Aav-mediated subcellular targeting of heterologous rhodopsins in retinal ganglion cells

Abstract

Microbial type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2/ChR2) or the ion pump halorhodopsin (HaloR) are expressed in retinal ganglion cells upon transduction using recombinant AAV vectors. Selective targeting of these transgenes for expression in discrete subcellular regions or sites is achieved by including a sorting motif in the vector that can target either the central area or surround (off-center) area of these cells. Nucleic acid molecules comprising nucleotide sequences encoding such rhodopsins and sorting motifs and their use in methods of differential expression of the transgene are disclosed. These compositions and methods provide significant improvements for restoring visual perception and various aspects of vision, particular in patients with retinal disease.

1 - 38 . (canceled) 
     
     
         39 . A polynucleotide molecule comprising a nucleic acid sequence encoding a rhodopsin for differential expression in subcellular regions of a retinal neuron, comprising:
 (a) a first nucleotide sequence encoding a light-gated channel rhodopsin or a light-driven ion pump rhodopsin;   (b) linked in frame to (a), a second nucleotide sequence encoding a peptide or polypeptide sorting motif, wherein the second nucleotide sequence is selected from:
 (1) a nucleotide sequence encoding nicotinic acetylcholine receptor α7 subunit (nAchR) comprising SEQ ID NO:9, 
 (2) a nucleotide sequence encoding voltage-gated potassium channel 4.2 (Kv4.2) comprising SEQ ID NO:11, 
 (3) a nucleotide sequence encoding telencephalin (TLCN) comprising SEQ ID NO:13, and 
 (4) a nucleotide sequence encoding AMPA receptor GluR1 subunit comprising SEQ ID NO:15; 
   (c) operatively linked to (a) and (b), a promoter sequence; and   (d) a polyadenylation sequence.   
     
     
         40 . The polynucleotide molecule of  claim 39 , wherein the promoter sequence is a cytomegalovirus enhancer/chicken β-actin promoter (CAG), and wherein the polyadenylation sequence is selected from:
 (i) a polyadenylation sequence from bovine growth hormone (bGHpolyA), and 
 (ii) a SV40-derived polyadenylation sequence. 
 
     
     
         41 . The polynucleotide molecule of  claim 39 , wherein (c) further comprises a transcriptional regulatory sequence, and wherein the transcriptional regulatory sequence is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 
     
     
         42 . The polynucleotide molecule of  claim 39 , further comprising, linked in frame with (a) and (b), a third nucleotide sequence encoding a reporter polypeptide. 
     
     
         43 . The polynucleotide molecule of  claim 39 , wherein the sorting motif targets the rhodopsin to the surround or off-center of the neuron's receptive field. 
     
     
         44 . The polynucleotide molecule of  claim 39 , wherein the sorting motif targets the rhodopsin to the somatodendritic region of the neurons. 
     
     
         45 . A recombinant adeno-associated virus-2 (rAAV2) expression vector comprising a nucleic acid molecule encoding a rhodopsin for differential expression in subcellular regions of a retinal neuron comprising:
 (a) a first nucleotide sequence encoding a light-gated channel rhodopsin or a light-driven ion pump rhodopsin;   (b) linked in frame to (a), a second nucleotide sequence encoding a peptide or polypeptide sorting motif, wherein the second nucleotide sequence is selected from:
 (1) a nucleotide sequence encoding nicotinic acetylcholine receptor α7 subunit (nAchR) comprising SEQ ID NO:9, 
 (2) a nucleotide sequence encoding voltage-gated potassium channel 4.2 (Kv4.2) comprising SEQ ID NO:11, 
 (3) a nucleotide sequence encoding telencephalin (TLCN) comprising SEQ ID NO:13, and 
 (4) a nucleotide sequence encoding AMPA receptor GluR1 subunit comprising SEQ ID NO:15; 
   (c) operatively linked to (a) and (b), a promoter sequence; and   (d) a polyadenylation sequence,   wherein the nucleic acid molecule is flanked at its 5′ end by a 5′ inverted terminal repeat (ITR) and at its 3′ end by a 3′ ITR of the AAV2, wherein nucleotide sequence of the 5′-ITR is set forth in SEQ ID NO: 17, and wherein nucleotide sequence of the 3′-ITR is set forth in SEQ ID NO: 18.   
     
     
         46 . The rAAV2 expression vector of  claim 45 , wherein the promoter sequence is a cytomegalovirus enhancer/chicken β-actin promoter (CAG), and wherein the polyadenylation sequence is selected from:
 (i) a polyadenylation sequence from bovine growth hormone (bGHpolyA), and 
 (ii) a SV40-derived polyadenylation sequence. 
 
     
     
         47 . The rAAV2 expression vector of  claim 45 , further comprising a transcriptional regulatory sequence, and wherein the transcriptional regulatory sequence is woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 
     
     
         48 . The rAAV2 expression vector of  claim 45 , further comprising, linked in frame with (a) and (b), a third nucleotide sequence encoding a reporter polypeptide. 
     
     
         49 . The rAAV2 expression vector of  claim 45 , further comprising the AAV vector backbone nucleotide sequence set forth in SEQ ID NO:29 linked to the 3′ end of the AAV 3′-ITR sequence. 
     
     
         50 . A recombinant adeno-associated virus-2 (rAAV2) expression vector comprising a schematic structure selected from:
 (a) 5′-ITR-CAG-ChR2-GFP-{Motif}-WPRE-bGHpolyA-ITR-3′,   (b) 5′-ITR-CAG-ChR2-{Motif}-WPRE-bGHpolyA-ITR-3′,   (c) 5′-ITR-CAG-HalOR-GFP-{Motif}-WPRE-bGHpolyA-ITR-3′, and   (d) 5′-ITR-CAG-HaloR-{Motif}-WPRE-bGHpolyA-ITR-3′;   wherein:   ITR is a AAV2 inverted terminal repeat,   Motif is a nucleotide sequence encoding a sorting motif, and   wherein any two or more of the ChR2, GFP, and Motif, or HaloR, GFP, and Motif, are linked in frame.   
     
     
         51 . The rAAV2 expression vector of  claim 50 , wherein the vector comprises a nucleotide sequence set forth in SEQ ID NO:30 or 31. 
     
     
         52 . The rAAV2 expression vector of  claim 50 , wherein the nucleotide sequence encoding a sorting motif is selected from:
 (1) a nucleotide sequence encoding nicotinic acetylcholine receptor α7 subunit (nAchR) comprising SEQ ID NO:9,   (2) a nucleotide sequence encoding voltage-gated potassium channel 4.2 (Kv4.2) comprising SEQ ID NO:11,   (3) a nucleotide sequence encoding telencephalin (TLCN) comprising SEQ ID NO:13, and   (4) a nucleotide sequence encoding AMPA receptor GluR1 subunit comprising SEQ ID NO:15.   
     
     
         53 . The rAAV2 expression vector of  claim 50 , further comprising the AAV vector backbone nucleotide sequence set forth in SEQ ID NO:29 linked to the 3′ end of the AAV 3′-ITR sequence. 
     
     
         54 . A method of restoring light sensitivity to a retina, comprising:
 (a) delivering to a retinal neuron the polynucleotide molecule comprising a nucleic acid sequence encoding a rhodopsin of  claim 39 , and   (b) expressing the polynucleotide molecule in the retinal neuron,   wherein the expression of the polynucleotide molecule results in selected expression of the rhodopsin in subcellular regions of the retinal neuron, thereby restoring light sensitivity.   
     
     
         55 . A method of selectively expressing a light-gated channel rhodopsin or a light-driven ion pump rhodopsin in a desired subcellular site or sites of a retinal ganglion cell (RGC), comprising:
 (a) delivering to the RGC the polynucleotide molecule of  claim 39 ; and   (b) expressing the polynucleotide molecule in the desired sites of said RGC.   
     
     
         56 . A method of restoring light sensitivity to a retina, comprising:
 (a) delivering to a retinal neuron the rAAV2 expression vector comprising a nucleic acid molecule encoding a rhodopsin for differential expression in subcellular regions of the retinal neuron of  claim 45 , and   (b) expressing the rAAV2 expression vector in the retinal neuron,   wherein the expression of the rAAV2 expression vector results in selected expression of the rhodopsin in subcellular regions of the retinal neuron, thereby restoring light sensitivity.   
     
     
         57 . A method of selectively expressing a light-gated channel rhodopsin or a light-driven ion pump rhodopsin in a desired subcellular site or sites of a retinal ganglion cell (RGC), comprising:
 (a) delivering to the RGC the rAAV2 expression vector of  claim 45 ; and   (b) expressing the vector in the desired sites of said RGC.   
     
     
         58 . A method of restoring light sensitivity to a retina, comprising:
 (a) delivering to a retinal neuron the rAAV2 expression vector comprising a nucleic acid molecule encoding a rhodopsin for differential expression in subcellular regions of the retinal neuron of  claim 50 , and   (b) expressing the rAAV2 expression vector in the retinal neuron,   wherein the expression of the rAAV2 expression vector results in selected expression of the rhodopsin in subcellular regions of the retinal neuron, thereby restoring light sensitivity.   
     
     
         59 . A method of selectively expressing a light-gated channel rhodopsin or a light-driven ion pump rhodopsin in a desired subcellular site or sites of a retinal ganglion cell (RGC), comprising:
 (a) delivering to the RGC the rAAV2 expression vector of  claim 50 ; and   (b) expressing the vector in the desired sites of said RGC.