Treatment of amd using aav sflt-1

Abstract

The present disclosure provides compositions and methods for the prevention or treatment of ocular neovascularization, such as AMD, in a human subject, by administering subretinally a pharmaceutical composition comprising a pharmaceutically effective amount of a vector comprising a nucleic acid encoding soluble Fms-related tyrosine kinase-1 (sFlt-1) protein to the human subject.

What is claimed is: 
     
         1 . A method for the treatment or prophylaxis of ocular neovascularization in a human subject comprising: administering to one or more subretinal sites a pharmaceutically effective amount of a pharmaceutical composition comprising a nucleic acid encoding sFLT-1 to a human subject in need of treatment. 
     
     
         2 . The method of  claim 1 , wherein the human subject has or is suspected of having one or more conditions selected from the group consisting of: age-related macular degeneration (AMD), wet-AMD, dry-AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy. 
     
     
         3 . The method of  claim 1 , wherein the human subject has or is suspected of having one or more conditions selected from the group consisting of: proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branched retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema. 
     
     
         4 . The method of  claim 1 , wherein the pharmaceutical composition comprises a recombinant virus, the virus selected from the group consisting of: adeno-associated virus (AAV), adenovirus, helper-dependent adenovirus, retrovirus, herpes simplex virus, lentivirus, poxvirus, hemagglutinatin virus of Japan-liposome (HVJ) complex, Moloney murine leukemia virus, and HIV-based virus. 
     
     
         5 . The method of  claim 1 , wherein the sFLT-1 nucleic acid encodes at least 1 dimerization domain. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid does not contain a prokaryotic regulatory sequence. 
     
     
         7 . The method of  claim 1 , further comprising administration of one or more treatments of a VEGF inhibitor to the human subject. 
     
     
         8 . The method of  claim 7 , wherein the VEGF inhibitor is administered within 30, 90, or 180 days of administration of the pharmaceutical composition. 
     
     
         9 . The method of  claim 8 , wherein the pharmaceutical composition and the VEGF inhibitor are administered at least 24 hours apart. 
     
     
         10 . The method of  claim 1 , wherein the administering is performed outside the fovea. 
     
     
         11 . The method of  claim 1 , wherein the best corrected visual acuity (BCVA) of the human subject, improves by at least 1, 2, 3, 4 or 5 lines as measured by ETDRS (Early Treatment Diabetic Retinopathy Study) letters following the administering of the pharmaceutical composition. 
     
     
         12 . The method of  claim 1 , wherein the best corrected visual acuity (BCVA) decreases by fewer than 15 letters as measured by ETDRS (Early Treatment Diabetic Retinopathy Study) following the administering of the pharmaceutical composition. 
     
     
         13 . The method of  claim 1 , wherein administering the pharmaceutical composition is performed under conditions selected from the group consisting of: administering the pharmaceutical composition in one eye, administering the pharmaceutical composition sequentially in two eyes, and administering the pharmaceutical composition simultaneously in two eyes. 
     
     
         14 . The method of  claim 1 , wherein a reduction in neovascularization, as observed by a Fluorscein Angiography (FA), follows the administering of the pharmaceutical composition. 
     
     
         15 . The method of  claim 1 , wherein no superficial, anterior segment or vitreous inflammatory signs are present in the human subject at least 1 week after injection. 
     
     
         16 . The method of  claim 1 , wherein the human subject does not require rescue treatment for at least 30, 60, 90, 120, 180, 270 or 365 days after the administering of the pharmaceutical composition. 
     
     
         17 . The method of  claim 1 , wherein the human subject has sensitivity or allergy to penicillin. 
     
     
         18 . The method of  claim 1 , wherein the administration does not increase systemic levels of sFlt-1 in the human subject. 
     
     
         19 . The method of  claim 4 , wherein no increased anti-AAV cytotoxic T cell response is measured following the administering step. 
     
     
         20 . The method of  claim 4 , wherein no virus is detected in the human subject's blood, saliva or urine samples, 3, 7, 14, 21 or 30 days after administering the pharmaceutical composition. 
     
     
         21 . The method of  claim 1  wherein the sFLT-1 protein level in the vitreous of the human subject is about 500-5,000 pg/ml, 7, 14, 21, 30, 60, 90, 120, 150, 180, 270 or 365 days after administering the pharmaceutical composition in the human subject. 
     
     
         22 . The method of  claim 1 , wherein the human subject has received one or more treatments with a VEGF inhibitor prior to the administering of the pharmaceutical composition. 
     
     
         23 . The method of  claim 1 , wherein the human subject has not previously received treatment with a VEGF inhibitor before the administering of the pharmaceutical composition. 
     
     
         24 . The method of  claim 1 , wherein the administering of the pharmaceutical composition is performed at a frequency less than 3 times a year in the human subject. 
     
     
         25 . The method of  claim 1 , wherein the administering of the pharmaceutical composition reduces the frequency of administration of additional VEGF inhibitor treatments in the human subject. 
     
     
         26 . The method of  claim 1 , wherein the concentration of sFLT-1 protein in the vitreous of the human subject is elevated when measured at 7, 14, 21, 30, 60, 90, 120, 150, 180, 270 or 365 days after the administering of the pharmaceutical composition. 
     
     
         27 . The method of  claim 1 , wherein the human subject has the vitreous gel removed prior to or within one week of the administration of said agent. 
     
     
         28 . The method of  claim 1 , wherein said pharmacological agent is administered using a vitrectomy system that is smaller than 20 gauge. 
     
     
         29 . The method of  claim 1 , wherein said pharmacological agent is administered using a cannula tip that is smaller than 39 gauge. 
     
     
         30 . The method of  claim 1 , wherein the central retinal thickness of said subject does not increase by more than 50 microns, 100 microns, or 250 microns within 12 months following treatment with said pharmacological agent. 
     
     
         31 . A pharmaceutical composition comprising recombinant viruses or plasmids comprising a nucleic acid comprising at least 1 promoter sequence operatively linked to an sFLT-1 transgene sequence, wherein the promoter sequence and the sFLT-1 transgene sequence are separated by a UTR sequence. 
     
     
         32 . The pharmaceutical composition of  claim 31 , wherein the promoter sequence and the sFLT-1 transgene sequence are separated by a sequence greater than 300 base pairs. 
     
     
         33 . The pharmaceutical composition of  claim 31 , wherein the nucleic acid sequence comprises at least 3 linker sequences each comprising at least 50 base pairs. 
     
     
         34 . A pharmaceutical composition comprising nucleic acid elements in the following order:
 a. a promoter sequence selected from the group consisting of SEQ ID No. 1, 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, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No.11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, 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 and SEQ ID No. 32;   b. a sequence encoding a VEGF inhibitor selected from the group consisting of SEQ ID No. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108 and SEQ ID No. 122;   c. an intron sequence consisting of SEQ ID No. 48, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119 and SEQ ID No. 120;   d. a UTR sequence selected from the group consisting of SEQ ID No. 91, SEQ ID No. 2, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99, SEQ ID No.100, and SEQ ID No. 101; and   e. a termination sequence selected from the group consisting of SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, and SEQ ID No. 55.   
     
     
         35 . A pharmaceutical composition comprising nucleic acid elements in the following order:
 a. an ITR sequence selected from the group consisting of SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59;   b. a linker sequence selected from the group consisting of SEQ ID No. 60, SEQ ID No. 63, SEQ ID No. 68, SEQ ID No. 72, SEQ ID No. 76, SEQ ID No. 77, SEQ ID No. 84;   c. a promoter sequence selected from the group consisting of SEQ ID No. 1, 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, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No.11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, 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 and SEQ ID No. 32;   d. a linker sequence selected from the group consisting of SEQ ID No. 60, SEQ ID No. 64, SEQ ID No. 70, SEQ ID No. 71, SEQ ID No. 73, SEQ ID No. 74, SEQ ID No. 87;   e. an intron sequence consisting of SEQ ID No. 48, SEQ ID No. 115, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 118, and SEQ ID No. 119;   f. a linker sequence selected from the group consisting of SEQ ID No. 60, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 75, SEQ ID No. 79, SEQ ID No. 81, SEQ ID No. 82, SEQ ID No. 89;   g. a UTR sequence selected from the group consisting of SEQ ID No. 91, SEQ ID No. 2, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99, SEQ ID No.100, and SEQ ID No. 101; and   h. a sequence encoding a VEGF inhibitor selected from the group consisting of SEQ ID No. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 107, SEQ ID No. 108 and SEQ ID No. 122;   i. a UTR sequence selected from the group consisting of SEQ ID No. 91, SEQ ID No. 2, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 95, SEQ ID No. 96, SEQ ID No. 97, SEQ ID No. 98, SEQ ID No. 99, SEQ ID No.100, and SEQ ID No. 101;   j. a linker sequence selected from the group consisting of SEQ ID No. 60, SEQ ID No. 62, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 69, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 85, SEQ ID No. 86;   k. a termination sequence selected from the group consisting of SEQ ID No. 49, SEQ ID No. 50, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, and SEQ ID No. 55;   l. a linker sequence selected from the group consisting of SEQ ID No. 60, SEQ ID No. 61, SEQ ID No. 80, SEQ ID No. 87, SEQ ID No. 88, SEQ ID No. 89, SEQ ID No. 90; and   m. an ITR sequence selected from the group consisting of SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, and SEQ ID No. 59.   
     
     
         36 . A unit dose of a pharmaceutical composition comprising recombinant viruses of 1×10 10  to 3×10 12  vector genomes, wherein the recombinant viruses comprise a nucleic acid encoding sFLT-1 operatively linked to a promoter. 
     
     
         37 . A method of generating a recombinant virus in a cell, the method comprising:
 a. introducing into a cell, a nucleic acid comprising at least 1 promoter sequence operatively linked to an sFLT-1 transgene sequence, an ITR sequence, and UTR sequence; and   b. purifying the recombinant virus.   
     
     
         38 . The method of  claim 37 , wherein said nucleic acid sequence does not contain a beta-lactam antibiotic resistance sequence. 
     
     
         39 . The method of  claim 37 , wherein the recombinant virus produces sFLT-1 protein in the range of 100-10,000 pg/mL when measured at 72 hours following transduction of HEK293 cells at multiplicity of infection of 1×10 4 -1×10 6 . 
     
     
         40 . The method of  claim 37 , wherein the recombinant virus inhibits proliferation of human umbilical vascular endothelial (HUVEC) cells. 
     
     
         41 . A cell for generating a recombinant viral vector, the cell comprising at least 1 promoter polynucleotide sequence operatively linked to a sFLT-1 transgene sequence, an ITR polynucleotide sequence, and a UTR polynucleotide sequence.