AAV-MEDIATED DELIVERY OF THERAPEUTIC ANTIBODIES TO THE INNER EAR
Provided herein are methods that include introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that includes a nucleotide sequence encoding (a) a polypeptide including an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide including an antibody light chain variable domain operably linked to a signal peptide; (b) a polypeptide including an antigen-binding antibody fragment operably linked to a signal peptide; or (c) a soluble vascular endothelial growth factor receptor operably linked to a signal peptide.
1 . A method comprising introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that comprises a nucleotide sequence encoding
(a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or
(b) a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.
2 . A method for increasing the level of an antibody or an antigen-binding antibody fragment in an inner ear of a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding
(a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or
(b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide;
wherein the introducing results in an increase in the level of the antibody or the antigen-binding antibody fragment in the inner ear of the mammal.
3 . The method of claim 1 or 2 , wherein the antibody or the antigen-binding antibody fragment binds specifically to vascular endothelial growth factor (VEGF).
4 . The method of claim 3 , wherein the antibody or antigen-binding antibody fragment decreases VEGF activity.
5 . The method of any one of claims 1 - 4 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.
6 . The method of claim 5 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
7 . The method of any one of claims 1 - 6 , wherein the AAV vector further comprises a polyadenylation signal sequence.
8 . The method of any one of claims 1 - 7 , wherein the mammal is a human.
9 . The method of any one of claims 1 - 8 , wherein the mammal has been identified as having an inner ear disorder.
10 . The method of any one of claims 1 - 8 , wherein the mammal has been diagnosed as having an inner ear disorder.
11 . The method of any one of claims 1 - 10 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.
12 . The method of any one of claims 1 - 10 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal.
13 . A method for treating an inner ear disorder in a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding:
(a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or
(b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide;
wherein the introducing results in the treatment of the inner ear disorder in the mammal.
14 . The method of claim 13 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.
15 . The method of claim 14 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
16 . The method of any one of claims 13 - 15 , wherein the AAV vector further comprises a polyadenylation signal sequence.
17 . The method of any one of claims 13 - 16 , wherein the mammal is a human.
18 . The method of any one of claims 13 - 17 , wherein the mammal has been identified as having an inner ear disorder.
19 . The method of any one of claims 13 - 17 , wherein the mammal has been diagnosed as having an inner ear disorder.
20 . The method of any one of claims 13 - 19 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.
21 . The method of any one of claims 13 - 19 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal.
22 . A method of reducing VEGF activity in an inner ear of a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding
(a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or
(b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide;
wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity;
wherein the introducing results in a reduction in VEGF activity in the inner ear of the mammal.
23 . The method of claim 22 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.
24 . The method of claim 23 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
25 . The method of any one of claims 22 - 24 , wherein the AAV vector further comprises a polyadenylation signal sequence.
26 . The method of any one of claims 22 - 25 , wherein the mammal is a human.
27 . The method of any one of claims 22 - 26 , wherein the mammal has been identified or diagnosed as having an acoustic neuroma.
28 . The method of any one of claims 22 - 26 , wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.
29 . The method of any one of claims 22 - 26 , wherein the mammal has been identified or diagnosed as having a neurofibromatosis type 2.
30 . The method of any one of claims 22 - 29 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.
31 . The method of any one of claims 22 - 29 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.
32 . A method of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding
(a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide; or
(b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide;
wherein the polypeptide of (a) encodes an antibody that binds specifically to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that binds specifically to VEGF and reduces VEGF activity;
wherein the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.
33 . The method of claim 32 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment.
34 . The method of claim 33 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
35 . The method of any one of claims 32 - 34 , wherein the AAV vector further comprises a polyadenylation signal sequence.
36 . The method of any one of claims 32 - 35 , wherein the mammal is a human.
37 . The method of any one of claims 32 - 36 , wherein the mammal has been identified or diagnosed as having an acoustic neuroma.
38 . The method of any one of claims 32 - 36 , wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.
39 . The method of any one of claims 32 - 36 , wherein the mammal has been identified or diagnosed as having neurofibromatosis type 2.
40 . The method of any one of claims 32 - 39 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide and a polypeptide comprising an antibody light chain variable domain operably linked to a signal peptide.
41 . The method of any one of claims 32 - 40 , wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.
42 . The method of any one of claims 1 - 41 , wherein the antibody comprises a Fc region that includes one or more amino acid substitutions that decreases the half-life of the antibody in a mammal as compared to a control antibody; or
the antigen-binding antibody fragment thereof has a decreased in vivo half-life as compared to a control antigen-binding antibody fragment.
43 . A method comprising introducing into an inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide.
44 . A method for increasing the level of a soluble vascular endothelial growth factor (VEGF) receptor in an inner ear of a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide;
wherein the introducing results in an increase in the level of the soluble VEGF receptor in the inner ear of the mammal.
45 . The method of claim 43 or 44 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-1 (VEGFR-1).
46 . The method of claim 45 , wherein the portion of the extracellular region of VEGFR-1 comprises a contiguous sequence from wildtype human VEGFR-1.
47 . The method of claim 46 , wherein the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1.
48 . The method of claim 45 , wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.
49 . The method of claim 43 or 44 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-2 (VEGFR-2).
50 . The method of claim 49 , wherein the portion of the extracellular region of VEGFR-2 comprises a contiguous sequence from wildtype human VEGFR-2.
51 . The method of claim 50 , wherein the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.
52 . The method of claim 49 , wherein the portion of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.
53 . The method of claim 43 or 44 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2.
54 . The method of claim 53 , wherein:
the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and
the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.
55 . The method of claim 54 , wherein the soluble VEGF receptor is aflibercept.
56 . The method of claim 43 or 44 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-3 (VEGFR-3).
57 . The method of claim 56 , wherein the portion of the extracellular region of VEGFR-3 comprises a contiguous sequence from wildtype human VEGFR-3.
58 . The method of claim 57 , wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3.
59 . The method of claim 56 , wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3.
60 . The method of any one of claims 43 - 59 , wherein the soluble VEGF receptor comprises a Fc domain.
61 . The method of claim 60 , wherein the Fc domain is an IgG1 Fc domain.
62 . The method of claim 61 , wherein the IgG1 Fc domain is a human wildtype IgG1 Fc domain.
63 . The method of any one of claims 43 - 62 , wherein the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.
64 . The method of any one of claims 43 - 63 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.
65 . The method of claim 64 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
66 . The method of any one of claims 43 - 65 , wherein the AAV vector further comprises a polyadenylation signal sequence.
67 . The method of any one of claims 43 - 66 , wherein the mammal is a human.
68 . The method of any one of claims 43 - 67 , wherein the mammal has been identified as having an inner ear disorder.
69 . The method of any one of claims 43 - 67 , wherein the mammal has been diagnosed as having an inner ear disorder.
70 . A method for treating an inner ear disorder in a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide;
wherein the introducing results in the treatment of the inner ear disorder in the mammal.
71 . The method of claim 70 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.
72 . The method of claim 71 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
73 . The method of any one of claims 70 - 72 , wherein the AAV vector further comprises a polyadenylation signal sequence.
74 . The method of any one of claims 70 - 73 , wherein the mammal is a human.
75 . The method of any one of claims 70 - 74 , wherein the mammal has been identified as having an inner ear disorder.
76 . The method of any one of claims 70 - 74 , wherein the mammal has been diagnosed as having an inner ear disorder.
77 . A method of reducing a VEGF activity in an inner ear of a mammal in need thereof, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide;
wherein the introducing results in a reduction in the VEGF activity in the inner ear of the mammal.
78 . The method of claim 77 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.
79 . The method of claim 78 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
80 . The method of any one of claims 77 - 79 , wherein the AAV vector further comprises a polyadenylation signal sequence.
81 . The method of any one of claims 77 - 80 , wherein the mammal is a human.
82 . The method of any one of claims 77 - 81 , wherein the mammal has been identified or diagnosed as having an acoustic neuroma.
83 . The method of any one of claims 77 - 81 , wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.
84 . The method of any one of claims 77 - 81 , wherein the mammal has been identified or diagnosed as having a neurofibromatosis type 2.
85 . A method of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in an inner ear of a mammal, the method comprising:
introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector that comprises a nucleotide sequence encoding a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide;
wherein the introducing results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II, respectively, in the inner ear of the mammal.
86 . The method of claim 85 , wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence that are operably linked to the sequence encoding the soluble VEGF receptor.
87 . The method of claim 86 , wherein the AAV vector comprises a promoter selected from the group consisting of: an inducible promoter, a constitutive promoter, and a tissue-specific promoter.
88 . The method of any one of claims 85 - 87 , wherein the AAV vector further comprises a polyadenylation signal sequence.
89 . The method of any one of claims 85 - 88 , wherein the mammal is a human.
90 . The method of any one of claims 85 - 89 , wherein the mammal has been identified or diagnosed as having an acoustic neuroma.
91 . The method of any one of claims 85 - 89 , wherein the mammal has been identified or diagnosed as having a vestibular schwannoma.
92 . The method of any one of claims 85 - 89 , wherein the mammal has been identified or diagnosed as having neurofibromatosis type 2.
93 . The method of any one of claims 70 - 92 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-1 (VEGFR-1).
94 . The method of claim 93 , wherein the portion of the extracellular region of VEGFR-1 comprises a contiguous sequence from wildtype human VEGFR-1.
95 . The method of claim 94 , wherein the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1.
96 . The method of claim 93 , wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-1.
97 . The method of any one of claims 70 - 92 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-2 (VEGFR-2).
98 . The method of claim 97 , wherein the portion of the extracellular region of VEGFR-2 comprises a contiguous sequence from wildtype human VEGFR-2.
99 . The method of claim 98 , wherein the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.
100 . The method of claim 97 , wherein the portion of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-2.
101 . The method of any one of claims 70 - 92 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGFR-1 and a portion of an extracellular region of VEGFR-2.
102 . The method of claim 101 , wherein:
the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-1; and
the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-2.
103 . The method of claim 102 , wherein the soluble VEGF receptor is aflibercept.
104 . The method of any one of claims 70 - 92 , wherein the soluble VEGF receptor comprises a portion of an extracellular region of VEGF receptor-3 (VEGFR-3).
105 . The method of claim 104 , wherein the portion of the extracellular region of VEGFR-3 comprises a contiguous sequence from wildtype human VEGFR-3.
106 . The method of claim 105 , wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region from wildtype human VEGFR-3.
107 . The method of claim 104 , wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a contiguous sequence from wildtype human VEGFR-3.
108 . The method of any one of claims 70 - 107 , wherein the soluble VEGF receptor comprises a Fc domain.
109 . The method of claim 108 , wherein the Fc domain is an IgG1 Fc domain.
110 . The method of claim 109 , wherein the IgG1 Fc domain is a human wildtype IgG1 Fc domain.
111 . The method of any one of claims 70 - 110 , wherein the soluble VEGF receptor decreases the ability of a VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.
112 . The method of any one of claims 43 - 111 , wherein the AAV vector further comprises a secretion sequence.