Compositions for treatment of wet age-related macular degeneration

Abstract

A recombinant adeno-associated virus (rAAV) having an AAV8 capsid which is suitable for intra-retinal injection is provided herein. The rAAV comprises a vector genome packaged within the capsid which contains, operably linked to regulatory elements which direct expression of anti-human vascular endothelial growth factor (VEGF) antigen binding antibody fragment (aVEGF), a coding sequence for aVEGF, wherein the coding sequence is operably linked to regulatory elements which direct expression of the anti-VEGF Fab in the eye. Also provided herein are liquid suspensions containing these rAAV8.aVEGF and methods of using same for treatment of wet AMD and other ocular conditions.

1 . A recombinant adeno-associated virus (rAAV) having an AAV8 capsid which is suitable for subretinal and/or intra-retinal injection, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising:
 (a) an AAV inverted terminal repeat (ITR);   (b) a coding sequence for an anti-human vascular endothelial growth factor (VEGF) antigen binding antibody fragment (Fab) having an exogenous leader sequence, a heavy immunoglobulin chain, a linker, and a light immunoglobulin chain having an exogenous leader sequence, wherein the coding sequence is operably linked to regulatory elements which direct expression of the anti-VEGF Fab in the eye;   (c) regulatory elements which direct expression of the heavy and light immunoglobulin chains of the anti-VEGF Fab which comprise a promoter selected from a chicken beta-actin promoter or a ubiquitin C promoter; and   (d) an AAV ITR.   
     
     
         2 . The rAAV according to  claim 1 , wherein the linker is an F2A linker. 
     
     
         3 . The rAAV according to  claim 1 , wherein the heterologous leader sequence is an IL2 leader. 
     
     
         4 . The rAAV according to  claim 1 , wherein the regulatory elements further comprise a UTR sequence. 
     
     
         5 . The rAAV according to  claim 1 , wherein the regulatory elements further comprise an enhancer and an intron. 
     
     
         6 . The rAAV according to  claim 5 , wherein the regulatory elements comprise a cytomegalovirus immediate-early enhancer, a CB7 promoter, and a chicken B-actin intron. 
     
     
         7 . The rAAV according to  claim 1 , wherein the coding sequence for the anti-VEGF Fab heavy and light chain variable regions are selected from:
 (a) aVEGFv3 (SEQ ID NO: 24);   (b) aVEGFv2 (SEQ ID NO: 3); or   (c) aVEGFv1 (SEQ ID NO: 19);   (d) aVEGFv4 (SEQ ID NO: 35);   (e) aVEGFv5 (SEQ ID NO: 36);   (f) aVEGFv6 (SEQ ID NO: 37);   (g) aVEGFv7 (SEQ ID NO: 38);   (h) aVEGFv8 (SEQ ID NO: 39);   (i) aVEGF v9 (SEQ ID NO: 40);   (j) aVEGFv10 (SEQ ID NO: 41);   (k) aVEGFv11 (SEQ ID NO: 42);   (l) aVEGFv12 (SEQ ID NO: 43); or   (m) aVEGFv13 (SEQ ID NO: 44).   
     
     
         8 . A plasmid comprising an expression cassette within a vector genome, wherein said vector genome is selected from:
 (a) ITR-CB7-CI-aVEGFv3-rBG-ITR (SEQ ID NO: 14);   (b) ITR-CB7-CI-aVEGFv2-rBG-ITR (SEQ ID NO: 3);   (c) ITR-UbC-CI-aVEGFv2-SV40-ITR (SEQ ID NO: 9);   (d) ITR-UbC-PI-aVEGFv3-SV40-ITR (SEQ ID NO: 19);   (e) ITR-UbC-PI-aVEGFv1-SV40-ITR (SEQ ID NO: 24);   (f) ITR-CB7.CI.aVEGFv4.rBG-ITR (SEQ ID NO: 35);   (g) ITR-CB7.CI.aVEGFv5.rBG-ITR (SEQ ID NO: 36);   (h) ITR-CB7.CI.aVEGFv6.rBG-ITR (SEQ ID NO: 37);   (i) ITR-CB7.CI.aVEGFv7.rBG-ITR (SEQ ID NO: 38);   (j) ITR-CB7.CI.aVEGFv8.rBG-ITR (SEQ ID NO: 39);   (k) ITR-CB7.CI.aVEGFv9.rBG-ITR (SEQ ID NO: 40);   (l) ITR-CB7.CI.aVEGFv10.rBG-ITR (SEQ ID NO: 41);   (m) ITR-CB7.CI.aVEGFv11.rBG-ITR (SEQ ID NO: 42);   (n) ITR-CB7.CI.aVEGFv13.rBG-ITR (SEQ ID NO: 43);   (o) ITR-CB7.CI.aVEGFv14.rBG-ITR (SEQ ID NO: 44);   (p) SEQ ID NO: 45;   (q) SEQ ID NO: 46; or   (r) SEQ ID NO: 47.   
     
     
         9 . A liquid suspension suitable for sub-retinal and/or intra-retinal injection, said composition comprising an aqueous liquid and recombinant adeno-associated virus (rAAV) and according to  claim 1  and optionally one or more excipients, preservatives, and/or surfactants. 
     
     
         10 . A method for administering an anti-VEGF Fab to a patient having wet age-related macular degeneration, the method comprising subretinally injecting a patient's eye with a liquid suspension according to  claim 9 . 
     
     
         11 . The method according to  claim 14  wherein the injection comprises about 1×108 genome copies (GC) per eye to about 1.5×10 12  GC per eye, wherein GC is as determined using digital droplet PCR. 
     
     
         12 . The method according to  claim 15 , wherein the dose is about 5×10 8  GC/eye to 2×10 11  GC/eye. 
     
     
         13 . The method according to  claim 15 , wherein the dose is about 7×109 GC/eye to 2×10 10  GC/eye. 
     
     
         14 . The method according to  claim 14 , wherein the rAAV are delivered in a volume of about 75 μL to about 150 μL of the suspension. 
     
     
         15 . The method according to  claim 14 , wherein the rAAV are delivered in a volume of about 100 μL of the suspension. 
     
     
         16 . A product comprising: (a) a first container comprising an rAAV according to  claim 1  and an aqueous liquid, (b) optionally a second container comprising a diluent, and (c) a needle for injection. 
     
     
         17 . The rAAV according to  claim 1 , wherein the vector genome comprises a 5′ AAV inverted terminal repeat, an expression cassette comprising a promoter, an anti-VEGF immunoglobulin construct transgene, and poly-adenylation signal, and a 3′ inverted terminal repeat, wherein the expression cassette is selected from:
 (a) CB7-CI-aVEGFv3-rBG (nt 198 to nt 3733 of SEQ ID NO: 14); 
 (b) CB7-CI-aVEGFv2-rBG (nt 198 to nt 3739 of SEQ ID NO: 3); 
 (c) UbC-CI-aVEGFv2-SV40 (nt 207 to nt 3576 of SEQ ID NO: 9); 
 (d) UbC-PI-aVEGFv3-SV40 (nt 207 to nt 3569 of SEQ ID NO: 19); 
 (e) UbC-PI-aVEGFv1-SV40 (nt 207 to nt 3570 of SEQ ID NO: 24); 
 (f) CB7.CI.aVEGFv4.rBG (nt 198 to nt 3733 of SEQ ID NO: 35); 
 (g) CB7.CI.aVEGFv5.rBG (nt 198 to nt 3733 of SEQ ID NO: 36); 
 (h) CB7.CI.aVEGFv6.rBG (nt 198 to nt 3733 of SEQ ID NO: 37); 
 (i) CB7.CI.aVEGFv7.rBG (nt 198 to nt 3733 of SEQ ID NO: 38); 
 (j) CB7.CI.aVEGFv8.rBG (nt 198 to nt 3733 of SEQ ID NO: 39); 
 (k) CB7.CI.aVEGFv9.rBG (nt 198 to nt 3739 of SEQ ID NO: 40); 
 (l) CB7.CI.aVEGFv10.rBG (nt 198 to nt 3733 of SEQ ID NO: 41); 
 (m) CB7.CI.aVEGFv11.rBG (nt 198 to nt 3733 of SEQ ID NO: 42); 
 (n) CB7.CI.aVEGFv13.rBG (nt 198 to nt 3733 of SEQ ID NO: 43); 
 (o) CB7.CI.aVEGFv14.rBG (nt 198 to nt 3733 of SEQ ID NO: 44); 
 (p) nt 191 to nt 3615 of SEQ ID NO: 45; 
 (q) nt 191 to nt 3492 of SEQ ID NO: 46; or 
 (r) SEQ ID NO: 47. 
 
     
     
         18 . The plasmid according to  claim 8 , wherein the plasmid is suitable for use in replication and in packaging host cell. 
     
     
         19 . A host cell comprising a plasmid according to  claim 8 . 
     
     
         20 . A method of manufacturing recombinant adeno-associated virus (rAAV) particles in host cells comprising: culturing host cells according to claim  23 , wherein the expression cassette is flanked by AAV inverted terminal repeat (ITR), (b) rep sequences operably linked to regulatory sequences which direct expression of rep proteins thereof in the host cells; and (c) nucleic acid sequences encoding an AAV capsid protein operably linked to regulatory sequences which direct expression of the AAV capsid in the host cells, and sufficient helper sequences to package the expression cassette into the AAV capsid, wherein the host cells express the rep and capsid proteins and package the expression cassette into the assembled capsid to produce the rAAV particles.