO: 6) 
               
               
                   
                   
               
            
           
         
       
     
     Another object of the present invention is a carrier including a vector of the present invention. 
     According to a particular embodiment of the present invention, the carrier can include a vector comprising a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a functional derivative thereof as described above and a vector comprising a nucleotide sequence encoding a mammalian cone opsin. For example, the mammalian cone opsin is a short wavelength cone opsin (SWO), e.g. from  Mus musculus  or human cone opsin. According to an embodiment, the mammalian cone opsin is human Long-wave-sensitive opsin 1 (SEQ ID NO: 16). 
     
       
         
           
               
            
               
                 Long-wave-sensitive opsin 1 
               
               
                 (OPN1LW)  homo sapiens   
               
               
                 (SEQ ID NO: 16) 
               
               
                 MVLKAEHTRSPSATLPSNVPSCRSLSSSEDGPSGP 
               
               
                   
               
               
                 SSLADGGLAHNLQDSVRHRILYLSEQLRVEKASRD 
               
               
                   
               
               
                 GNTVSYLKLVSKADRHQVPHIQQAFEKVNQRASAT 
               
               
                   
               
               
                 IAQIEHRLHQCHQQLQELEEGCRPEGLLLMAESDP 
               
               
                   
               
               
                 ANCEPPSEKALLSEPPEPGGEDGPVNLPHASRPFI 
               
               
                   
               
               
                 LESRFQSLQQGTCLETEDVAQQQNLLLQKVKAELE 
               
               
                   
               
               
                 EAKRFHISLQESYHSLKERSLTDLQLLLESLQEEK 
               
               
                   
               
               
                 CRQALMEEQVNGRLQGQLNEIYNLKHNLACSEERM 
               
               
                   
               
               
                 AYLSYERAKEIWEITETFKSRISKLEMLQQVTQLE 
               
               
                   
               
               
                 AAEHLQSRPPQMLFKFLSPRLSLATVLLVFVSTLC 
               
               
                   
               
               
                 ACPSSLISSRLCTCTMLMLIGLGVLAWQRWRAIPA 
               
               
                   
               
               
                 TDWQEWWPSRCRLYSKDSGPPADGP 
               
            
           
         
       
     
     According to a particular embodiment of the present invention, the carrier is for example chosen from solid-lipid nanoparticles, chitosan nanoparticles, liposome, lipoplex or cationic polymer. 
     According to a particular embodiment of the present invention, the vector of the present invention is a virus, chosen from an adeno-associated virus (AAV), an adenovirus, a lentivirus, an SV40 viral vector. According to a particular embodiment of the present invention, the present invention is equal to or less than 30 nm in size. For example it is an adeno-associated virus (AAV), preferably an AAV8, or an AAV2-7m8 or AAV9-7m8 capsid variant as described in the international application WO 2012145601 [32]. 
     An AAV2-7m8 or AAV9-7m8 capsid variant is an AAV2 or AAV9 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 amino acid sequence LGETTRP (SEQ ID NO: 7). 
     The genomic and polypeptide sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits including VP1 protein are known in the art. Such sequences may be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB). See, e.g., GenBank and PDB AF043303 and 1 LP3 (AAV-2), AY530579 and 3UX1 (AAV-9 (isolate hu.14)), the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. Exemplary amino acid sequence of wild-type VP1 for AAV9 and AAV2 are shown in SEQ ID NO: 8 and SEQ ID NO:9, respectively. 
     
       
         
           
               
               
            
               
                   
                 wild-type AAV9 VP1 capsid protein 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKAN 
               
               
                   
               
               
                   
                 QQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADA 
               
               
                   
               
               
                   
                 AALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLK 
               
               
                   
               
               
                   
                 EDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAP 
               
               
                   
               
               
                   
                 GKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQ 
               
               
                   
               
               
                   
                 TGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAP 
               
               
                   
               
               
                   
                 VADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTR 
               
               
                   
               
               
                   
                 TWALPTYNNHLYKQISNSTSGGSSNDNAYGYSTPW 
               
               
                   
               
               
                   
                 GYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFK 
               
               
                   
               
               
                   
                 LFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQ 
               
               
                   
               
               
                   
                 LPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGS 
               
               
                   
               
               
                   
                 QAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVP 
               
               
                   
               
               
                   
                 FHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQ 
               
               
                   
               
               
                   
                 NQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVST 
               
               
                   
               
               
                   
                 TVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMAS 
               
               
                   
               
               
                   
                 HKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMIT 
               
               
                   
               
               
                   
                 NEEEIKTTNPVATESYGQVATNHQSA Q   588   Q   589 A 
               
               
                   
               
               
                   
                 QTGWWQNQGILPGMVWQDRDVYLQGPIWAKIPHT 
               
               
                   
               
               
                   
                 DGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT 
               
               
                   
               
               
                   
                 AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRW 
               
               
                   
               
               
                   
                 NPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTR 
               
               
                   
               
               
                   
                 YLTR 
               
               
                   
               
               
                   
                 wild-type AAV2 VP1 capsid protein 
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPA 
               
               
                   
               
               
                   
                 ERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADA 
               
               
                   
               
               
                   
                 AALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLK 
               
               
                   
               
               
                   
                 EDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAP 
               
               
                   
               
               
                   
                 GKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQ 
               
               
                   
               
               
                   
                 TGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAP 
               
               
                   
               
               
                   
                 MADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTR 
               
               
                   
               
               
                   
                 TWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWG 
               
               
                   
               
               
                   
                 YFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKL 
               
               
                   
               
               
                   
                 FNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQL 
               
               
                   
               
               
                   
                 PYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQ 
               
               
                   
               
               
                   
                 AVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPF 
               
               
                   
               
               
                   
                 HSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTT 
               
               
                   
               
               
                   
                 TQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSK 
               
               
                   
               
               
                   
                 TSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMAS 
               
               
                   
               
               
                   
                 HKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMIT 
               
               
                   
               
               
                   
                 DEEEIRTTNPVATEQYGSVSTNLQRG N   587   R   588 QAA 
               
               
                   
               
               
                   
                 TADVNTQGVLPGIVIVWQDRDVYLQGPIWAKI 
               
               
                   
               
               
                   
                 PHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPAN 
               
               
                   
               
               
                   
                 PSTTFSAAKFASFITQYSTGQVSVEIEWELQKENS 
               
               
                   
               
               
                   
                 KRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPI 
               
               
                   
               
               
                   
                 GTRYLTRNL 
               
            
           
         
       
     
     Preferably, the insertion site of the insertion peptide in the GH loop of the VP1 capsid protein is between amino acids 587 and 588 of AAV2 wild-type VP1 capsid protein, between amino acids 588 and 589 of AAV9 wild-type VP1 capsid protein. 
     According to some embodiments, the insertion peptide has a length of 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids. 
     The insertion peptide may comprise one or more spacer amino acids at the N- and/or C-terminus of amino acid sequence LGETTRP (SEQ ID NO: 7). Preferably, the spacer amino acids are selected from the group consisting of Ala, Leu, Gly, Ser, and Thr, more preferably from the group consisting of Ala, Leu, and Gly. 
     According to an embodiment, the insertion peptide comprises or consists of sequence AALGETTRPA (SEQ ID NO: 10), LALGETTRPA (SEQ ID NO: 11), or GLGETTRPA (SEQ ID NO: 12), preferably comprises or consists of sequence AALGETTRPA (SEQ ID NO: 10) or LALGETTRPA (SEQ ID NO: 11). 
     According to a particular embodiment, the viral vector, in particular AAV, AAV8, AAV2-7m8 or AAV9-7m8, comprises the polynucleotide of interest (nucleotide sequence encoding GIRK2 or a functional derivative thereof, and/or nucleotide sequence encoding mammalian cone opsin) under the control of a cone-specific promoter, preferably a pR1.7 or a functional variant thereof, or a minimal M-opsin promoter, in particular in a pMNTC expression cassette. In said AAV, the polynucleotide of interest which is operatively linked to the cone-specific promoter, e.g. promoter pR1.7, minimal M-opsin promoter or pMNTC, is preferably flanked by two adeno-associated virus inverted terminal repeats (AAV ITRs). 
     pR1.7 is a 1.7 kilobases synthetic promoter based on the human red opsin promoter sequence described in Hum Gene Ther. 2016 January; 27(1):72-82. As used herein, “pR1.7” denotes the promoter of sequence SEQ ID NO:13 and functional variants thereof. “Functional variants” of the pR1.7 promoter typically have one or more nucleotide mutations (such as a nucleotide deletion, addition, and/or substitution) relative to the native pR1.7 promoter (SEQ ID NO: 13), which do not significantly alter the transcription of the polynucleotide of interest. In the context of the present invention, said functional variants retain the capacity to drive a strong expression, in cone photoreceptors, of the polynucleotide of interest. Such capacity can be tested as described by Ye et al. (2016) [33] and Khabou et al. (20183) [34]. 
     Another example of cone-specific promoter which may be used is a minimal M-opsin promoter region such as disclosed in International application WO 2015142941 [35], in particular in SEQ ID NO:55 or SEQ ID NO: 93 as is disclosed in WO 2015142941 [35]. Instant sequence SEQ ID NO: 14 is identical to SEQ ID NO: 93 of WO 2015142941 [35]. 
     In an embodiment, the polynucleotide of interest which is placed under the control the minimal M-opsin promoter region, is inserted in a pMNTC expression cassette comprising an optimized enhancer, optimized promoter, optimized 5′UTR, optimized intron, optimized kozak and optimized polyA region (SEQ ID NO:95 of WO 2015142941 [35]). 
     
       
         
           
               
               
            
               
                   
                 pR1.7 promoter 
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 ggaggctgaggggtggggaaagggcatgggtgttt 
               
               
                   
                   
               
               
                   
                 catgaggacagagcttccgtttcatgcaatgaaaa 
               
               
                   
                   
               
               
                   
                 gagtttggagacggatggtggtgactggactatac 
               
               
                   
                   
               
               
                   
                 acttacacacggtagcgatggtacactttgtatta 
               
               
                   
                   
               
               
                   
                 tgtatattttaccacgatctttttaaagtgtcaaa 
               
               
                   
                   
               
               
                   
                 ggcaaatggccaaatggttccttgtcctatagctg 
               
               
                   
                   
               
               
                   
                 tagcagccatcggctgttagtgacaaagcccctga 
               
               
                   
                   
               
               
                   
                 gtcaagatgacagcagcccccataactcctaatcg 
               
               
                   
                   
               
               
                   
                 gctctcccgcgtggagtcatttaggagtagtcgca 
               
               
                   
                   
               
               
                   
                 ttagagacaagtccaacatctaatcttccaccctg 
               
               
                   
                   
               
               
                   
                 gccagggccccagctggcagcgagggtgggagact 
               
               
                   
                   
               
               
                   
                 ccgggcagagcagagggcgctgacattggggcccg 
               
               
                   
                   
               
               
                   
                 gcctggcttgggtccctctggcctttccccagggg 
               
               
                   
                   
               
               
                   
                 ccctctttccttggggctttcttgggccgccactg 
               
               
                   
                   
               
               
                   
                 ctcccgctcctctccccccatcccaccccctcacc 
               
               
                   
                   
               
               
                   
                 ccctcgttcttcatatccttctctagtgctccctc 
               
               
                   
                   
               
               
                   
                 cactttcatccacccttctgcaagagtgtgggacc 
               
               
                   
                   
               
               
                   
                 acaaatgagttttcacctggcctggggacacacgt 
               
               
                   
                   
               
               
                   
                 gcccccacaggtgctgagtgactttctaggacagt 
               
               
                   
                   
               
               
                   
                 aatctgctttaggctaaaatgggacttgatcttct 
               
               
                   
                   
               
               
                   
                 gttagccctaatcatcaattagcagagccggtgaa 
               
               
                   
                   
               
               
                   
                 ggtgcagaacctaccgcctttccaggcctcctccc 
               
               
                   
                   
               
               
                   
                 acctctgccacctccactctccttcctgggatgtg 
               
               
                   
                   
               
               
                   
                 ggggctggcacacgtgtggcccagggcattggtgg 
               
               
                   
                   
               
               
                   
                 gattgcactgagctgggtcattagcgtaatcctgg 
               
               
                   
                   
               
               
                   
                 acaagggcagacagggcgagcggagggccagctcc 
               
               
                   
                   
               
               
                   
                 ggggctcaggcaaggctgggggcttcccccagaca 
               
               
                   
                   
               
               
                   
                 ccccactcctcctctgctggacccccacttcatag 
               
               
                   
                   
               
               
                   
                 ggcacttcgtgttctcaaagggcttccaaatagca 
               
               
                   
                   
               
               
                   
                 tggtggccttggatgcccagggaagcctcagagtt 
               
               
                   
                   
               
               
                   
                 gcttatctccctctagacagaaggggaatctcggt 
               
               
                   
                   
               
               
                   
                 caagagggagaggtcgccctgttcaaggccaccca 
               
               
                   
                   
               
               
                   
                 gccagctcatggcggtaatgggacaaggctggcca 
               
               
                   
                   
               
               
                   
                 gccatcccaccctcagaagggacccggtggggcag 
               
               
                   
                   
               
               
                   
                 gtgatctcagaggaggctcacttctgggtctcaca 
               
               
                   
                   
               
               
                   
                 ttcttggatccggttccaggcctcggccctaaata 
               
               
                   
                   
               
               
                   
                 gtctccctgggctttcaagagaaccacatgagaaa 
               
               
                   
                   
               
               
                   
                 ggaggattcgggctctgagcagtttcaccacccac 
               
               
                   
                   
               
               
                   
                 cccccagtctgcaaatcctgacccgtgggtccacc 
               
               
                   
                   
               
               
                   
                 tgccccaaaggcggacgcaggacagtagaagggaa 
               
               
                   
                   
               
               
                   
                 cagagaacacataaacacagagagggccacagcgg 
               
               
                   
                   
               
               
                   
                 ctcccacagtcaccgccaccttcctggcggggatg 
               
               
                   
                   
               
               
                   
                 ggtggggcgtctgagtttggttcccagcaaatccc 
               
               
                   
                   
               
               
                   
                 tctgagccgcccttgcgggctcgcctcaggagcag 
               
               
                   
                   
               
               
                   
                 gggagcaagaggtgggaggaggaggtctaagtccc 
               
               
                   
                   
               
               
                   
                 aggcccaattaagagatcaggtagtgtagggtttg 
               
               
                   
                   
               
               
                   
                 ggagcttttaaggtgaagaggcccgggctgatccc 
               
               
                   
                   
               
               
                   
                 acaggccagtataaagcgccgtgaccctcaggtga 
               
               
                   
                   
               
               
                   
                 tgcgccagggccggctgccgtcggggacagggctt 
               
               
                   
                   
               
               
                   
                 tccatagcc 
               
               
                   
                   
               
               
                   
                 minimal M-opsin promoter region 
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 Ccagcaaatccctctgagccgcccccgggggctcg 
               
               
                   
                   
               
               
                   
                 cctcaggagcaaggaagcaaggggtgggaggagga 
               
               
                   
                   
               
               
                   
                 ggtctaagtcccaggcccaattaagagatcagatg 
               
               
                   
                   
               
               
                   
                 gtgtaggatttgggagcttttaaggtgaagaggcc 
               
               
                   
                   
               
               
                   
                 cgggctgatcccactggccggtataaagcaccgtg 
               
               
                   
                   
               
               
                   
                 accctcaggtgacgcaccagggccggctgccgtcg 
               
               
                   
                   
               
               
                   
                 gggacagggctttccatagcccag 
               
               
                   
                   
               
               
                   
                 pMNTC 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 cctacagcagccagggtgagattatgaggctgagc 
               
               
                   
                   
               
               
                   
                 tgagaatatcaagactgtaccgagtagggggcctt 
               
               
                   
                   
               
               
                   
                 ggcaagtgtggagagcccggcagctggggcagagg 
               
               
                   
                   
               
               
                   
                 gcggagtacggtgtgcgtttacggacctcttcaaa 
               
               
                   
                   
               
               
                   
                 cgaggtaggaaggtcagaagtcaaaaagggaacaa 
               
               
                   
                   
               
               
                   
                 atgatgtttaaccacacaaaaatgaaaatccaatg 
               
               
                   
                   
               
               
                   
                 gttggatatccattccaaatacacaaaggcaacgg 
               
               
                   
                   
               
               
                   
                 ataagtgatccgggccaggcacagaaggccatgca 
               
               
                   
                   
               
               
                   
                 cccgtaggattgcactcagagctcccaaatgcata 
               
               
                   
                   
               
               
                   
                 ggaatagaagggtgggtgcaggaggctgaggggtg 
               
               
                   
                   
               
               
                   
                 gggaaagggcatgggtgtttcatgaggacagagct 
               
               
                   
                   
               
               
                   
                 tccgtttcatgcaatgaaaagagtttggagacgga 
               
               
                   
                   
               
               
                   
                 tggtggtgactggactatacacttacacacggtag 
               
               
                   
                   
               
               
                   
                 cgatggtacactttgtattatgtatattttaccac 
               
               
                   
                   
               
               
                   
                 gatctttttaaagtgtcaaaggcaaatggccaaat 
               
               
                   
                   
               
               
                   
                 ggttccttgtcctatagctgtagcagccatcggct 
               
               
                   
                   
               
               
                   
                 gttagtgacaaagcccctgagtcaagatgacagca 
               
               
                   
                   
               
               
                   
                 gcccccataactcctaatcggctctcccgcgtgga 
               
               
                   
                   
               
               
                   
                 gtcatttaggagtagtcgcattagagacaagtcca 
               
               
                   
                   
               
               
                   
                 acatctaatcttccaccctggccagggccccagct 
               
               
                   
                   
               
               
                   
                 ggcagcgagggtgggagactccgggcagagcagag 
               
               
                   
                   
               
               
                   
                 ggcgctgacattggggcccggcctggcttgggtcc 
               
               
                   
                   
               
               
                   
                 ctctggcctttccccaggggccctctttccttggg 
               
               
                   
                   
               
               
                   
                 gctttcttgggccgccactgctcccgctcctctcc 
               
               
                   
                   
               
               
                   
                 ccccatcccaccccctcaccccctcgttcttcata 
               
               
                   
                   
               
               
                   
                 tccttctctagtgctccctccactttcatccaccc 
               
               
                   
                   
               
               
                   
                 ttctgcaagagtgtgggaccacaaatgagttttca 
               
               
                   
                   
               
               
                   
                 cctggcctggggacacacgtgcccccacaggtgct 
               
               
                   
                   
               
               
                   
                 gagtgactttctaggacagtaatctgctttaggct 
               
               
                   
                   
               
               
                   
                 aaaatgggacttgatcttctgttagccctaatcat 
               
               
                   
                   
               
               
                   
                 caattagcagagccggtgaaggtgcagaacctacc 
               
               
                   
                   
               
               
                   
                 gcctttccaggcctcctcccacctctgccacctcc 
               
               
                   
                   
               
               
                   
                 actctccttcctgggatgtgggggctggcacacgt 
               
               
                   
                   
               
               
                   
                 gtggcccagggcattggtgggattgcactgagctg 
               
               
                   
                   
               
               
                   
                 ggtcattagcgtaatcctggacaagggcagacagg 
               
               
                   
                   
               
               
                   
                 gcgagcggagggccagctccggggctcaggcaagg 
               
               
                   
                   
               
               
                   
                 ctgggggcttcccccagacaccccactcctcctct 
               
               
                   
                   
               
               
                   
                 gctggacccccacttcatagggcacttcgtgttct 
               
               
                   
                   
               
               
                   
                 caaagggcttccaaatagcatggtggccttggatg 
               
               
                   
                   
               
               
                   
                 cccagggaagcctcagagttgcttatctccctcta 
               
               
                   
                   
               
               
                   
                 gacagaaggggaatctcggtcaagagggagaggtc 
               
               
                   
                   
               
               
                   
                 gccctgttcaaggccacccagccagctcatggcgg 
               
               
                   
                   
               
               
                   
                 taatgggacaaggctggccagccatcccaccctca 
               
               
                   
                   
               
               
                   
                 gaagggacccggtggggcaggtgatctcagaggag 
               
               
                   
                   
               
               
                   
                 gctcacttctgggtctcacattcttccagcaaatc 
               
               
                   
                   
               
               
                   
                 cctctgagccgcccccgggggctcgcctcaggagc 
               
               
                   
                   
               
               
                   
                 aaggaagcaaggggtgggaggaggaggtctaagtc 
               
               
                   
                   
               
               
                   
                 ccaggcccaattaagagatcagatggtgtaggatt 
               
               
                   
                   
               
               
                   
                 tgggagcttttaaggtgaagaggcccgggctgatc 
               
               
                   
                   
               
               
                   
                 ccactggccggtataaagcaccgtgaccctcaggt 
               
               
                   
                   
               
               
                   
                 gacgcaccagggccggctgccgtcggggacagggc 
               
               
                   
                   
               
               
                   
                 tttccatagcccaggtaagtatcaaggttacaaga 
               
               
                   
                   
               
               
                   
                 caggtttaaggagaccaatagaaactgggcttgtc 
               
               
                   
                   
               
               
                   
                 gagacagagaagactcttgcgtttctgataggcac 
               
               
                   
                   
               
               
                   
                 ctattggtcttactgacatccactttgcctttctc 
               
               
                   
                   
               
               
                   
                 tccacaggcccagagaggagacaggccgccacc 
               
            
           
         
       
     
     The promoter and the polynucleotide of interest are operatively linked. As used herein, the term “operatively linked” refers to two or more nucleic acid or amino acid sequence elements that are physically linked in such a way that they are in a functional relationship with each other. For instance, a promoter is operatively linked to a coding sequence if the promoter is able to initiate or otherwise control/regulate the transcription and/or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of” the promoter. Generally, when two nucleic acid sequences are operatively linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may not be required. 
     According to an embodiment, the vector is an AAV9 (AAV9-7m8-pR1.7) comprising:
         a VP1 capsid protein in which 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 amino acid sequence LGETTRP (SEQ ID NO: 7); and   the polynucleotide of interest (nucleotide sequence encoding GIRK2 or a functional derivative thereof and/or nucleotide sequence encoding mammalian cone opsin) under the control of a pR1.7 promoter.       

     In said AAV9-7m8, the insertion peptide has a length of 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids. Preferably, the insertion peptide comprises one or more spacer amino acids at the N- and/or C-terminus of amino acid sequence LGETTRP (SEQ ID NO: 7). Preferably, the spacer amino acids are selected from the group consisting of Ala, Leu, Gly, Ser, and Thr, more preferably from the group consisting of Ala, Leu, and Gly. According to an embodiment, the insertion peptide comprises or consists of sequence AALGETTRPA (SEQ ID NO: 10), LALGETTRPA (SEQ ID NO: 11), or GLGETTRPA (SEQ ID NO: 12); preferably comprises or consists of sequence AALGETTRPA (SEQ ID NO: 10) or LALGETTRPA (SEQ ID NO: 11). 
     The vectors of the invention are produced using methods known in the art. In short, the methods generally involve (a) the introduction of the AAV vector into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the AAV vector and (c) introducing a helper virus into the host cell. All functions for AAV virion replication and packaging need to be present, to achieve replication and packaging of the AAV vector into AAV virions. The introduction into the host cell can be carried out using standard virology techniques simultaneously or sequentially. Finally, the host cells are cultured to produce AAV virions and are purified using standard techniques such as iodixanol or CsCl gradients or other purification methods. The purified AAV virion is then ready for use. 
     Another object of the present invention is a pharmaceutical composition comprising the vector or the carrier of the present invention, with a pharmaceutically acceptable carrier, diluent or excipient. 
     Another object of the present invention is a vector, a carrier or a pharmaceutical composition of the present invention, for use in treating rod-cone dystrophy (RCD). 
     Rod-cone dystrophy (RCD) is a heterogeneous group of diseases such as Retinitis Pigmentosa (RP), in particular non-syndromic X-linked Retinitis Pigmentosa (XLRP), autosomal recessive RP, autosomal dominant RP. The most common syndromic forms of RCD include Usher syndrome, Bardet-Biedl syndrome, Refsum disease, Bassen-Kornzweig syndrome and Batten disease. 
     The RCD subject to be treated is a mammal, in particular a non-human or human primate, preferably a human. The RCD in the mammal may be at an early, intermediate or advanced stage of the disease. In RCD subjects at intermediate or advanced stage of the disease, transduction of the subjects&#39; cones with a nucleotide sequence GIRK2 or a functional derivative thereof is sufficient to achieve vision restoration provided cone opsin and cone arrestin are still expressed in the patients&#39; cone cell bodies. In RCD subjects whose cone cell bodies no longer express cone opsin, transduction of the subjects&#39; cones with a nucleotide sequence GIRK2 or a functional derivative thereof and a mammalian cone opsin is required. 
     Treatment of RCD may be implemented by administering the vector(s), carrier or pharmaceutical composition of the present invention to the mammal, so as to achieve transduction of cones with the GIRK2 transgene, or GIRK 2 and mammalian cone opsin transgenes. 
     In other words, another object of the present invention is a method of treating a RCD in a mammal in need thereof, the method comprising administering to the mammal an effective amount of the vector or the carrier of the pharmaceutical composition of the present invention. 
     Accordingly, in a first embodiment, the vector comprising a nucleotide sequence encoding GIRK2 or a functional derivative thereof, carrier including said vector, or a pharmaceutical composition comprising the vector or carrier is for use in treating rod-cone dystrophy in a RCD mammalian subject whose cone cells still express endogenous cone opsin. According to an embodiment, the vector further comprises a nucleotide sequence encoding a mammalian cone opsin. According to another embodiment, the vector does not comprise a nucleotide sequence encoding a mammalian cone opsin. According to an embodiment, the carrier further includes a vector comprising a nucleotide sequence encoding a mammalian cone opsin. According to another embodiment, the carrier does not include a vector comprising a nucleotide sequence encoding a mammalian cone opsin. 
     In a second embodiment, the vector comprising a nucleotide sequence encoding GIRK2 or a functional derivative thereof, carrier including said vector, or a pharmaceutical composition comprising the vector or carrier is for use in treating rod-cone dystrophy in a RCD mammalian subject whose cone cells no longer express endogenous cone opsin. According to this embodiment, the vector further comprises a nucleotide sequence encoding a mammalian cone opsin, or the carrier further includes a vector comprising a nucleotide sequence encoding a mammalian cone opsin. 
     Treatment of RCD may also be implemented by transducing a mammalian cone precursor cell with vector(s), carrier or pharmaceutical composition of the present invention, and administering the transduced mammalian cone precursor cell to the retina, in particular to the fovea region, of the RCD mammal. 
     In other words, another object of the present invention is a method of treating a RCD in a mammal in need thereof, the method comprising administering to the mammal an effective amount of mammalian cone precursor cell transduced with the vector or the carrier of the pharmaceutical composition of the present invention. 
     The invention also relates to a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof, or encoding GIRK2 or a functional derivative thereof and a mammalian cone opsin, for use in treating a RCD. Accordingly, it is also provided a method of treating a RCD in a mammal in need thereof, the method comprising administering to the mammal a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof, or encoding GIRK2 or a functional derivative thereof and a mammalian cone opsin. As used herein, the term «heterologous nucleic acid» refers to a gene, polynucleotide or nucleic acid sequence that is not in its natural environment. 
     Cone precursor cells are not-fully differentiated, non-dividing cells committed to differentiate into cone cells. 
     In an embodiment, cone precursor cells are obtained from retina of donor (e.g. cadaver eye donor) or from the RCD subject to be treated, preferably from the RCD subject to be treated. In another embodiment, cone precursor cells are obtained from stem cells, in particular embryonic stem cells, induced pluripotent stem (iPS cells), adult stem cells or fetal stem cells. In another embodiment, cone precursor cells are obtained from differentiated embryonic stem cells. According to one embodiment, embryonic stem cells are non-human embryonic stem cells. According to another embodiment, human embryonic stem cells may be used with the proviso that the method itself or any related acts do not include destruction of human embryos. Preferably cone precursor cells are obtained by differentiation of stem cells, preferably from differentiation of adult stem cells or induced pluripotent stem cells, more preferably from differentiation of induced pluripotent stem cells obtained from somatic cells, e.g. fibroblasts, of the RCD subject to be treated. 
     Embryonic stem cells are able to maintain an undifferentiated state or can be directed to mature along lineages deriving from all three germ layers, ectoderm, endoderm and mesoderm. Embryonic stem cells can be reprogrammed towards cone photoreceptors by manipulation of key developmental signaling pathways as described in the international application WO 2018055131 [36]. For example, it may be used antagonists of the nodal and wnt pathway in addition to activin-A and serum (Watanabe K et al, 2005) [37], or inhibition of the Notch signaling pathway can be implemented (Osakada F et al., 2009) [38]. Cone precursor cells can be obtained from embryonic stem cells using any protocol known by the skilled person (Osakada F et al., 2008; Amirpour N et al., 2012; Nakano T et al., 2012; Zhu Y et al., 2013; Yanai A et al., 2013; Kuwahara A et al., 2015; Mellough C B et al., 2015; Singh K et al., 2015) [39-46]. 
     Preferably, cone precursor cells are obtained from iPS cells or adult stem cells, more preferably from iPS cells. Induced pluripotent stem (iPS) cells are derived from a non-pluripotent cell, typically an adult somatic cell, by a process known as reprogramming, where the introduction of only a few specific genes are necessary to render the cells pluripotent (e.g. OCT4, SOX2, KLF4 and C-MYC in human cells). One benefit of use of iPS cells is the avoidance of the use of embryonic cells altogether and hence any ethical questions thereof. Photoreceptor precursor cells can be obtained from iPS cells using any differentiation method known by the skilled person. 
     In particular, photoreceptor precursor cells can be obtained from human iPS cells by a method as disclosed in Garita-Hernandez et al. (2019) [47]. Human iPS are expanded to confluence in iPS medium (e.g. Essential 8™ medium, GIBCO, Life Technologies). After 80% confluence, the medium was switched to a proneural medium (e.g. Essential 6™ medium supplemented with 1% N2 supplement (100×); GIBCO, Life Technologies). The medium was changed every 2-3 days. After 4 weeks of differentiation, neural retina-like structures grew out of the cultures and were mechanically isolated. Pigmented parts, giving rise to RPE were carefully removed. The extended 3D culture in Maturation medium (DMEM/F-12 medium supplemented with 2% B-27™ Supplement (50×), serum free, and 1% MEM Non-Essential Amino Acids Solution (100×); GIBCO, Life Technologies) allowed the formation of retinal organoids. Addition of 10 ng/ml Fibroblast growth factor 2 (FGF2, Preprotech) at this point favoured the growth of retinal organoids and the commitment towards retinal neurons instead of RPE lineage. In order to promote the commitment of retinal progenitors towards photoreceptors, Notch signalling was specifically blocked for a week starting at day 42 of differentiation using the gamma secretase inhibitor DAPT (10 μM, Selleckchem). Floating organoids were cultured in 6 well-plates (10 organoids per well) and medium was changed every 2 days. 
     Photoreceptor precursor cells can also be obtained from human iPS cells using any other protocol known by the skilled person (Lamba, Osakada and colleagues: Lamba et al., 2006; Lamba et al., 2010; Osakada et al., 2009; Meyer J S et al., 2009; Meyer J S et al., 2011; Mellough C B et al., 2012; Boucherie C et al., 2013; Sridhar A et al., 2013; Tucker B A et al., 2013; Tucker B A et al., 2013; Eichman S et al., 2014; Zhong X et al., 2014; Wang X et al., 2015) [48, 49, 38, 50-59]. 
     The cone precursor cells comprise a heterologous nucleic acid encoding i) GIRK2 or a functional derivative thereof, or ii) encoding GIRK2 or a functional derivative thereof and a mammalian cone opsin. Where the cone precursor cells comprise a heterologous nucleic acid encoding GIRK2, or a functional derivative thereof, and a mammalian cone opsin, the cone precursor cells either comprise i) a heterologous nucleic acid encoding both GIRK2, or a functional derivative thereof, and a mammalian cone opsin, or ii) a heterologous nucleic acid encoding GIRK2, or a functional derivative thereof, and another heterologous nucleic acid encoding a mammalian cone opsin. 
     Said cone precursor cells may be prepared by introducing into said cone precursor cells said heterologous nucleic acid(s), or an expression cassette or vector comprising said nucleic acid(s), by any method known to the skilled person. According to an embodiment, a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof, or encoding GIRK2 or a functional derivative thereof and a mammalian cone opsin, is prepared by infecting the cone precursor cell with a viral vector as described above, in particular with an AAV vector, preferably the AAV8, AAV2-7m8 or AAV9-7m8. 
     In another aspect, the invention therefore further refers to a method of preparing a cone precursor cell comprising a heterologous nucleic acid encoding GIRK2 or a functional derivative thereof, or encoding GIRK2 or a functional derivative thereof and a mammalian cone opsin, said method comprising infecting cone precursor cells with a viral vector or carrier according to the invention, and recovering infected cone precursor cells. 
     The vector, carrier, or pharmaceutical composition, or cone precursor cells may be administered by any suitable route known to the skilled person in particular by intravitreal or subretinal administration. 
     The fovea is a small region in the central retina of primates of approximately equal to or less than 0.5 mm in diameter that contains only cone photoreceptor cells, and highest density of cones in the whole retina. The fovea dominates the visual perception of primates by providing high-acuity color vision. The highest density of cones is found at the center of the fovea (&lt;0.3 mm from the foveal center), devoid of rod photoreceptors. Cone density decreases by up to 100-fold with distance from the fovea. 
     Cone cells in the fovea are the primary targets of gene therapies aiming to treat inherited retinal diseases like retinitis pigmentosa. Usually, viral vectors encoding therapeutic proteins are injected “subretinally”, i.e. into the subretinal space between the photoreceptors and the retinal pigment epithelium (RPE) cells in order to provide gene delivery to cones. 
     The subretinal delivery leads to the formation of a “bleb”, which refers to a fluid-filled pocket within the subretinal space of the injected eye. In this approach, gene delivery is limited to cells that contact the local bleb of injected fluid. Retinal detachment, and in particular foveal detachment, that occurs during subretinal injections is a concern in eyes with retinal degeneration. 
     Advantageously, when the vector is an AAV9-7m8 vector (in particular AAV9-7m8-pR1.7 vector), the vector (or carrier of pharmaceutical composition comprising said vector) can be administered by a distal subretinal injection, or in the periphery of the fovea, and then spread laterally to reach the foveal region. According to an embodiment the bleb formed is greater than or equal to 0.5 millimeters away from the center of the fovea, without detaching the foveal region. 
     In particular, subretinal injection of AAV9-7m8 vector (in particular AAV9-7m8-pR1.7 vector) can be performed a) in a region adjacent to the superior or inferior temporal branch of retinal artery; b) at a distance of 2-3 optic disk diameter away from the center of the fovea; and c) at a position localized in the geometric shape, preferably quadrilateral, delineated by the branches of temporal retinal artery and temporal retinal vein, usually between the 3 rd  and 4 th  anterior venous crossings (see  FIG.  13   ). Preferably, injection is performed at a position forming an angle comprised between −10° and +10° with the vertical axis of the retina passing through the center of the fovea. In an embodiment, said AAV9-7m8 viral vector is formulated in a solution and 50 to 100 μL of solution are injected continuously in 20 to 30 seconds. In an embodiment, said AAV9-7m8 viral vector is formulated in a solution at a concentration of 1×10 10  to 1×10 12  vg/mL (viral genome/mL), preferably of 0.5×10 11  to 5×10 11  vg/mL, still preferably of 1×10 11  vg/mL. 
     Preferably, the cone precursor cells are administered by intraocular injection, preferably by subretinal space injection, more preferably by injection between the neural retina and the overlying PE. The amount of cone precursor cells to be administered may be determined by standard procedure well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight) and type and severity of the disease being treated have to be taken into account to determine the appropriate dosage. The cone precursor cells may be administered as a single dose or in multiple doses. In particular, each unit dosage may contain, from 100,000 to 300,000 cone precursor cells per μl, preferably from 200,000 to 300,000 cone precursor cells per μl. 
     Another object of the present invention is a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a derivative thereof as described above, for use as a medicament. In particular said nucleotide sequence is useful for treating rod-cone dystrophy (RCD). In other words, another object of the present invention is a method of treating a RCD in a mammal in need thereof, the method comprising administering to the mammal an effective amount of a nucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a derivative thereof as described above. According to an embodiment, the polynucleotide sequence encoding subunit 2 of G-protein-gated inwardly rectifying potassium channel (GIRK2) or a derivative thereof is under the control of the pR1.7 promoter or of a functional variant of said promoter. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         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. 
         FIG.  2    represents alignments of GIRK2 (A) rat truncated GIRK2 vs mouse GIRK2 (B) mouse GIRK2 vs human GIRK2. 
         FIG.  3    represents plasmids (A) CMV-GIRK2-GFP and (B) CMV-SWO-mCherry. 
         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. 
         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). 
         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 SWO+GIRK2 =0.0381 and P 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. 
         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 (P50-P365) =0.0022. (D) Linear regression correlation between the ERG amplitudes and the number of cones in rd10 mice (n=6). Pvalue non-injected =0.0482. Pvalue AAV-GIRK2-GFP =0,0007. Pvalue PBS =0.0104. 
         FIG.  8    represents what remained in the phototransduction cascade in huP347S +/−  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 +/−  mouse at P14 stained with (E) opsin, (F) transducin, (G) PDE and (H) cone arrestin. (I-L) retinal cross-section of a huP347S +/−  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. 
         FIG.  9    represents universality of the approach. (A) Photopic ERG amplitude in huP347S +/−  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 +/−  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 +/−  mice over time (n=6). Pvalue (P50-P365) =0.0022. (D) Linear regression correlation between the ERG amplitudes and the number of cones in huP347S +/−  mice (n=5). Pvalue non-injected =0.0313. Pvalue AAV-GIRK2-GFP =0, 0146. Pvalue PBS =0.0497. 
         FIG.  10    represents the efficiency of the mouse GIRK2 in HEK cells transfected with two plasmids: CMV-SWO-mCherry and CMV-GIRK2-GFP. 
         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&#39;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. 
         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. 
         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 
     1. Animals 
     C57BL/6j 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 +/−  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. 
     2. AAV Injections 
     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. 
     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: 
     
       
         
           
               
            
               
                   
               
               
                 Injection Table 
               
            
           
           
               
               
               
               
               
            
               
                 Mice 
                 Eyes 
                 viral vector injected 
                 viral vector titration 
                 volume injected 
               
               
                   
               
               
                 rd10 
                 both 
                 AAV8-mCAR-GFP 
                 10 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 8  rAAV 
               
               
                   
                   
                   
                 particles 
               
               
                 huRhoP347S +/−   
                 right 
                 PBS 
               
               
                 huRhoP347S +/−   
                 left 
                 AAV8-PR1.7-GIRK2-GFP 
                 5.10 8  rAAV 
               
               
                   
                   
                   
                 particles 
               
               
                   
               
            
           
         
       
     
     3. Eye Fundus Examination 
     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. 
     4. Electroretinography (ERG) Recordings 
     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. 
     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). 
     Graph and statistical analysis were performed using GraphPad. 
     5. Optokinetic Test 
     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. 
     6. Immunohistochemistry and Confocal Imaging 
     Animals were sacrificed by CO 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. 
     
       
         
           
               
            
               
                   
               
               
                 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 
               
               
                   
               
            
           
         
       
     
     7. Cell Counts 
     Flat mount retinas of rd10 and huRhoP347S +/−  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 2  at different ages. 
     8. In Vitro Test of the Efficiency of Mouse GIRK2 
     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. 
     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). 
     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. 
     9. Patient Eye Fundus Imaging 
     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 
     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. 
       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 
     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&#39;s response to light. Thus two AAVs were injected subretinally to degenerating rd10 mouse retinas at p15 ( FIG.  5 A ). This led to a significant increase in phototpic ERG amplitudes in treated eyes compared to controls ( FIG.  5 B ). Flicker ERGs confirmed that the recovery mechanism was still active in these cone cells expressing GIRK2 allowing them to follow a fast stimulus ( FIG.  5 C ). The rd10 animals treated with GIRK2 showed also an improved optokinetic reflex compared to controls ( FIG.  5 D ). 
     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.  6 A-B ). Flicker ERGs were also robustly amplified with this approach ( FIG.  6 C ). 
     3. GIRK2-Mediated Vision Restoration: Long-Term Efficacy 
     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.  7 A ). Moreover, these results are consistent with the optokinetic test, both controls and treated eyes with GIRK2 show a decreased optokinetic reflex over time ( FIG.  7 B ). This decline was to be expected as cone numbers also decreased over time in the rd10 mice ( FIG.  7 C ). 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.  7 D ). 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 
     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 −/− -huRhoP347S +/−  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. 
     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.  9 A ). 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.  9 B ). This decline is to be expected as cone numbers also decreases over time in this RCD mouse model ( FIG.  9 C ). The decrease in time in ERG amplitudes also correlated with a decrease in cone numbers in this model ( FIG.  9 D ). 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 
     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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