Restoration of Visual Responses by In Vivo Delivery of Rhodopsin Nucleic Acids

Abstract

Nucleic acid vectors encoding light-gated cation-selective membrane channels, in particular channelrhodopsin-2 (Chop2), converted inner retinal neurons to photosensitive cells in photoreceptor-degenerated retina in an animal model. Such treatment restored visual perception and various aspects of vision. A method of restoring light sensitivity to a retina of a subject suffering from vision loss due to photoreceptor degeneration, as in retinitis pigmentosa or macular degeneration, is provided. The method comprises delivering to the subject by intravitreal or subretinal injection, the above nucleic acid vector which comprises an open reading frame encoding a rhodopsin, to which is operatively linked a promoter and transcriptional regulatory sequences, so that the nucleic acid is expressed in inner retinal neurons. These cells, normally light-insensitive, are converted to a light-sensitive state and transmit visual information to the brain, compensating for the loss, and leading to restoration of various visual capabilities.

We claim: 
     
         1 . A method of restoring light sensitivity to a retina, comprising:
 (a) delivering to retinal neurons a nucleic acid expression vector that encodes a light-gated channel rhodopsin or a light-driven ion pump rhodopsin expressible in said neurons, which vector comprises an open reading frame encoding the rhodopsin, and operatively linked thereto, a promoter sequence, and optionally, transcriptional regulatory sequences; and   (b) expressing said vector in said neurons,   thereby restoring light sensitivity.   
     
     
         2 . The method of  claim 1  wherein the rhodopsin is channelrhodopsin-2 (Chop2) with the sequence SEQ ID NO:6, or a biologically active fragment thereof, preferably SEQ ID NO:3, or a conservative amino acid substitution variant thereof. 
     
     
         3 . The method of  claim 1  wherein the vector is a rAAV viral vector. 
     
     
         4 . The method of  claim 1  wherein the promoter is a constitutive promoter. 
     
     
         5 . The method of  claim 4  wherein the constitutive promoter is a hybrid CMV enhancer/chicken 13-actin promoter (CAG) 
     
     
         6 . The method of  claim 2  wherein the promoter is a hybrid CAG. 
     
     
         7 . The method of  claim 4  wherein the constitutive promoter is a CMV promoter. 
     
     
         8 . The method of  claim 1  wherein the promoter is an inducible and/or a cell type-specific promoter. 
     
     
         9 . The method of  claim 8  wherein the cell type-specific promoter is selected from the group consisting of a mGluR6 promoter, a Pcp2 (L7) promoter or a neurokinin-3 (NK-3) promoter. 
     
     
         10 . The method of  claim 9  wherein the promoter is the mGlu6 promoter and is part of a promoter sequence SEQ ID NO:7. 
     
     
         11 . The method of  claim 1  wherein the vector comprises hybrid CMV enhancer/chicken (3-actin (CAG) promoter, a woodchuck posttranscriptional regulatory element (WPRE), and a human or bovine growth hormone polyadenylation sequence. 
     
     
         12 . The method of  claim 2  wherein the vector comprises a CAG promoter, a woodchuck post-transcriptional regulatory element (WPRE), and a human or bovine growth hormone polyadenylation sequence. 
     
     
         13 . The method of  claim 1  wherein the retinal neurons are selected from ON- and OFF-type retinal ganglion cells, retinal rod bipolar cells, All amacrine cells and ON and OFF retinal cone bipolar cells. 
     
     
         14 . The method of  claim 13  wherein the vector is targeted to and expressed in ON type ganglion cells and/or ON type bipolar cells. 
     
     
         15 . The method of  claim 14  wherein the vector comprises a mGluR6 promoter 
     
     
         16 . The method of  claim 15  wherein the mGluR6 promoter is part of a promoter sequence SEQ ID NO:7. 
     
     
         17 . The method of  claim 9  wherein the promoter is an NK-3 promoter and the vector is targeted to OFF cone bipolar cells. 
     
     
         18 . A method of restoring photosensitivity to retinal neurons of a subject suffering from vision loss or blindness in whom retinal photoreceptor cells are degenerating or have degenerated and died, which method comprises:
 (a) delivering to the retina of said subject a nucleic acid vector that encodes a light-gated channel rhodopsin or a light-driven ion pump rhodopsin expressible in said neurons; which vector comprises an open reading frame encoding the rhodopsin, and operatively linked thereto, a promoter sequence, and optionally, transcriptional regulatory sequences;   (b) expressing said vector in said neurons, wherein the expression of the rhodopsin renders said neurons photosensitive, thereby restoring of photosensitivity to said retina.   
     
     
         19 . The method of  claim 18  wherein the rhodopsin is Chop2 or a biologically active fragment or conservative amino acid substitution variant thereof. 
     
     
         20 . The method of  claim 18  wherein the vector is a rAAV viral vector. 
     
     
         21 . The method of  claim 18  wherein the promoter is a constitutive promoter. 
     
     
         22 . The method of  claim 21  wherein the constitutive promoter is a hybrid CAG promoter. 
     
     
         23 . The method of  claim 19  wherein the promoter is a hybrid CAG promoter. 
     
     
         24 . The method of  claim 21  wherein the constitutive promoter is a CMV promoter. 
     
     
         25 . The method of  claim 18  wherein the promoter is an inducible or a cell type-specific promoter. 
     
     
         26 . The method of  claim 25  wherein the cell type-specific promoter is selected from the group consisting of a mGluR6 promoter, a Pcp2 (L7) promoter or a neurokinin-3 (NK-3) promoter. 
     
     
         27 . The method of  claim 26  wherein the promoter is the mGlu6 promoter and is part of a promoter sequence SEQ ID NO:7. 
     
     
         28 . The method of  claim 18  wherein the retinal neurons are ON-type retinal ganglion cells, OFF-type retinal ganglion cells, retinal rod bipolar cells, All amacrine cells, ON-type retinal cone bipolar cells or OFF-type retinal cone bipolar cells. 
     
     
         29 . The method of  claim 28  wherein the vector is targeted to and expressed in ON type ganglion cells and/or ON type bipolar cells. 
     
     
         30 . The method of  claim 29  wherein the vector comprises a mGluR6 promoter 
     
     
         31 . The method of  claim 30  wherein the mGluR6 promoter is part of a promoter sequence SEQ ID NO:7. 
     
     
         32 . The method of  claim 26  wherein the promoter is the NK-3 promoter and the vector is targeted to OFF cone bipolar cells. 
     
     
         33 . The method  claim 18  wherein the restoration of photosensitivity results in restoration of vision in said subject. 34 The method of  claim 33  wherein said vision is measured by one or more of the following methods:
 (i) a light detection response by the subject after exposure to a light stimulus 
 (ii) a light projection response by the subject after exposure to a light stimulus; 
 (iii) light resolution by the subject of a light versus a dark patterned visual stimulus; 
 (iv) electrical recording of a response in the visual cortex to a light flash stimulus or a pattern visual stimulus 
 
     
     
         35 . The method of  claim 18  wherein said vision loss or blindness is a result of a degenerative disease. 
     
     
         36 . The method of  claim 35  wherein said disease is retinitis pigmentosa or age-related macular degeneration. 
     
     
         37 . The method of  claim 18  wherein the subject is also provided with a visual prosthesis before, at the same time as, or after delivery of said vector. 
     
     
         38 . The method of  claim 37 , wherein the visual prosthesis is a retinal implant, a cortical implant, a lateral geniculate nucleus implant, or an optic nerve implant. 
     
     
         39 . The method  claim 18 , wherein the subject's visual response is subjected to training using one or more visual stimuli. 
     
     
         40 . The method of  claim 38  wherein the subject's visual response is subjected to training using one or more visual stimuli. 
     
     
         41 . The method of  claim 39  wherein said training is achieved by one of more of the following methods:
 (a) habituation training characterized by training the subject to recognize (i) varying levels of light and/or pattern stimulation, and/or (ii) environmental stimulation from a common light source or object; and 
 (b) orientation and mobility training characterized by training the subject to detect visually local objects and move among said objects more effectively than without the training