Reagents and methods for modulating cone photoreceptor activity

Abstract

The present invention provides reagents and methods for modulating cone photoreceptor activity, and devices for assessment of cone photoreceptor activity.

We claim 
     
         1 . A method for cone cell gene therapy in a primate, comprising administering to the eye of a primate in need of cone cell gene therapy a recombinant gene delivery vector comprising
 (a) a promoter region, wherein the promoter region is specific for retinal cone cells; and   (b) a gene encoding a therapeutic, wherein the gene is operatively linked to the promoter region;   wherein in vivo expression of the therapeutic in cone cells of the primate serves to treat the primate in need of cone cell gene therapy.   
     
     
         2 . The method of  claim 1 , wherein the gene therapy serves to treat a cone cell disorder. 
     
     
         3 . The method of  claim 2 , wherein the cone cell disorder is selected from the group consisting of color blindness, blue cone monochomacy, achromatopsia, incomplete achromatopsia, rod-cone degeneration, retinitis pigmentosa (RP), macular degeneration, cone dystrophy, blindness, Stargardt's Disease, and Leber's congenital amaurosis. 
     
     
         4 . The method of  claim 1  wherein the primate is of the Parvorder Catarrhini. 
     
     
         5 . The method of  claim 1  wherein the promoter comprises a sequence selected from the group consisting of the L opsin promoter (SEQ ID NO: 1), the M opsin promoter (SEQ ID NO: 2), and the S opsin promoter (SEQ ID NO: 3), 
     
     
         6 . The method of  claim 1 , wherein the gene delivery vector further comprises an enhancer element upstream of the promoter, wherein the gene is operatively linked to the enhancer element. 
     
     
         7 . The method of  claim 6 , the enhancer element is specific for primate retinal cone cells. 
     
     
         8 . The method of  claim 7 , wherein the enhancer element comprises the nucleic acid sequence of SEQ ID NO: 51. 
     
     
         9 . The method of  claim 1 , wherein the gene delivery vector further comprises an intron comprising a splice donor/acceptor region, wherein the intron is located downstream of the promoter region and is located upstream of the gene. 
     
     
         10 . The method of  claim 1 , wherein the gene encodes a therapeutic protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and a polymorph of SEQ ID NO: 11 selected from the group consisting of:
 (i) Thr65Ile   (ii) Ile111Val   (iii) Ser116Tyr   (iv) Leu153Met   (v) Ile171Val   (vi) Ala174Val   (vii) Ile178Val   (viii) Ser180Ala   (ix) Ile230Thr   (x) Ala233Ser   (xi) Val236Met   (xii) Ile274Val   (xiii) Phe275Leu   (xiv) Tyr277Phe   (xv) Val279Phe   (xvi) Thr285Ala   (xvii) Pro298Ala; and   (xviii) Tyr309Phe.   
     
     
         11 . The method of  claim 1 , wherein the gene delivery vector comprises a recombinant adeno-associated virus (AAV) gene delivery vector. 
     
     
         12 . The method of  claim 11 , wherein the AAV gene vector comprises rAAV2/5 vector. 
     
     
         13 . The method of  claim 1 , wherein the method restores visual capacity in the primate. 
     
     
         14 . The method of  claim 1 , wherein the primate suffers from color blindness, and the primate is able to visualize new colors as a result of the therapy. 
     
     
         15 . The method of  claim 14 , wherein the promoter comprises a sequence selected from the group consisting of L opsin promoter (SEQ ID NO: 1), M opsin promoter (SEQ ID NO: 2), and S opsin promoter (SEQ ID NO: 3); and
 the gene encodes one or more polypeptides comprising a sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11; and a polymorph of SEQ ID NO: 11 selected from the group consisting of   (i) Thr65Ile   (ii) Ile111Val   (iii) Ser116Tyr   (iv) Leu153Met   (v) Ile171Val   (vi) Ala174Val   (vii) Ile178Val   (viii) Ser180Ala   (ix) Ile230Thr   (x) Ala233Ser   (xi) Val236Met   (xii) Ile274Val   (xiii) Phe275Leu   (xiv) Tyr277Phe   (xv) Val279Phe   (xvi) Thr285Ala   (xvii) Pro298Ala; and   (xviii) Tyr309Phe.   
     
     
         16 . The method of  claim 1 , wherein the primate has a vision disorder in which its photoreceptors are healthy. 
     
     
         17 . The method of  claims 1 , wherein the primate is an adult primate. 
     
     
         18 . An isolated nucleic acid expression vector comprising:
 (a) a promoter region, wherein the promoter region is specific for primate retinal cone cells; and   (b) a gene encoding a therapeutic, wherein the gene is operatively linked to the promoter region.   
     
     
         19 . The isolated nucleic acid expression vector of  claim 18 , wherein the promoter comprises a nucleic acid selected from the group consisting of L opsin promoter (SEQ ID NO: 1), M opsin promoter (SEQ ID NO: 2), and S opsin promoter (SEQ ID NO: 3). 
     
     
         20 . The isolated nucleic acid expression vector of  claim 18 , wherein the isolated nucleic acid expression vector further comprises an enhancer element upstream of the promoter, wherein the gene is operatively linked to the enhancer element. 
     
     
         21 . The isolated nucleic acid expression vector of  claim 20 , wherein the enhancer element comprises a nucleic acid sequence according to SEQ ID NO: 51. 
     
     
         22 . The isolated nucleic acid expression vector of  claim 18 , further comprising an intron comprising a splice donor/acceptor region, wherein the intron is located downstream of the promoter region and is located upstream of the gene. 
     
     
         23 . The isolated nucleic acid expression vector of  claim 18 , wherein the gene encodes a therapeutic protein selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, and a polymorph of SEQ ID NO: 11 selected from the group consisting of:
 (i) Thr65Ile   (ii) Ile111Val   (iii) Ser116Tyr   (iv) Leu153Met   (v) Ile171Val   (vi) Ala174Val   (vii) Ile178Val   (viii) Ser180Ala   (ix) Ile230Thr   (x) Ala233Ser   (xi) Val236Met   (xii) Ile274Val   (xiii) Phe275Leu   (xiv) Tyr277Phe   (xv) Val279Phe   (xvi) Thr285Ala   (xvii) Pro298Ala; and   (xviii) Tyr309Phe.   
     
     
         24 . The isolated nucleic acid expression vector of  claim 18 , wherein the vector comprises an AAV vector. 
     
     
         25 . A formulation comprising packaged viral particles comprising the nucleic acid expression vectors of  claim 18 . 
     
     
         26 . A recombinant host cells transected or transduced with the nucleic acid expression vector of  claim 18 . 
     
     
         27 . A wide field, color multi-focal electroretinogram (mf-ERG) comprising:
 (a) an electroretinogram (ERG) comprising
 (i) a recording electrode that is (A) designed for placement on at least one of a cornea and a sclera of at least one eye of a subject and (B) arranged to output at least one signal generated by the at least one eye; and 
 (ii) a computing system communicatively coupled to the recording electrode, the computing system comprising (A) at least one processor and (B) data storage containing instructions executable by the at least one processor to carry out a set of functions, the set of functions including processing and saving the at least one signal generated by the at least one eye; 
   (b) a retinal stimulator comprising matched light sources selected from the group consisting of red, green, blue, and ultraviolet light sources, wherein the matched light sources are connected to the ERG and in operation can be independently frequency modulated at rates between about 1 Hz and about 60 Hz, inclusive, wherein the stimulator in operation is capable of stimulating a retinal field of a subject throughout an operating radius of at least about 70 degrees;   (c) one or more constant current integrated circuit chips arranged to drive the stimulator; and   (d) a pulse-frequency modulator connected to the retinal stimulator, wherein in operation the pulse-frequency modulator is capable of controlling individual stimulator segments while keeping relative spectral content of the light constant.   
     
     
         28 . The mf-ERG of  claim 27 , wherein the matched light sources are paired red and green light sources. 
     
     
         29 . The mf-ERG of  claim 27 , wherein the matched light sources are triplets of red, green, and blue light sources. 
     
     
         30 . The mf-ERG of  claim 27 , wherein the matched light sources are quartets of red, green, blue, and ultraviolet light sources. 
     
     
         31 . The mf-ERG of  claim 27 , wherein the matched light sources comprise matched light emitting diodes (LEDs). 
     
     
         32 . The mf-ERG of  claim 27 , wherein the retinal stimulator comprises a concave surface comprising a series of trapezoidal-shaped circuit boards placed edge-to-edge, wherein the concave surface positions the matched light sources so in operation they are held equidistantly from and pointing toward a single focal point where a subject's pupil can be positioned. 
     
     
         33 . The mf-ERG of  claim 32 , wherein the concave surface comprises a geodesic dome. 
     
     
         34 . The mf-ERG of  claim 27 , wherein the set of functions further comprises coding and decoding topographical regions on the recording electrode using a cyclic summation technique. 
     
     
         35 . The mf-ERG of  claim 27 , wherein the ERG further comprises an amplifier; and wherein the computing system is communicatively coupled to the amplifier. 
     
     
         36 . A method for determining a location of functioning opsin expression in a subject, comprising use of the mf-ERG of  claim 27 , wherein the recording electrode is placed on at least one of a cornea and a sclera of at least one eye of a subject; stimulation of the subject's retinal field with the retinal stimulator; and determining responses of different areas of the subject's retina to different stimulation frequencies to generate a map of retinal responses, wherein the map provides a location of functioning opsin expression in a subject. 
     
     
         37 . (canceled)