Modified soluble vegf receptor-1 genes and vectors for gene therapy

Abstract

The present invention relates to a modified and optimized sFlt1 nucleic acid for inclusion in a virus vector. Use of such vectors can be used for treatment of ocular disorders causing neovascularization, such as macular degeneration.

1 . A method of preventing or reducing ocular neovascularization in a subject in need thereof comprising, directly administering to an eye of the subject a therapeutic amount of a vector recombinant adeno-associated viral (rAAV) vector comprising an optimized, modified coding sequence for soluble fms-like tyrosine kinase-1 (sFlt1) peptide operably linked to a promoter sequence to thereby deliver the vector to retinal cells of the subject, wherein the optimized, modified coding sequence has increased GC content and reduced cis motifs relative to a wild type sFlt1 coding sequence, wherein the optimized, modified coding sequence is expressed at a level which produces a therapeutically effective amount of the sFlt1 peptide in the retinal cells, to thereby prevent or reduce ocular neovascularization in the subject. 
     
     
         2 . The method of  claim 1 , wherein the optimized, modified coding sequence has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or compliment thereof. 
     
     
         3 . The method of  claim 1 , wherein the vector is a viral vector. 
     
     
         4 . The method of  claim 13  wherein the viral vector is a recombinant adeno-associated viral (rAAV) vector. 
     
     
         5 . The method of  claim 4 , wherein the rAAV vector has a capsid of an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and combinations thereof. 
     
     
         6 . The method of  claim 4 , wherein the rAAV vector has a capsid that is a chimera of 2 or more AAV serotype capsids. 
     
     
         7 . The method of  claim 1 , wherein the subject has exudative age-related macular degeneration, diabetic retinopathy, corneal neovascularization, choroidal neovascularization, neovascular glaucoma, cyclitis, Hippel-Lindau Disease, retinopathy of prematurity, pterygium, histoplasmosis, iris neovascularization, macular edema, glaucoma-associated neovascularization macular edema, diabetes, uveitis, or has received cataract surgery. 
     
     
         8 . A method for inhibiting VEGF in a cell comprising administering to the cell an optimized, modified coding sequence for soluble fms-like tyrosine kinase-1 (sFlt1) peptide operably linked to a promoter sequence, wherein the optimized, modified coding sequence has increased GC content and reduced cis motifs relative to a wild type sFlt1 coding sequence. 
     
     
         9 . The method of  claim 8 , wherein the optimized, modified coding sequence has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or compliment thereof. 
     
     
         10 . The method of  claim 8 , wherein the vector is a viral vector. 
     
     
         11 . The method of  claim 10 , wherein the viral vector is a recombinant adeno-associated viral (rAAV) vector. 
     
     
         12 . The method of  claim 11 , wherein the rAAV vector has a capsid of an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and combinations thereof. 
     
     
         13 . The method of  claim 11 , wherein the rAAV vector has a capsid that is a chimera of 2 or more AAV serotype capsids. 
     
     
         14 . A method for preventing or reducing choroidal neovascularization in a subject in need thereof comprising, directly administering to an eye of the subject a therapeutically effective amount of a vector comprising an optimized, modified coding sequence for soluble fms-like tyrosine kinase-1 (sFlt1) peptide operably linked to a promoter sequence to thereby deliver the vector to retinal cells of the subject, wherein the optimized, modified coding sequence has increased GC content and reduced cis motifs relative to a wild type sFlt1 coding sequence, wherein the optimized, modified coding sequence is expressed at a level which produces a therapeutically effective amount of the sFlt1 peptide in the retinal cells to thereby prevent or reduce choroidal neovascularization in the subject. 
     
     
         15 . The method of  claim 14 , wherein the optimized, modified coding sequence has the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or compliment thereof. 
     
     
         16 . The method of  claim 14 , wherein the vector is a viral vector. 
     
     
         17 . The method of  claim 16 , wherein the viral vector is a recombinant adeno-associated viral (rAAV) vector. 
     
     
         18 . The method of  claim 17 , wherein the rAAV vector has a capsid of an AAV selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and combinations thereof. 
     
     
         19 . The method of  claim 17 , wherein the rAAV vector has a capsid that is a chimera of 2 or more AAV serotype capsids.