Vectors for delivery of light sensitive proteins and methods of use

Abstract

Provided herein are compositions and methods for gene and etiology-nonspecific and circuit-specific treatment of diseases, utilizing vectors for delivery of light-sensitive proteins to diseased and normal cells and tissues of interest.

What is claimed is: 
     
         1 . A recombinant nucleic acid comprising a nucleic acid encoding a light-sensitive protein operatively linked to a metabotropic glutamate receptor 6 (mGluR6) regulatory sequence or fragment thereof. 
     
     
         2 . The nucleic acid of  claim 1  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         3 . The nucleic acid of  claim 1  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         4 . The nucleic acid of  claim 1  wherein the mGluR6 regulatory sequence fragment comprises less than about 1000, less than about 750, less than about 500, less than about 250, or less than about 100 base pairs. 
     
     
         5 . The nucleic acid of  claim 1  wherein the mGluR6 regulatory sequence or fragment thereof is an mGluR6 promoter or enhancer. 
     
     
         6 . The nucleic acid of  claim 1  further comprising a green fluorescent protein 
     
     
         7 . The nucleic acid of  claim 1  wherein the nucleic acid is encapsidated within a recombinant adeno-associated virus (AAV) 
     
     
         8 . The nucleic acid of  claim 7  wherein the recombinant adeno-associated virus is of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. 
     
     
         9 . The nucleic acid of  claim 7  wherein the recombinant adeno-associated virus is of a serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, and hybrids thereof. 
     
     
         10 . The nucleic acid of  claim 1  wherein the nucleic acid is encapsidated within a recombinant virus selected from the group consisting of recombinant adeno-associated virus (AAV), recombinant retrovirus, recombinant lentivirus, and recombinant poxvirus. 
     
     
         11 . A vector comprising a nucleic acid encoding a light-sensitive protein, said nucleic acid operatively linked to a metabotropic glutamate receptor 6 (mGluR6) regulatory sequence or fragment thereof. 
     
     
         12 . The vector of  claim 11  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         13 . The nucleic acid of  claim 11 , wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         14 . The vector of  claim 11  wherein the mGluR6 regulatory sequence fragment is less than about 1000, less than about 750, less than about 500, less than about 250, or less than about 100 base pairs. 
     
     
         15 . The vector of  claim 11  wherein the mGluR6 regulatory sequence fragment is represented by the sequence in  FIG. 6 . 
     
     
         16 . The vector of  claim 11  wherein the vector comprises a recombinant adeno-associated virus (AAV). 
     
     
         17 . The vector of  claim 11  wherein the vector comprises a recombinant virus selected from the group consisting of recombinant adeno-associated virus (AAV), recombinant retrovirus, recombinant lentivirus, and recombinant poxvirus. 
     
     
         18 . The vector of  claim 16  wherein the AAV is of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. 
     
     
         19 . The vector of  claim 18  wherein the AAV comprises mutated capsid protein. 
     
     
         20 . The vector of  claim 19  wherein the capsid protein comprises a mutated tyrosine residue. 
     
     
         21 . The vector of  claim 20  wherein the mutated tyrosine residue is selected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. 
     
     
         22 . The vector of  claim 20  wherein the mutated capsid protein comprises a tyrosine residue mutated to a phenylalanine. 
     
     
         23 . A method of treating a subject suffering from a disease or disorder of the eye comprising introducing into an affected eye a recombinant adeno-associated virus (AAV) comprising a light-sensitive protein operatively linked to a metabotropic glutamate receptor 6 regulatory sequence (mGluR6 regulatory sequence) or fragment thereof. 
     
     
         24 . The method of  claim 23  wherein the disease or disorder of the eye is caused by photoreceptor cell degeneration. 
     
     
         25 . The method of  claim 23  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         26 . The method of  claim 23  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         27 . The method of  claim 23  wherein the mGluR6 regulatory sequence fragment is less than about 1000, less than about 750, less than about 500, less than about 250, or less than about 100 base pairs. 
     
     
         28 . The method of  claim 27  wherein the mGluR6 regulatory sequence fragment is represented by the sequence in  FIG. 6 . 
     
     
         29 . The method of  claim 23  wherein the AAV is of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. 
     
     
         30 . The method of  claim 29  wherein the AAV comprises a mutated capsid protein. 
     
     
         31 . The method of  claim 30  wherein the capsid protein comprises a mutated tyrosine residue. 
     
     
         32 . The method of  claim 31  wherein the mutated tyrosine residue is selected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. 
     
     
         33 . The vector of  claim 31  wherein the mutated capsid protein comprises a tyrosine residue mutated to a phenylalanine. 
     
     
         34 . The method of  claim 23  wherein the AAV is introduced using intravitreal injection, subretinal injection and/or ILM peel. 
     
     
         35 . The method of  claim 23  wherein the AAV is introduced into a retinal bipolar cell. 
     
     
         36 . The method of  claim 23  wherein the method further comprises using a light-generating device external to the eye. 
     
     
         37 . A method of expressing an exogenous nucleic acid in a retinal bipolar cell comprising introducing into a retina a vector comprising the exogenous nucleic operatively linked to a retinal bipolar cell-specific regulatory sequence wherein the method results in at least about a 25-30% transduction efficiency. 
     
     
         38 . The method of  claim 36  wherein the method results in at least about a 40%, 50%, 60%, 70%, 80%, or 90% transduction efficiency. 
     
     
         39 . The method of  claim 36  wherein the transduction efficiency is measured by quantifying the total number of retinal bipolar cells infected. 
     
     
         40 . The method of  claim 36  wherein the exogenous nucleic acid comprises a light-sensitive protein. 
     
     
         41 . The method of  claim 40  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         42 . The nucleic acid of  claim 40  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         43 . The method of  claim 36  wherein the regulatory sequence comprises a metabotropic glutamate receptor 6 regulatory sequence (mGluR6) or a fragment thereof. 
     
     
         44 . The method of  claim 43  wherein the mGluR6 regulatory sequence fragment is less than about 1000, less than about 750, less than about 500, less than about 250, or less than about 100 base pairs. 
     
     
         45 . The method of  claim 43  wherein the mGluR6 regulatory sequence fragment is represented by the sequence in  FIG. 6 . 
     
     
         46 . The method of  claim 36  wherein the exogenous nucleic acid is introduced using a recombinant adeno-associated viral vector (AAV). 
     
     
         47 . The method of  claim 46  wherein the AAV comprises a mutated capsid protein. 
     
     
         48 . The method of  claim 47  wherein the capsid protein comprises a mutated tyrosine residue. 
     
     
         49 . The method of  claim 48  wherein the mutated tyrosine residue is selected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. 
     
     
         50 . The vector of  claim 48  wherein the mutated capsid protein comprises a tyrosine residue mutated to a phenylalanine. 
     
     
         51 . The method of  claim 46  wherein the exogenous nucleic acid is introduced using intravitreal injection, subretinal injection, and/or ILM peel. 
     
     
         52 . The method of  claim 46  wherein the AAV is of a serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. 
     
     
         53 . A method of introducing an exogenous nucleic acid into the nucleus of a retinal cell comprising introducing a vector comprising an exogenous nucleic acid operatively linked to a retinal cell-specific regulatory sequence into a retinal cell, wherein the vector is specifically designed to avoid ubiquitin-mediated protein degradation. 
     
     
         54 . The method of  claim 53  wherein the degradation is proteasome-mediated. 
     
     
         55 . The method of  claim 53  wherein the exogenous nucleic acid comprises a light-sensitive protein. 
     
     
         56 . The method of  claim 55  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         57 . The nucleic acid of  claim 55  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         58 . The method of  claim 53  wherein the retinal cell is a retinal bipolar cell. 
     
     
         59 . The method of  claim 58  wherein the regulatory sequence comprises a metabotropic glutamate receptor 6 regulatory sequence (mGluR6) or fragment thereof. 
     
     
         60 . The method of  claim 59  wherein the mGluR6 fragment is less than 1000, 750, 500, 250, or 100 base pairs. 
     
     
         61 . The method of  claim 59  wherein the mGluR6 regulatory sequence fragment is represented by the sequence in  FIG. 6 . 
     
     
         62 . The method of  claim 53  wherein the vector is selected from the group consisting of recombinant adeno-associated virus (AAV), recombinant retrovirus, recombinant lentivirus, and recombinant poxvirus. 
     
     
         63 . The method of  claim 53  the vector is a recombinant adeno-associated viral vector (AAV). 
     
     
         64 . The method of  claim 63  wherein the AAV is of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof. 
     
     
         65 . The method of  claim 63  wherein the AAV comprises a mutated capsid protein. 
     
     
         66 . The method of  claim 65  wherein the capsid protein comprises a mutated tyrosine residue. 
     
     
         67 . The method of  claim 66  wherein the mutated tyrosine residue is selected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. 
     
     
         68 . The vector of  claim 66  wherein the mutated capsid protein comprises a tyrosine residue mutated to a phenylalanine. 
     
     
         69 . The method of  claim 53  wherein the vector is introduced using intravitreal injection, subretinal injection, and/or ILM peel. 
     
     
         70 . A method of transducing a retinal bipolar cell comprising introducing into a retina a vector comprising an exogenous nucleic acid operatively linked to a regulatory sequence. 
     
     
         71 . The method of  claim 70  wherein the regulatory sequence is a non-cell type specific promoter. 
     
     
         72 . The method of  claim 70 , wherein the regulatory sequence is a guanine nucleotide binding protein alpha activating activity polypeptide O (GNAO1) promoter or a fusion of the cytomegalovirus (CMV) immediate early enhancer and the bovine β-actin promoter plus intron1-exon1 junction (CBA, smCBA). 
     
     
         73 . The method of  claim 70  wherein the exogenous nucleic acid comprises a light-sensitive protein. 
     
     
         74 . The method of  claim 73  wherein the light-sensitive protein is selected from the group consisting of ChR1, ChR2, VChR1, ChR2 C128A, ChR2 C128S, ChR2 C128T, ChR1-ChR2 hybrids/chimeras, ChD, ChEF, ChF, ChIEF, NpHR, eNpHR, melanopsin, and variants thereof. 
     
     
         75 . The nucleic acid of  claim 73  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70%, at least about 80%, at least about 90% or at least about 95% identical to ChR2. 
     
     
         76 . The method of  claim 70  wherein the vector is selected from the group consisting of recombinant adeno-associated virus (AAV), recombinant retrovirus, recombinant lentivirus, and recombinant poxvirus. 
     
     
         77 . The method of  claim 70  the vector is a recombinant adeno-associated viral vector (AAV). 
     
     
         78 . The method of  77  wherein the AAV is of a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and
 hybrids thereof. 
 
     
     
         79 . The method of  77  wherein the AAV comprises a mutated capsid protein. 
     
     
         80 . The method of  claim 79  wherein the capsid protein comprises a mutated tyrosine residue. 
     
     
         81 . The method of  claim 79  wherein the mutated tyrosine residue is selected from the group consisting of Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F and Y720F. 
     
     
         82 . The vector of  claim 80  wherein the mutated capsid protein comprises a tyrosine residue mutated to a phenylalanine. 
     
     
         83 . The method of  claim 70  wherein the vector is introduced using intravitreal injection, subretinal injection, and/or ILM peel.