US2012093772A1PendingUtilityA1

Vectors for delivery of light sensitive proteins and methods of use

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Assignee: HORSAGER ALANPriority: May 20, 2008Filed: May 20, 2009Published: Apr 19, 2012
Est. expiryMay 20, 2028(~1.9 yrs left)· nominal 20-yr term from priority
Inventors:Alan HorsagerWilliam W. HauswirthJianwen LiuBenjamin C. MatteoEdward S. Boyden
A61P 27/00A61P 27/02C12N 2830/008C12N 2799/025C07K 14/70571C07K 14/723A61K 48/005C12N 2710/10043A61K 48/0058A61K 45/06C12N 15/86C12N 15/63C12N 15/11A61K 48/00
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Claims

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.

Claims

exact text as granted — not AI-modified
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% identical to ChR2. 
     
     
         4 . The nucleic acid of  claim 1  wherein the mGluR6 regulatory sequence fragment comprises less than about 1000 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 - 22 . (canceled) 
     
     
         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 . (canceled) 
     
     
         26 . The method of  claim 23  wherein the light-sensitive protein is ChR2 or a light-sensitive protein that is at least about 70% identical to ChR2. 
     
     
         27 . The method of  claim 23  wherein the mGluR6 regulatory sequence fragment is less than about 1000 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 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 10% transduction efficiency. 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 37  wherein the exogenous nucleic acid comprises a light-sensitive protein. 
     
     
         41 . (canceled) 
     
     
         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% identical to ChR2. 
     
     
         43 . The method of  claim 37  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 base pairs. 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 36  wherein the exogenous nucleic acid is introduced using a recombinant adeno-associated viral vector (AAV). 
     
     
         47 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 . (canceled) 
     
     
         55 . The method of  claim 53  wherein the exogenous nucleic acid comprises a light-sensitive protein. 
     
     
         56 . (canceled) 
     
     
         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% 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 base pairs. 
     
     
         61 . The method of  claim 59  wherein the mGluR6 regulatory sequence fragment is represented by the sequence in  FIG. 6 . 
     
     
         62 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 - 83 . (canceled)

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