AAV-mediated delivery of therapeutic antibodies to inner ear

Abstract

To provide an adeno-associated virus (AAV) vector for treating hearing loss in human subjects.SOLUTION: 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.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 607,665, filed on December 19, 2017, the entire contents of which are incorporated herein by reference.

[0002] This disclosure generally relates to the use of nucleic acids for treating hearing loss in a human subject.

Background Art

[0003] Sensorineural hearing loss is hearing loss caused by the malfunction of cells (e.g., hair cells) in the inner ear of mammals. Non - limiting causes of sensorineural hearing loss include exposure to loud sounds, head trauma, viral infections, autoimmune inner ear diseases, hereditary hearing loss, aging, inner ear malformations, Meniere's disease, otosclerosis, and tumors.

Summary of the Invention

Means for Solving the Problems

[0004] The present invention relates to a method comprising introducing a therapeutically effective amount of any adeno - associated virus (AAV) vector comprising a nucleotide sequence encoding a polypeptide comprising (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, into the inner ear of a mammal (e.g., a human).

[0005] The inner ear of a mammal in need of an increase in the level of an antibody or antigen - binding antibody fragment in the inner ear ​​​​​​​​A method for increasing the level of an antibody or antigen-binding antibody fragment in a mammalian inner ear, the method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy-chain variable domain of an antibody operably linked to a signal peptide and a light-chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in an increase in the level of the antibody or antigen-binding antibody fragment in the mammalian inner ear. In some embodiments, the antibody or antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment reduces VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of the methods described herein, the mammalian is a human. In some embodiments of any of the methods described herein, the mammalian is a non-human mammal. A method for increasing the level of an antibody or antigen-binding antibody fragment in a mammalian inner ear, the method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy-chain variable domain of an antibody operably linked to a signal peptide and a light-chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in an increase in the level of the antibody or antigen-binding antibody fragment in the mammalian inner ear. In some embodiments, the antibody or antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment reduces VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence.

[0006] In some embodiments of any of the methods described herein, the mammalian is a human. In some embodiments of any of the methods described herein, the mammalian is a non-human mammal. A method for increasing the level of an antibody or antigen-binding antibody fragment in a mammalian inner ear, the method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy-chain variable domain of an antibody operably linked to a signal peptide and a light-chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in an increase in the level of the antibody or antigen-binding antibody fragment in the mammalian inner ear. In some embodiments, the antibody or antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment reduces VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of the methods described herein, the mammalian is a human. In some embodiments of any of the methods described herein, the mammalian is a non-human mammal.

[0007] A method for increasing the level of an antibody or antigen-binding antibody fragment in a mammalian inner ear, the method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy-chain variable domain of an antibody operably linked to a signal peptide and a light-chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in an increase in the level of the antibody or antigen-binding antibody fragment in the mammalian inner ear. In some embodiments, the antibody or antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment reduces VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of the methods described herein, the mammalian is a human. In some embodiments of any of the methods described herein, the mammalian is a non-human mammal.

[0008] A method for increasing the level of an antibody or antigen-binding antibody fragment in a mammalian inner ear, the method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy-chain variable domain of an antibody operably linked to a signal peptide and a light-chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in an increase in the level of the antibody or antigen-binding antibody fragment in the mammalian inner ear. In some embodiments, the antibody or antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). In some embodiments, the antibody or antigen-binding antibody fragment reduces VEGF activity. In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or antigen-binding antibody fragment. and has been identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal has been

[0009] In some embodiments of any of the methods described herein, 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.

[0010] In some embodiments of any of the methods described herein, the AAV vector comprises a nucleic acid sequence encoding a polypeptide

[0011] A method for treating an inner ear disorder in a mammal in need of treatment of the inner ear disorder, the method comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising 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

[0012] comprising an antigen-binding antibody fragment operably linked to a signal peptide, wherein introduction thereof effects treatment of the inner ear disorder in the mammal. A method is also provided herein.

[0013] In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. and a polyadenylation signal sequence.

[0014] In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence.

[0015] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal is identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal is diagnosed as having an inner ear disorder.

[0016] In some embodiments of any of the methods described herein, 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. and a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal. encoding nucleic acid sequence.

[0017] In some embodiments of any of the methods described herein, the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal. encoding nucleic acid sequence.

[0018] A method of reducing VEGF activity in the inner ear of a mammal in need of reducing VEGF activity in the inner ear, the method comprising administering to the inner ear of the mammal: (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. linked to the inner ear of the mammal, (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. linked thereto. A polypeptide comprising the obtained antibody light chain variable domain, or (b) a nucleotide sequence encoding a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide An AAV vector comprising a therapeutically effective amount of an introduced polypeptide, wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and the introduction results in a reduction of VEGF activity in the inner ear of a mammal, is also provided herein In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding an antibody or an antigen-binding antibody fragment In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence In some embodiments of any of the methods described herein, the mammal is a human In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma

[0019] In some embodiments of any of the methods described herein, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding an antibody or an antigen-binding antibody fragment In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence

[0020] In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence

[0021] In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence In some embodiments of any of the methods described herein, the mammal is a human In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having an acoustic neuroma

[0022] In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having an acoustic neuroma In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma In some embodiments, the mammal is identified or diagnosed as having neurofibromatosis type 2. It has been.

[0023] In some embodiments of any of the methods described herein, the AAV vector comprises 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. It contains a nucleic acid sequence encoding.

[0024] In some embodiments of any of the methods described herein, the AAV vector contains a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide. It contains.

[0025] A method of treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in the inner ear of a mammal, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising 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 specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and said introduction results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively. A method is also provided herein. It contains. respectively, wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and said introduction results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively. A method is also provided herein. treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively. A method is also provided herein. treatment is provided.

[0026] In some embodiments of any of the methods described herein, the AAV vector is further comprises one or both of a promoter operably linked to a sequence encoding an antibody or an antigen-binding antibody fragment and a Kozak sequence.

[0027] In some embodiments of any of the methods described herein, the AAV vector is comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

[0028] In some embodiments of any of the methods described herein, the AAV vector is further comprises a polyadenylation signal sequence.

[0029] In some embodiments of any of the methods described herein, the mammal is a human. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having an acoustic neuroma. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having neurofibromatosis type 2.

[0030] In some embodiments of any of the methods described herein, the AAV vector is 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.

[0031] In some embodiments of any of the methods described herein, the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide.

[0032] In some embodiments of any of the methods described herein, the antibody comprises an Fc region comprising one or more amino acid substitutions that decrease 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.

[0033] Also provided herein is a method comprising introducing into the inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide.

[0034] A method for increasing the level of soluble vascular endothelial growth factor (VEGF) receptor in the inner ear of a mammal in need thereof, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide, wherein introduction thereof results in an increase in the level of soluble VEGF receptor in the inner ear of the mammal.

[0035] In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-1 (VEGFR-1). ​​​​​​​​​In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-1 includes an adjacent sequence from wild-type human VEGFR-1. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-1 includes one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-1. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR -1 includes a sequence that is at least 9 0% identical to an adjacent sequence from wild-type human VEGFR-1.

[0036] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of the extracellular region of VEGF receptor-2 (VEGFR-2). In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-2 includes an adjacent sequence from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-2 includes one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR -2 includes a sequence that is at least 9 0% identical to an adjacent sequence from wild-type human VEGFR-2.

[0037] In some embodiments of any of the methods described herein, the soluble VEGF receptor includes a portion of the extracellular region of VEGFR-1 and a portion of the extracellular region of VEGFR-2. In some embodiments of any of the methods described herein, VEGFR A portion of the extracellular region of -1 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-1, and a portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, the soluble VEG F receptor is aflibercept. In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-3 (VEGFR-3). In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-3 comprises a contiguous sequence derived from wild-type human VEGFR-3. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-3. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a contiguous sequence derived from wild-type human VEGFR-3. In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises an Fc domain. In some embodiments of any of the methods described herein, the Fc domain is an IgG1 Fc domain. In some embodiments of any of the methods described herein, the IgG1 Fc domain is a human wild-type IgG1 Fc domain.

[0038]

[0039] ​​​​​​​​​​​​​​​In some embodiments of any of the methods described herein, the soluble VEGF receptor reduces the ability of VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3.

[0040] In some embodiments of any of the methods described herein, the AAV vector further comprises a promoter and one or both of the Kozak sequences operably linked to a sequence encoding a soluble VEGF receptor. In some embodiments of any of the methods described herein, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of the methods described herein, the mammal is human.

[0041] In some embodiments of any of the methods described herein, the mammal is identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal is diagnosed as having an inner ear disorder.

[0042] 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 comprising 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 the inner ear disorder in the mammal, is also provided herein.

[0043] In some embodiments of any of the methods described herein, the AAV vector is further comprised of a promoter operably linked to a sequence encoding a soluble VEGF receptor and one or both of the Ko zak sequences. In some embodiments of any of the methods described herein, the AAV vector is comprised of a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein, the AAV vector is further comprised of a polyadenylation signal sequence.

[0044] In some embodiments of any of the methods described herein, the mammal is human. In some embodiments of any of the methods described herein, the mammal is identified as having an inner ear disorder. In some embodiments of any of the methods described herein, the mammal is diagnosed as having an inner ear disorder.

[0045] Also provided herein is a method of reducing VEGF activity in the inner ear of a mammal in need of reducing VEGF activity in the inner ear, the method comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide, whereby reduction of VEGF activity in the inner ear of the mammal is effected.

[0046] In some embodiments of any of the methods described herein, the AAV vector is further comprised of a promoter operably linked to a sequence encoding a soluble VEGF receptor and one or both of the Ko further comprises one or both of the zak arrays. Any of the methods described herein In some embodiments, the AAV vector comprises a promoter selected from the group of inducible promoters, constitutive promoters, and tissue-specific promoters. In some embodiments of any of the methods described herein, the AAV vector further comprises a polyadenylation signal sequence.

[0047] In some embodiments of any of the methods described herein, the mammal is a human . In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma . In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having neurofibromatosis type 2.

[0048] A method of treating a vestibular schwannoma, acoustic neuroma, or neurofibromatosis type 2 in the inner ear of a mammal comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide wherein said introduction results in treatment of a vestibular schwannoma, acoustic neuroma, or neurofibromatosis type II in the inner ear of the mammal, respectively, is also provided herein. nucleotide sequence, wherein said introduction results in treatment of a vestibular schwannoma, acoustic neuroma, or neurofibromatosis type II in the inner ear of the mammal, respectively, is also provided herein. respectively, a vestibular schwannoma, acoustic neuroma, or neurofibromatosis type II in the inner ear of the mammal treatment is provided.

[0049] In some embodiments of any of the methods described herein, the AAV vector is , a promoter operably linked to a sequence encoding a soluble VEGF receptor and Ko zak sequence, either or both. Any of the methods described herein In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of the methods described herein the AAV vector further comprises a polyadenylation signal sequence.

[0050] In some embodiments of any of the methods described herein, the mammal is a human being. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having a vestibular schwannoma being. In some embodiments of any of the methods described herein, the mammal is identified or diagnosed as having neurofibromatosis type 2.

[0051] In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-1 (VEGFR-1). In some embodiments of any of the methods described herein a portion of the extracellular region of VEGFR-1 comprises adjacent sequences from wild-type human VEGFR-1. In some embodiments of any of the methods described herein a portion of the extracellular region of VEGFR-1 is wild type human VEGFR-1-derived extracellular region contains one or more immunoglobulin-like domains is included. In some embodiments of any of the methods described herein, VEGFR a portion of the extracellular region of -1 contains a sequence that is at least 9 0% identical to the adjacent sequence from wild-type human VEGFR-1.

[0052] In some embodiments of any of the methods described herein, the soluble VEGF receptor contains a portion of the extracellular region of VEGF receptor-2 (VEGFR-2). In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-2 contains the adjacent sequence from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR-2 contains one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR -2 contains a sequence that is at least 9 0% identical to the adjacent sequence from wild-type human VEGFR-2. 0% identical to the adjacent sequence from wild-type human VEGFR-2.

[0053] In some embodiments of any of the methods described herein, the soluble VEGF receptor contains a portion of the extracellular region of VEGFR-1 and a portion of the extracellular region of VEGFR-2 In some embodiments of any of the methods described herein, a portion of the extracellular region of VEGFR -1 contains one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-1, and a portion of the extracellular region of VEGFR-2 contains one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, the soluble VEG receptor contains one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-1, and a portion of the extracellular region of VEGFR-2 contains one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2. In some embodiments of any of the methods described herein, the soluble VEG The F receptor is aflibercept.

[0054] In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-3 (VEGFR-3). In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 comprises flanking sequences from wild-type human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-3. In some embodiments of any of the methods described herein, the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to the flanking sequences from wild-type human

[0055] In some embodiments of any of the methods described herein, the soluble VEGF receptor comprises an Fc domain. In some embodiments of any of the methods described herein, the Fc domain is an IgG1 Fc domain. In some embodiments of any of the methods described herein, the IgG1 Fc domain is a human wild-type IgG1 Fc domain.

[0056] In some embodiments of any of the methods described herein, the soluble VEGF receptor reduces the ability of VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3. In some embodiments of any of the methods described herein, the AAV vector further comprises a secretion sequence.

[0057] ​ Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes degenerate versions of each other, and thus includes all nucleotide sequences encoding the same amino acid sequence.

[0058] The term "isolated" means that it has been modified or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that has been partially or completely separated from its co-existing materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or in a non-natural environment, such as a host cell, etc.

[0059] The terms "transfected", "transformed", or "transduced" refer to the process by which an exogenous nucleic acid is introduced or transferred into a cell. A "transfected", "transformed", or "transduced" mammalian cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid.

[0060] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence encoding a protein.

[0061] The term "transient expression" refers to the expression of an unintegrated coding sequence over a short period (e.g., several hours or days). A coding sequence transiently expressed in a cell (e.g., a mammalian cell) is lost by multiple cell divisions.

[0062] The term "subject" is intended to include any mammal. In some embodiments ​​​​​​​​​​​​In this state, the subject is a rodent (e.g., rat or mouse), rabbit, sheep, goat, pig, dog, cat, non-human primate, or human. In some embodiments, the subject has or is at risk of developing non-symptomatic hearing loss. In some embodiments, the subject has been previously identified as having an inner ear disorder. In some embodiments, the subject has been previously diagnosed as having an inner ear disorder. In some embodiments, the subject has been identified as having drug-induced hearing loss. In some embodiments, the subject is an infant (e.g., a human infant). Treatment is "therapeutically effective" if it results in a reduction in one or more of the number, severity, and frequency of symptoms of one or more diseases (e.g., asymptomatic sensorineural hearing loss) in a subject (e.g., a human). The term "nucleic acid" or "polynucleotide" refers to either single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof. Unless otherwise explicitly limited, this term encompasses nucleic acids containing known analogs of natural nucleotides having similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses the complementary sequence as well as the explicitly shown sequence. In some embodiments of any of the nucleic acids described herein, the nucleic acid is DNA. In some embodiments of any of the nucleic acids described herein, the nucleic acid is RNA. The term "signal peptide" is present at the N-terminus of a nascent secreted protein but is naturally

[0063]

[0064]

[0065] ​​​​​​​​​​​​​​​Refers to sequences that do not exist in the mature protein present in. "Signal peptide" is the signal peptide is cleaved by a protease (e.g., signal peptidase) after being translated. Signal peptides are known in the art. Non-limiting examples of signal peptides include MEFFKKTALAALVMGFSGAALA (SEQ ID NO: 9) and M KYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 10).

[0066] The term "inner ear disorder" refers to a disorder caused by the malfunction of cells (e.g., hair cells, supporting cells, spiral ganglion neurons, macrophages, or Schwann cells) within or around the inner ear of a mammal. Non-limiting examples of inner ear disorders include, for example, sensorineural hearing loss (SNHL), noise-induced hearing loss, drug-induced hearing loss, age-related hearing loss, acoustic neuroma, neurofibromatosis type 2, auditory neuropathy, noise-induced cochlear synaptopathy without hair cell loss, age-related cochlear synaptopathy, acquired sensorineural hearing loss, and vestibular schwannoma. See, for example, Kujaw a et al., Hear Res 330(0 0):191-199,2015, and Suzuki et al., Scientific Reports 6:249 07. Non-limiting examples of inner ear disorders are described herein, and further examples of inner ear disorders are known in the art.

[0067] The term "antibody" means a complex of two or more single polypeptide chains that interact to form at least one antigen-binding domain. Non-limiting examples of antibodies include monoclonal nal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal nal antibodies, ​​​​​​Examples include monoclonal antibodies, polyvalent antibodies, and multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc., as long as they exhibit the desired biological activity). The antibodies can be human antibodies, humanized antibodies, and / or affinity matured antibodies. The term "antigen-binding antibody fragment" refers to a single polypeptide that contains all the amino acids that make up at least one antigen-binding domain (e.g., scFv).

[0068]

[0069] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigen. Further, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0070] Also included within the meaning of monoclonal antibodies herein are "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of those chain(s) is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies as long as they exhibit the desired biological activity (e.g., U.S. Patent No. 4,816,567, and Morrison et al, Proc. Natl. Acad. Sci. USA 81:68 51-6855(1984)).

[0071] An "antigen-binding domain" refers to a polypeptide that is formed from amino acids from a single polypeptide (e.g., a polypeptide that is or derived from two or more polypeptides (e.g., the same or different polypeptides). one or more antigens capable of specifically binding to one or more different antigens formed from the amino acids In some examples, the antigen-binding domain is a protein domain or domains on the antigen binding domain. It binds to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain may comprise an alternative scaffold. Non-limiting examples of binding domains are described herein. Examples are known in the art. In some examples, the antigen binding domain is can be combined.

[0072] "Affinity" refers to the affinity of an antigen-binding site to its binding partner (e.g., an antigen or epitope) Unless otherwise indicated, as used herein, the term "covalent interaction strength" refers to the total strength of non-covalent interactions between a molecule and a molecule. As used herein, "affinity" refers to the affinity of a member of an antigen-binding domain to an antigen or epitope. It refers to the intrinsic binding affinity that reflects the 1:1 interaction between a molecule X and its partner Y. The affinity for a molecule is expressed as the equilibrium dissociation constant (K D Affinity can be expressed as It can be measured by common methods known in the art, including the methods described in Affinity can be measured, for example, by surface plasmon resonance (SPR) techniques (e.g., BIACORE). (Trademark)) or biolayer interferometry (e.g., FORTEBIO®) can be determined. The affinity between the antigen-binding domain and its corresponding antigen or epitope Additional methods for determining are known in the art.

[0073] The term "half-life" refers to the half-life of an antibody, its antigen-binding antibody fragment, or soluble VEG F receptor in the circulation (e.g., blood) of a mammal (e.g., any of the mammals described herein), and is repre sented by the time required for 50% of the antibody, its antigen-binding antibody fragment, or soluble VEGF re ceptor to be removed from the circulation. In some embodiments a change in half-life (e.g., a decrease in the half-life of an antibody, its antigen-binding antibody fragment, or so luble VEGF receptor) is determined by comparing the half-life of the antibody, antigen-binding antibody fragment, or soluble VEGF receptor in a subject to the half-life of a control antibody, control antigen-binding antibody fragment, or control soluble VEGF receptor in a similar mammal.

[0074] In some embodiments, the half-life of an antibody, its antigen-binding antibody fragment, or soluble VEG F receptor in a mammal is determined by measuring the level of the antibody, its antigen-binding antibody fragment, or soluble VEGF receptor in a sample obtained from the subject (e.g., a blood sample) at different tim es after systemic administration (e.g., intravenous) of any of the AAV vectors described herein. In some em bodiments, the level of the antibody, its antigen-binding antibody fragment, or soluble VEGF receptor present in a sample obtained from a mammal is determined using an enzyme-linked immunosorbent assay (ELISA) or an other assay known in the art, and the determination of the antibody, its antigen-binding antibody fragment, or soluble VEGF receptor present in the sample The determined levels are plotted as a function of time using a software program (e.g., GraphPad Prism).

[0075] The term "VEGF activity" refers to one or more known activities of the VEGF protein. For example, one activity of the VEGF protein is the ability to bind to one or more VEGF receptors Another example, one activity of the VEGF protein is the ability of VEGF to induce downstream signaling pathway(s) in mammalian cells expressing VEGF receptor Methods for detecting one or more activities of VEGF are known in the art.

[0076] The term "soluble VEGF receptor" refers to a polypeptide comprising a portion of the extracellular region of one or more mammalian VEGF receptors (e.g., VEGFR-1, VEGFR-2, and VEGFR-3) operably linked to a signal peptide, and the soluble VEGF receptor can specifically bind to one or more mammalian VEGF proteins (e.g., one or more of VEGF-A, VEGF-B , VEGF-C, and VEGF-D). In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1 ( e.g., an adjacent sequence from wild-type human VEGFR-1 (e.g., one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-1 or a sequence that is at least 90% identical to an adjacent sequence from wild-type human VEGFR-1). In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-2 (e.g., an adjacent sequence from wild-type human V EGFR-2 (e.g., one or more immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2 or a sequence that is at least 90% identical to an adjacent sequence from wild-type human VEGFR-2). In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-2 (e.g., an adjacent sequence from wild-type human V EGFR-2 (e.g., an adjacent sequence from wild-type human VEGFR-2 in the extracellular region ​​one or more immunoglobulin-like domains) or adjacent to those derived from wild-type human VEGFR-2 and comprises a sequence that is at least 90% identical to the sequence. In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1 and a portion of the extracellular region of VEGFR-2 (e.g., for example, one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-1 and one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-2 (e.g., aflibercept). In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-3 (e.g., an adjacent sequence derived from wild-type human VEGFR- 3 (e.g., one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR- 3) or a sequence that is at least 90% identical to an adjacent sequence derived from wild-type human VEGFR-3).

[0077] In some examples, the soluble VEGF receptor further comprises a stabilizing domain (e.g., an IgG1 F c domain (e.g., an Fc domain such as the human wild-type IgG1 Fc domain). In some examples, the soluble VEGF receptor reduces the ability to bind VEGF to one or more (e.g., two or three) of VEGFR-1, VEGFR-2 and VEGFR-3. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and distributions mentioned herein are incorporated by reference in their entirety. ​​​​​ Columns, database entries, and other references are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control. BRIEF DESCRIPTION OF THE DRAWINGS

[0078]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 4C

Mode for Carrying Out the Invention

[0079] Introducing a therapeutically effective amount of an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a polypeptide comprising (a) a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein. A polypeptide comprising a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein. A polypeptide comprising a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein. A polypeptide comprising a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein. A polypeptide comprising a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein. A polypeptide comprising a heavy chain variable domain of an antibody operably linked to a signal peptide and a light chain variable domain of an antibody operably linked to a signal peptide, or (b) an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide into the inner ear of a mammal is provided herein.

[0080] Mammalian Inner Ear in Need of Increasing Levels of Antibody or Antigen-Binding Antibody Fragment in the Inner Ear 1. A method for increasing levels of an antibody or antigen-binding antibody fragment in a mammal, comprising: (a) an antibody heavy chain variable domain operably linked to a signal peptide in the inner ear of an animal; The present invention relates to 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. Nucleotide sequence encoding a polypeptide comprising a fragment (e.g., Fab or scFv) and administering to the mammal a therapeutically effective amount of an AAV vector comprising the Methods are also described herein that result in increased levels of antibodies or antigen-binding antibody fragments in the inner ear. provided in the book.

[0081] 1. A method for treating an inner ear disorder in a mammal in need thereof, comprising: (a) an antibody heavy chain variable domain operably linked to a signal peptide, An antibody light chain variable domain operably linked to a polypeptide comprising a signal peptide and or (b) a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide. A therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising introducing, which results in treatment of an inner ear disorder in a mammal. , a method is also provided.

[0082] VEGF activity in the inner ear of mammals requiring reduction of VEGF activity in the inner ear The method includes administering to the inner ear of a mammal (a) a nucleic acid sequence operably linked to a signal peptide. a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide; A polypeptide comprising the obtained antibody light chain variable domain, or (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) operably linked to a signal peptide Introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding the polypeptide wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and this introduction results in a reduction in VEGF activity in the inner ear of a mammal, a method is also provided herein. A method for treating a vestibular schwannoma, acoustic neuroma, or neurofibromatosis type II in the inner ear of a mammal, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding (a) a polypeptide comprising a 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 (e.g., Fab or scFv) operably linked to a signal peptide, wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and this introduction results in the treatment of an acoustic neuroma or vestibular schwannoma in the inner ear of the mammal, a method is also provided herein. Into the inner ear of a mammal, a soluble vascular endothelial growth factor operably linked to a signal peptide

[0083]

[0084] An adeno-associated virus (AAV) containing a nucleotide sequence encoding a (VEGF) receptor Also provided herein is a method comprising introducing a therapeutically effective amount of the vector.

[0085] Requiring an increase in the level of soluble vascular endothelial growth factor (VEGF) receptor in the inner ear A method for increasing the level of soluble VEGF receptor in the inner ear of a mammal, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector containing a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide wherein said introduction results in an increase in the level of soluble VEGF receptor in the inner ear of the mammal Also provided herein is a method.

[0086] A method for treating an inner ear disorder in a mammal in need of treatment for the inner ear disorder, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector containing a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide, wherein said introduction results in treatment of the inner ear disorder in the mammal Also provided herein is a method.

[0087] A method for reducing VEGF activity in the inner ear of a mammal in need of reduction of VEGF activity in the inner ear, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector containing a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide, wherein said introduction results in reduction of VEGF activity in the inner ear of the mammal Also provided herein is a method.

[0088] Treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in the inner ear of a mammal A method comprising introducing into the inner ear of a mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide wherein said introduction results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively, is also provided herein A kit comprising any of the AAV vectors described herein is also provided Additional non-limiting aspects of the compositions, kits, and methods are described herein and may be used in any combination without limitation Antibodies and antigen-binding antibody fragments

[0089] In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, or a multivalent antibody

[0090] In some embodiments, the antibody or antigen-binding antibody fragment can be an scFv-Fc, VH domain, VL domain, a(scFv)2, a minibody, or a BiTE In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut

[0091] H H domain, VL domain, a(scFv)2, a minibody, or a BiTE NAR In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut domain, VL domain, a(scFv)2, a minibody, or a BiTE In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut In some embodiments, the antibody or antigen-binding antibody fragment can be a DVD-Ig, and a dual affinity retargeting antibody (DART), a triomab, a kih IgG having a common LC, a cross mab, an ortho-Fab IgG, a two-in-one IgG, an IgG-ScFv, an scFv2-Fc, a bi-nanobody, a tandem antibody, a DART-Fc, an scFv-HAS-scFv, a DNL-Fab3, a DAF (two-in-one or four-in-one), a Dut aMab, DT-IgG, common LC of nob-in-hole, nob-in-hole assembly, electric charged antibody, Fab arm exchange antibody, SEED body, triomab, LUZ-Y, Fcab , kλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-( H)IgG, IgG(L)-scFv, scFv-(L)-IgG, IgG(L,H)- Fc, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, K IH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-I g, Zybody, DVI-IgG, nanobody, nanobody-HSA, diabody, TandAb, sc diabody, sc diabody-CH3, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 K IH, Fab-scFv, scFv-CH-CL-scFv, F(ab’)2-scFV 2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-F c, diabody-Fc, tandem scFv-Fc, intrabody, dock-and- lock bispecific antibody, ImmTAC, HSAbody, sc diabody-HAS, ta ndem scFv, IgG-IgG, Cov-X-Body, and scFv1-PEG-s cFv2 can be.

[0092] Further examples of antibodies or antigen-binding antibody fragments include Fv fragments, Fab fragments, F(ab ’)2 fragments, and Fab’ fragments. Further examples of antibodies or antigen-binding antibody fragments include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or Ig G4) (e.g., human or humanized IgG, e.g., human or humanized I Antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA e.g., antigen-binding fragments of human or humanized IgA1 or IgA2), antigen-binding fragments of IgD binding fragments (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.g g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments of IgM (e.g ., antigen-binding fragments of human or humanized IgM).

[0093] Any of the antibodies or antigen-binding antibody fragments described herein specifically binds to VEGF binds.

[0094] V H The H domain is a single monomeric variable antibody domain found in camels. V N AR The domain is a single monomeric variable antibody domain found in cartilaginous fish. V H H dom ains and V NAR domains, non-limiting embodiments of which are described, for example, in Cromie et al., Curr.Top.Med.Chem.15:2543-2557,2016, De G enst et al.,Dev.Comp.Immunol.30:187-198, 2006, De Meyer et al.,Trends Biotechnol.3 2:263-270,2014, Kijanka et al.,Nanomedici ne 10:161-174,2015, Kovaleva et al.,Exper t.Opin.Biol.Ther.14:1527-1539,2014, Krah et al.,Immunopharmacol.Immunotoxicol.38: 21 - 28, 2016, Mujic - Delic et al., Trends Pha rmacol. Sci. 35:247 - 255, 2014, Muyldermans, J . Biotechnol. 74:277 - 302, 2001, Muyldermans et al., Trends Biochem. Sci. 26:230 - 235, 200 1, Muyldermans, Ann. Rev. Biochem. 82:775 - 797 , 2013, Rahbarizadeh et al., Immunol. Invest . 40:299 - 338, 2011, Van Audenhove et al., EB ioMedicine 8:40 - 48, 2016, Van Bockstaele e t al., Curr. Opin. Investig. Drugs 10:1212 - 1 224, 2009, Vincke et al., Methods Mol. Biol. 911:15 - 26, 2012, and Wesolowski et al., Med. M icrobiol. Immunol. 198:157 - 174, 2009 as described in it.

[0095] The "Fv" fragment contains a non - covalent dimer of one heavy - chain variable domain and one light - chain variable domain.

[0096] The "Fab" fragment contains, in addition to the heavy - chain variable domain and the light - chain variable domain of the Fv fragment, the constant domain of the light chain and the first constant domain (C ) of the heavy chain. H1

[0097] The "F(ab’)2" fragment contains two Fab fragments linked near the hinge region by a disulfide bond.

[0098] The term "dual variable domain immunoglobulin" or "DVD-Ig" refers to, for example, DiGiammarino et al., Methods Mol.Biol.899:145-156,2012, Jakob et al., MABs 5:358-363,2013, and U.S. Patent Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645, which are incorporated herein by reference in their entirety and refer to the multivalent multispecific binding proteins described therein.

[0099] DART is described, for example, in Garber, Nature Reviews Drug Di scovery 13:799-801,2014.

[0100] Further aspects of antibodies and antigen-binding antibody fragments are known in the art.

[0101] In some embodiments, any of the antibodies or antigen-binding antibody fragments described herein are, for example, surface plasmon resonance (SPR) of a VEGF protein (e.g., any of the VEGF proteins described herein, e.g., mature human VEGF-A, mature human VEGF-B, mature human VE GF-C, and mature human VEGF-D) measured in phosphate-buffered saline using less than 1×10 -5 M (e.g., 0. 5×10 -5 less than M, 1×10 -6 less than M, 0.5×10 -6 less than M, 1×10 -7 less than M less than 0.5×10 -7 less than M, 1×10​​-8 less than M, 0.5×10 -8 less than M, 1×10 -9 less than M, 0.5×10 -9 less than M, 1×10 -10 less than M, 0.5×10 -10 less than M , 1×10 -11 less than M, 0.5×10 -11 less than M, or 1×10 -12 less than M) has a dissociation constant (K D ).

[0102] In some embodiments, any one of the antibodies or antigen-binding antibody fragments described herein is, for example, surface plasmon resonance (SPR ) of, for example, a VEGF protein (e.g., any one of the VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VE GF-C, and mature human VEGF-D), measured in phosphate-buffered saline, from about 1×10 M to about 1×10 M, about 0.5×10 -12 M, about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0.5×10 -6 M, about 1×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, about 0.5×10 -8 M, about 1×10 -9 M, about 0.5×10 -9 M, about 1×10 -10 M, about 0.5×10 -10 M, about 1× 10 -11 M, or about 0.5×10 -11 M (including): about 0.5×10 -11 M to about 1 ×10 -5 M, about 0.5×10 -5 M, about 1×10 -6M, about 0.5×10 -6 M, about 1 ×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, about 0.5×10 -8 M, about 1 ×10 -9 M, about 0.5×10 -9 M, about 1×10 -10 M, about 0.5×10 -10 M, or about 1×10 -11 M (including): about 1×10 -11 M ~ about 1×10 -5 M, about 0.5 ×10 -5 M, about 1×10 -6 M, about 0.5×10 -6 M, about 1×10 -7 M, about 0.5 ×10 -7 M, about 1×10 -8 M, about 0.5×10 -8 M, about 1×10 -9 M, about 0.5 ×10 -9 M, about 1×10 -10 M, or about 0.5×10 -10 M (including): about 0.5 ×10 -10 M ~ about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0. 5×10 -6 M, about 1×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, about 0. 5×10 -8 M, about 1×10 -9 M, about 0.5×10 -9 M, or about 1×10 -10 M (including): about 1×10 -10 M ~ about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0.5×10 -6M, about 1×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, about 0.5×10 -8 M, about 1×10 -9 M, or about 0.5×10 -9 M (including ): about 0.5×10 -9 M ~ about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0.5×10 -6 M, about 1×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, about 0.5×10 -8 M, or about 1×10 -9 M (including): about 1×10 -9 M ~ about 1 ×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0.5×10 -6 M, about 1 ×10 -7 M, about 0.5×10 -7 M, about 1×10 -8 M, or about 0.5×10 -8 M (including): about 0.5×10 -8 M ~ about 1×10 -5 M, about 0.5×10 -5 M, about 1×1 0 -6 M, about 0.5×10 -6 M, about 1×10 -7 M, about 0.5×10 -7 M, or about 1×10 -8 M (including): about 1×10 -8 M ~ about 1×10 -5 M, about 0.5×10 -5 M , about 1×10 -6 M, about 0.5×10 -6 M, about 1×10 -7 M, or about 0.5×10 -7 M (including): about 0.5×10 -7 M to about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, about 0.5×10 -6 M, or about 1×10 -7 M (including): about 1×10 -7 M to about 1×10 -5 M, about 0.5×10 -5 M, about 1×10 -6 M, or about 0.5 ×10 -6 M (including): about 0.5×10 -6 M to about 1×10 -5 M, about 0.5×10 -5 M, or about 1×10 -6 M (including): about 1×10 -6 M to about 1×10 -5 M, or about 0.5×10 -5 M (including): or, for example, about 0.5×10 -5 M to about 1×10 -5 M (including) of K D has.

[0103] Using various different methods known in the art, any of the antibodies or antigens described herein The K value of the binding antibody fragment can also be determined (e.g., electrophoretic mobility D shift assay, filter binding assay, surface plasmon resonance, and biomolecular binding rate theory assay, etc.). assay, etc.).

[0104] In some embodiments of any of the antibodies and / or antigen-binding antibody fragments described herein In some embodiments, the half-life of the antibody and / or antigen-binding antibody fragment in a subject (e.g., human) is is that of a control antibody and / or control antigen-binding antibody fragment in a similar subject (e.g., herein Compared to the half-life of either the control antibody or the control antigen-binding antibody fragment described in [reference], about 0.5-fold to about 4-fold (e.g., about 0.5-fold to about 3.5-fold, about 0.5-fold to about 3-fold, about 0.5-fold to about 2.5-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 1.5-fold, about 0.5-fold to about 1-fold, about 1-fold to about 4-fold, about 1-fold to about 3.5-fold, about 1-fold to about 3-fold, about 1-fold to about 2.5-fold, about 1-fold to about 2-fold, about 1.5-fold to about 4-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 2-fold, about 2-fold to about 4-fold, about 2-fold to about 3.5-fold, about 2-fold to about 3-fold, about 2-fold to about 2.5-fold, about 2.5-fold to about 4-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 3-fold, about 3-fold to about 4-fold, about 3-fold to about 3.5-fold, or about 3.5-fold to about 4-fold) decrease. For example, see Leabman et al., MAbs. 5(6):896-903, 20 13. In some embodiments, the antibodies or antigen-binding antibody fragments described herein have one or more amino acid substitutions in the Fc region that decrease their half-life in a mammal, and the control antibody lacks at least one (e.g., all) of these one or more amino acid substitutions in the Fc region.

[0105] VEGF The VEGF gene encodes vascular endothelial growth factor (VEGF), which was previously known as fms-like tyrosine kinase (Flt-1). The VEGF protein is a heparin-binding protein that induces the migration and proliferation of vascular endothelial cells.

[0106] Non-limiting examples of nucleotide sequences encoding the protein and wild-type VEGF protein are shown below. Human VEGF transcript variant 1 protein sequence (SEQ ID NO: 1) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGG GVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAA RSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWT GEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPP HSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHH AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEES NITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE KKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPG PHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLEL NERTCRCDKPRR Human VEGF transcript variant 1c cDNA (SEQ ID NO: 2) ct gacggacaga cagacagaca ccgcccccag cccc agctac cacctcctcc ccggccggcg gcggacagtg gacgcggcgg cgagccgcgg gcaggggccg gagcccg cgc ccggaggcgg ggtggagggg gtcggggctc gcg gcgtcgc actgaaactt ttcgtccaac ttctgggctg ttctcgcttc ggaggagccg tggtccgcgc ggggga agccgagccgagcg gagccgcgag aagtgctagc tcg ggccggg aggagccgca gccggaggag ggggaggagg aagaagagaa ggaagaggag agggggccgc agtggc gact cggcgctcgg aagccgggct catggacggg tg aggcggcg gtgtgcgcag acagtgctcc agccgcgcg c gctccccagg ccctggcccg ggcctcgggc cgggg aggaa gagtagctcg ccgaggcgcc gaggagagcg g gccgcccca cagcccgagc cggagaggga gcgcgagc cg cgccggcccc ggtcgggcct ccgaaaccat gaac tttctg ctgtcttggg tgcattggag ccttgccttgc tgctctacc tccaccatgc caagtggtcc caggctgc ac ccatggcaga aggaggaggg cagaatcatc acga agtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca atcgagaccc tggtggacat cttccag gag taccctgatg agatcgagta catcttcaag cca tcctgtg tgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt gtgcccactg aggagt ccaa catcaccatg cagattatgc ggatcaaacc tc accaaggc cagcacatag gagagatgag cttcctaca g cacaacaaat gtgaatgcag accaaagaaa gatag agcaa gacaagaaaa aaaatcagtt cgaggaaagg g aaaggggca aaaacgaaag cgcaagaaat cccggtat aa gtcctggagc gtgtacgttg gtgcccgctg ctgt ctaatg ccctggagcc tccctggccc ccatccctgt gggccttgct cagagcggag aaagcatttg tttgtac aag atccgcagac gtgtaaatgt tcctgcaaaa aca cagactc gcgttgcaag gcgaggcagc ttgagttaaa cgaacgtact tgcagatgtg acaagccgag gcggtg a Human VEGF transcript variant 3 protein sequence (SEQ ID NO: 3) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGG GVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAA RSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWT GEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPP HSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHH AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVD IFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEES NITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQE KKSVRGKGKGQKRKRKKSRPCGPCSERRKHLFVQDPQTCK CSCKNTDSRCKARQLELNERTCRCDKPRR Human VEGF transcript variant 3 cDNA (SEQ ID NO: 4) ct gacggacaga cagacagaca ccgcccccag cccc agctac cacctcctcc ccggccggcg gcggacagtg gacgcggcgg cgagccgcgg gcaggggccg gagcccg cgc ccggaggcgg ggtggagggg gtcggggctc gcg gcgtcgc actgaaactt ttcgtccaac ttctgggctg ttctcgcttc ggaggagccg tggtccgcgc ggggga agccgagccgagcg gagccgcgag aagtgctagc tcg ggccggg aggagccgca gccggaggag ggggaggagg aagaagagaa ggaagaggag agggggccgc agtggc gact cggcgctcgg aagccgggct catggacggg tg aggcggcg gtgtgcgcag acagtgctcc agccgcgcg c gctccccagg ccctggcccg ggcctcgggc cgggg aggaa gagtagctcg ccgaggcgcc gaggagagcg g gccgcccca cagcccgagc cggagaggga gcgcgagc cg cgccggcccc ggtcgggcct ccgaaaccat gaac tttctg ctgtcttggg tgcattggag ccttgccttgc tgctctacc tccaccatgc caagtggtcc caggctgc ac ccatggcaga aggaggaggg cagaatcatc acga agtggt gaagttcatg gatgtctatc agcgcagcta ctgccatcca atcgagaccc tggtggacat cttccag gag taccctgatg agatcgagta catcttcaag cca tcctgtg tgcccctgat gcgatgcggg ggctgctgca atgacgaggg cctggagtgt gtgcccactg aggagt ccaa catcaccatg cagattatgc ggatcaaacc tc accaaggc cagcacatag gagagatgag cttcctaca g cacaacaaat gtgaatgcag accaaagaaa gatag agcaa gacaagaaaa aaaatcagtt cgaggaaagg g aaaggggca aaaacgaaag cgcaagaaat cccgtccc tg tgggccttgc tcagagcgga gaaagcattt gttt gtacaa gatccgcaga cgtgtaaatg ttcctgcaaa aacacagact cgcgttgcaa ggcgaggcag cttgagt taa acgaacgtac ttgcagatgt gacaagccga ggc ggtga Mature human VEGF-A (SEQ ID NO: 13) apma egggqnhhev vkfmdvyqrs ychpietlvd if qeypdeie yifkpscvpl mrcggccnde glecvptee s nitmqimrik phqgqhigem sflqhnkcec rpkkd rarqe kksvrgkgkg qkrkrkksry kswsvyvgar c clmpwslpg phpcgpcser rkhlfvqdpq tckcsckn td srckarqlel nertcrcdkp rr Mature human VEGF-B (SEQ ID NO: 14) pvsqpdapg hqrkvvswid vytratcqpr evvvpltv el mgtvakqlvp scvtvqrcgg ccpddglecv ptgq hqvrmq ilmirypssq lgemsleehs qcecrpkkkd savkpdraat phhrpqprsv pgwdsapgap spadith ptp apgpsahaap sttsaltpgp aaaaadaaas sva kgga Mature human VEGF-C (SEQ ID NO: 15) Ahynteilk sidnewrktq cmprevcidv gkefgvat nt ffkppcvsvy rcggccnseg lqcmntstsy lskt lfeitv plsqgpkpvt isfanhtscr cmskldvyrq vhsiirr Mature human VEGF-D (SEQ ID NO: 16) fa atfydietlk videewqrtq cspretcvev asel gkstnt ffkppcvnvf rcggccnees licmntstsy iskqlfeisv pltsvpelvp vkvanhtgck clptapr hpy siirr

[0107] In some examples of any of the antibodies and antigen-binding fragments thereof described herein, the anti bodies and antigen-binding fragments are VEGF antigens (e.g., any of the exemplary VEGF proteins described herein, e.g., mature human VEGF-A, mature human VEGF-B, mature human VEGF-A, mature human VEGF-B, mature can bind to one or more of human VEGF-C and mature human VEGF-D (e.g., any of the binding affinities described herein). (For example, any of the binding affinities described herein).

[0108] In some embodiments described herein, the antibody or antigen-binding antibody fragment can reduce the activity of VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and one or more of mature human VEGF-D). In some embodiments, the antibody or antigen-binding antibody fragment can prevent VEGF (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors). See, e.g., WO1998 / 045331, US9, 079,953, US2015 / 0147317, US2016 / 0289314, Plotkin et al., Otology & Neurotology 33:10 46 - 1052 (2012), and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328 - 335. In some embodiments, the antibody or antigen-binding antibody can inhibit downstream signaling (e.g., VEGF receptors, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., human VEGFR-1, human VEGFR-2, and human V (e.g., one or more of any of the exemplary VEGF proteins described herein, e.g., mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors). See, e.g., WO1998 / 045331, US9, 079,953, US2015 / 0147317, US2016 / 0289314, Plotkin et al., Otology & Neurotology 33:10 46 - 1052 (2012), and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328 - 335. In some embodiments, the antibody or antigen-binding antibody can inhibit downstream signaling (e.g., VEGF receptors, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., human VEGFR-1, human VEGFR-2, and human V EGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors). For example, see WO1998 / 045331, US9, 079,953, US2015 / 0147317, US2016 / 0289314, Plotkin et al., Otology & Neurotology 33:10 079,953, US2015 / 0147317, US2016 / 0289314, Plotkin et al., Otology & Neurotology 33:10 46 - 1052 (2012), and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328 - 335. In some embodiments, the antibody or antigen-binding antibody can inhibit downstream signaling (e.g., VEGF receptors, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., human VEGFR-1, human VEGFR-2, and human V 46 - 1052 (2012), and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328 - 335. In some embodiments, the antibody or antigen-binding antibody can inhibit downstream signaling (e.g., VEGF receptors, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., human VEGFR-1, human VEGFR-2, and human V EGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors). For example, see WO1998 / 045331, US9, 079,953, US2015 / 0147317, US2016 / 0289314, Plotkin et al., Otology & Neurotology 33:10 46 - 1052 (2012), and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328 - 335. In some embodiments, the antibody or antigen-binding antibody can inhibit downstream signaling (e.g., VEGF receptors, e.g., one or more of any of the exemplary VEGF receptors described herein, e.g., human VEGFR-1, human VEGFR-2, and human V EGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) from binding to one or more of its receptors (e.g., one or more VEGF receptors). For example, see WO1998 / 045331, US9, One or more of the downstream signal transmissions of EGFR-3 can be reduced. How many In some embodiments, the reduction in VEGF activity is, for example, the auditory nerve tumor, vestibular schwannoma, or neurofibromatosis type II in a mammal, respectively, compared to the auditory level or size before administration of any of the AAV vectors described herein. Or the size of the auditory nerve tumor, vestibular schwannoma, or neurofibromatosis type II in a mammal or the severity of one or more of its symptoms Degree of improvement in hearing (e.g., improvement in hearing of 1% to about 400% (or any of the sub-ranges within this range described herein)) or decrease (e.g., 1% to 99%, 1% to 95%, 1% to 90%, 1% to 85%, 1% to 80%, 1% to 75%, 1% to 70%, 1% to 65%, 1% to 60%, 1% to 55%, 1% to 50%, 1% to 45%, 1% to 40 %, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 99%, 5% to 95%, 5% to 90%, 5% to 85%, 5% to 80%, 5% to 75%, 5% to 70%, 5% to 65%, 5% to 60%, 5% to 55%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25 %, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 99%, 10% to 95%, 1 0% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 1 0% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 1 0% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 1 0% to 15%, 15% to 99%, 15% to 95%, 15% to 90%, 15% to 85%, 1 5% to 80%, 15% to 75%, 15% to 70%, 15% to 65%, 15% to 60%, 1 5% to 80%, 15% to 75%, 15% to 70%, 15% to 65%, 15% to 60%, 1 5% to 80%, 15% to 75%, 15% to 70%, 15% to 65%, 15% to 60%, 1 5%~55%、15%~50%、15%~45%、15%~40%、15%~35%、1 5%~30%、15%~25%、15%~20%、20%~99%、20%~95%、2 0%~90%、20%~85%、20%~80%、20%~75%、20%~70%、2 0%~65%、20%~60%、20%~55%、20%~50%、20%~45%、2 0%~40%、20%~35%、20%~30%、20%~25%、25%~99%、2 5%~95%、25%~90%、25%~85%、25%~80%、25%~75%、2 5%~70%、25%~65%、25%~60%、25%~55%、25%~50%、2 5%~45%、25%~40%、25%~35%、25%~30%、30%~99%、3 0%~95%、30%~90%、30%~85%、30%~80%、30%~75%、3 0%~70%、30%~65%、30%~60%、30%~55%、30%~50%、3 0%~45%、30%~40%、30%~35%、35%~99%、35%~95%、3 5%~90%、35%~85%、35%~80%、35%~75%、35%~70%、3 5%~65%、35%~60%、35%~55%、35%~50%、35%~45%、3 5%~40%、40%~99%、40%~95%、40%~90%、40%~85%、4 0%~80%、40%~75%、40%~70%、40%~65%、40%~60%、4 0%~55%、40%~50%、40%~45%、45%~99%、45%~95%、4 5%~90%、45%~85%、45%~80%、45%~75%、45%~70%、4 5%~65%、45%~60%、45%~55%、45%~50%、50%~99%、5 0% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 5 0% to 70%, 50% to 65%, 50% to 60%, 50% to 55%, 55% to 99%, 5 5% to 95%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 5 5% to 70%, 55% to 65%, 55% to 60%, 60% to 99%, 60% to 95%, 6 0% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 6 0% to 65%, 65% to 99%, 65% to 95%, 65% to 90%, 65% to 85%, 6 5% to 80%, 65% to 75%, 65% to 70%, 70% to 99%, 70% to 95%, 7 0% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 75% to 99%, 7 5% to 95%, 75% to 90%, 75% to 85%, 75% to 80%, 80% to 99%, 8 0% to 95%, 80% to 90%, 80% to 85%, 85% to 99%, 85% to 95%, 8 5% to 90%, 90% to 99%, 90% to 95%, or a decrease of 95% to 99%) thereby can be indirectly detected. In some embodiments, the reduction of VEGF activity is in vitro can be detected in an assay.

[0109] In some embodiments, an antibody that specifically binds to VEGF is bevacizumab (Ava statin (registered trademark)) or an antigen-binding fragment thereof. Bevacizumab (full-size antibody of approximately 150 kDa) inhibits all isoforms of VEGF-A. Bevacizumab was approved by the Food and Drug Administration (FDA) in 2004 for colorectal cancer for intravenous (IV) administration at 4.0 to 7.5 mg / kg every 2 to 3 weeks (plasma half-life of 21 days), and intravitreal (IVT) administration at 1.25 mg in 0.05 mL (half-life of 5.6 days). Bevacizumab is 5 Has a K for 8 pM of VEGF 165 (VEGF-A). For example, see WO201 D 7 / 050825. In some embodiments, an antibody that specifically binds to VEGF is ranibizumab (Lucentis®), or an antigen-binding fragment thereof. Ranibizumab (approximately 50 kDa) inhibits all isoforms of VEGF-A. Ranibizumab was approved by the FDA in 2006 for intravitreal use at 4.0 - 7.5 mg / kg intravenous (IV) administration (plasma half-life 0.5 days) over 2 - 3 weeks, 0.5 mg in 0.05 mL intravitreal (IVT) administration (half-life 3.2 days). Ranibizumab has a K for 4 6 pM of VEGF 165 (VEGF-A). For example, see WO201 4 / 178078. In some embodiments, an antibody that specifically binds to VEGF is sevastuzumab (APX003 / SIM-BD0801), or an antigen-binding fragment thereof. The amino acids encoding the light chain of bevacizumab (SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP GKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP D EDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The amino acids encoding the heavy chain of bevacizumab (SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA GKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC The amino acids encoding the heavy chain of bevacizumab (SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK

[0110] Some of the antibodies and antigen-binding fragments thereof that specifically bind to VEGF described in this specification In some embodiments, the antibody or its antigen-binding fragment is the variable light chain domain of bevacizumab or, or at least 80% identical thereto (e.g., at least 82%, at least 84% , at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical sequence including a variable light chain domain, and / or is the variable heavy chain domain of bevacizumab or, or at least 80% identical thereto (e.g., at least 82%, at least 84% , at least 86%, at least 88%, at least 90%, at least 92%, at least (94%, at least 96%, at least 98%, or at least 99%) identical The variable heavy domain comprises a sequence.

[0111] Some of the antibodies and antigen-binding fragments thereof that specifically bind VEGF described herein In embodiments, the antibody or antigen-binding fragment thereof is the variable light chain domain of bevacizumab. or a variable light chain domain comprising the same, and / or a variable heavy chain domain of bevacizumab. The VEGF-specific heavy chain domains described herein are or contain a variable heavy chain domain that is specific for VEGF. In some embodiments of the antibodies and antigen-binding fragments thereof, the antibody or its antigen The binding fragments are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, The variable light domain of bevacizumab, except that it contains 1 or 15 amino acid substitutions. or a variable light chain domain comprising the sequence thereof, and / or 1, 2, 3, 4, 5, Containing 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. A variable heavy chain domain that is or contains the variable heavy chain of bevacizumab, except for In some embodiments, the first antigen-binding domain comprises the light chain variable domain of bevacizumab. Three CDRs in the domain and / or three CDRs in the heavy chain variable domain of bevacizumab Includes. Amino acid coding for the light chain of ranibizumab (SEQ ID NO:7) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP GKAPKVLIYFTSSLHSGVPSRFSGSGGSGTDFTLTISSLQP EDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC Amino acids encoding the heavy chain of ranibizumab (SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQA PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY LQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

[0112] Some of the antibodies and antigen-binding fragments thereof that specifically bind to VEGF described herein In some embodiments, the antibody or antigen-binding fragment thereof is the variable light chain domain of ranibizumab or, or at least 80% identical thereto (e.g., at least 82%, at least 84% , at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical sequence including a variable light chain domain, and / or the variable heavy chain domain of ranibizumab or, or at least 80% identical thereto (e.g., at least 82%, at least 84% , at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical sequence The variable heavy domain comprises a sequence.

[0113] Some of the antibodies and antigen-binding fragments thereof that specifically bind VEGF described herein In embodiments, the antibody or antigen-binding fragment thereof is the variable light chain domain of ranibizumab. or a variable light chain domain comprising the same, and / or a variable heavy chain domain of ranibizumab The VEGF-specific heavy chain domains described herein are or contain a variable heavy chain domain that is specific for VEGF. In some embodiments of the antibodies and antigen-binding fragments thereof, the antibody or its antigen The binding fragments are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, The variable light domain of ranibizumab, except that it contains 1 or 15 amino acid substitutions. or a variable light chain domain comprising the sequence thereof, and / or 1, 2, 3, 4, 5, Containing 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions. A variable heavy chain domain that is or contains the variable heavy chain of ranibizumab, except for In some embodiments, the first antigen-binding domain comprises the light chain variable domain of ranibizumab. Three CDRs in the domain and / or three CDRs in the heavy chain variable domain of ranibizumab Includes.

[0114] Soluble VEGF receptors Soluble VEGF receptors can contain a signal peptide (e.g., an exemplary signal peptide described herein). one or more (e.g., two or more of the nucleic acid sequences) operably linked to 3) mammalian VEGF receptor(s) (e.g., VEGFR-1, VEGFR-2, and VEGFR-3), , a soluble VEGF receptor can specifically bind to one or more mammalian VEGF proteins (plural) (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D (e.g., two, three, or four), e.g., one or more of human wild-type VEGF-A, human wild-type VEGF -B, human wild-type VEGF-C, and human wild-type VEGF-D (e.g., two, three, or four)).

[0115] In some examples, the soluble VEGF receptor is the extracellular region of VEGFR-1 (e.g., adjacent sequences from wild-type human VEGFR-1 (e.g., the extracellular region from wild-type human VEGFR-1 (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6 in SEQ ID NO: 23) of the immunoglobulin-like domains in the extracellular region (e.g., adjacent sequences containing one or more (e.g., 1, 2, 3, 4, 5, 6 or 7) of the immunoglobulin-like domains in the extracellular region (e.g., adjacent sequences from wild-type human VEGFR-1), or adjacent sequences from wild-type human VEGFR R-1 that are at least 80% (e.g., at least 82%, at least 84 %, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the adjacent sequences in SEQ ID NO: 23, e.g., adjacent sequences in SEQ ID NO: 23 that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to the adjacent sequences in SEQ ID NO: 23), e.g., a portion (e.g., about 10 amino acids to about 732 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 55 0 amino acids, from about 10 amino acids to about 500 amino acids, from about 10 amino acids to about 450 amino acids, from about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, from about 10 amino acids to about 300 amino acids, about 10 ami no acids to about 250 amino acids, from about 10 amino acids to about 200 amino acids, about 10 amino acids to about 150 amino acids, from about 10 amino acids to about 100 amino acids, about 10 amino acids to about 50 amino acids, from about 50 amino acids to about 732 amino acids, about 5 0 amino acids to about 700 amino acids, from about 50 amino acids to about 650 amino acids, from about 50 amino acids to about 600 amino acids, from about 50 amino acids to about 550 amino acids, from about 50 amino acids to about 500 amino acids, from about 50 amino acids to about 450 a mino acids, from about 50 amino acids to about 400 amino acids, from about 50 amino acids to about 350 amino acids, from about 50 amino acids to about 300 amino acids, from about 50 amino acids to about 25 0 amino acids, from about 50 amino acids to about 200 amino acids, from about 50 amino acids to about 150 amino acids, from about 50 amino acids to about 100 amino acids, about 100 amino acids to about 732 amino acids, from about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, from about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, from about 100 amino acids to about 500 amino no acids, from about 100 amino acids to about 450 amino acids, from about 100 amino acids to about 400 amino acids, from about 100 amino acids to about 350 amino acids, from about 100 amino acids to about 300 amino acids, from about 100 amino acids to about 250 amino acids, about 100 a Amino acids - from about 200 amino acids, from about 100 amino acids to about 150 amino acids, about 15 0 amino acids to about 732 amino acids, from about 150 amino acids to about 700 amino acids , from about 150 amino acids to about 650 amino acids, from about 150 amino acids to about 600 amino acids, from about 150 amino acids to about 550 amino acids, from about 150 amino acids to about 5 00 amino acids, from about 150 amino acids to about 450 amino acids, from about 150 amino acids to about 400 amino acids, from about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, from about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, from about 200 amino acids to about 732 amino acids in, from about 200 amino acids to about 700 amino acids, from about 200 amino acids to about 650 amino acids, from about 200 amino acids to about 600 amino acids, from about 200 amino acids to about 550 amino acids, from about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, from about 200 amino acids to about 400 amino acids, about 20 0 amino acids to about 350 amino acids, from about 200 amino acids to about 300 amino acids , from about 200 amino acids to about 250 amino acids, from about 250 amino acids to about 732 amino acids, from about 250 amino acids to about 700 amino acids, from about 250 amino acids to about 6 50 amino acids, from about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, from about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, from about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids 300 to 732 amino acids, 300 to 700 amino acids 100 amino acids, about 300 amino acids ~ 650 amino acids, about 300 amino acids ~ Approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 30 0 to about 400 amino acids, about 300 to about 350 amino acids , about 350 amino acids to about 732 amino acids, about 350 amino acids to about 700 amino acids Amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 6 00 amino acids, about 350 amino acids to about 550 amino acids, about 350 amino acids ~About 500 amino acids, ~About 350 amino acids, ~About 450 amino acids, ~About 350 400 amino acids to about 400 amino acids, 400 amino acids to about 732 amino acids, 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids About 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 400 amino acids ~ 500 amino acids 400 amino acids ~ About 450 amino acids, about 450 amino acids to about 732 amino acids, about 450 amino acids ~ about 700 amino acids, about 450 amino acids ~ about 650 amino acids, about 45 0 to about 600 amino acids, about 450 to about 550 amino acids , about 450 amino acids to about 500 amino acids, about 500 amino acids to about 732 amino acids Amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 6 50 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 732 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 732 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 732 amino acids, about 650 amino acids to about 700 amino acids, or about 700 amino acids to about 732 amino acids).

[0116] In some examples, the soluble VEGF receptor is the extracellular region of VEGFR-2 (e.g., adjacent sequences from wild-type human VEGFR-2 (e.g., the extracellular region from wild-type human VEGFR-2 (e.g., one or more (e.g., 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region (e.g., SEQ ID NO: 26)), adjacent sequences, or sequences that are at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence from wild-type human VEGFR- 2, e.g., at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence in SEQ ID NO: 26), a portion (e.g., about 20 amino acids to about 745 amino acids) of the sequence e.g., at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence in SEQ ID NO: 26), at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence in SEQ ID NO: 26), at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence in SEQ ID NO: 26), at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to an adjacent sequence in SEQ ID NO: 26), comprises an acid or a partial range within this range described herein).

[0117] In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1 ( for example, any of the portions of the extracellular region of VEGFR-1 described herein) and V a portion of the extracellular region of VEGFR-2 (for example, any of the portions of the extracellular region of VEGFR-2 described herein). For example, the soluble VEGF receptor may comprise one or more (for example, 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region derived from wild-type human V EGFR-1, and one or more (for example, 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-2 (for example, aflibercept).

[0118] In some examples, the soluble VEGF receptor comprises the extracellular region of VEGFR-3 (for example, a flanking sequence derived from wild-type human VEGFR-3 (for example, one or more (for example, 1, 2, 3, 4, 5, 6 or 7) immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-3 (for example, SEQ ID NO: 29)), or a flanking sequence that is at least 80% (for example, at least 82%, at least 84 %, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical to a flanking sequence derived from wild-type human VEGF R-3, for example, a flanking sequence in SEQ ID NO: 29 that is at least 80% (for example, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, At least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) a portion of the same sequence (e.g., from about 20 amino acids to about 751 amino acids, or any of the sub-ranges within this range described herein).

[0119] Non-limiting examples of the extracellular regions of different mammalian VEGFR-1, different mammalian VEGFR-2, and different mammalian VEG FR-3 are described herein. Non-limiting examples of nucleotide sequences encoding proteins and wild type VEGF receptor proteins are shown below. As can be understood by those skilled in the art, while substitutions in amino acids conserved across species are likely to result in changes in protein function, substitutions at amino acid positions that are not conserved across species are less likely to affect protein function. Human VEGF receptor 1 isoform 2 protein sequence (SEQ ID NO: 17) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQ HIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSA CGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKET ESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPV KLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRR RIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFK ​SVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVK AFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPD PALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARC DFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMAS TLVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFH VNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTM HYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARN VYTGEEILQKKEITIRGEHCNKKAVFSRISKFKSTRNDCT TQSNVKH Human VEGF Receptor 1 Isoform 2 cDNA (SEQ ID NO: 18) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGC TGCTCAGCTGTCTGCTTCTCACAGGATCTAGTTCAGGTTC AAAATTAAAAGATCCTGAACTGAGTTTAAAAGGCACCCAG CACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCA GGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCC TGTGGAAGAAATGGCAAACAATTCTGCAGTACTTTAACCT TGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTG CAAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACA GAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACA CATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTT ACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCAC TTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGA CAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGC ATTTGTATAAGACAAACTATCTCACACATCGACAAACCAA TACAATCATAGATGTCCAAATAAGCACACCACGCCCAGTC AAATTACTTAGAGGCCATACTCTTGTCCTCAATTGTACTG CTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGA CGAATTGACCAAAGCAATTCCCATGCCAACATATTCTACA GTGTTCTTACTATTGACAAAATGCAGAACAAAGACAAAGG ACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAA TCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGT AGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGAAG GCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGT TACCTGCGACTGAGAAATCTGCTCGCTATTTGACTCGTGG CTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATG TGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAA ACCCCAGATTTACGAAAAGGCCGTGTCATCGTTTCCAGAC CCGGCTCTCTACCCACTGGGCAGCAGACAAATCCTGACTT GTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGT GACTTTTGTTCCAATAATGAAGAGTCCTTTATCCTGGATG CTGACAGCAACATGGGAAACAGAATTGAGAGCATCACTCA GCGCATGGCAATAATAGAAGGAAAGAATAAGATGGCTAGC ACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACA TTTGCATAGCTTCCAATAAAGTTGGGACTGTGGGAAGAAA CATAAGCTTTTATATCACAGATGTGCCAAATGGGTTTCAT GTTAACTTGGAAAAAATGCCGACGGAAGGAGAGGACCTGA AACTGTCTTGCACAGTTAACAAGTTCTTATACAGAGACGT TACTTGGATTTTACTGCGGACAGTTAATAACAGAACAATG CACTACAGTATTAGCAAGCAAAAAATGGCCATCACTAAGG AGCACTCCATCACTCTTAATCTTACCATCATGAATGTTTC CCTGCAAGATTCAGGCACCTATGCCTGCAGAGCCAGGAAT GTATACACAGGGGAAGAAATCCTCCAGAAGAAAGAAATTA CAATCAGAGGTGAGCACTGCAACAAAAAGGCTGTTTTCTC TCGGATCTCCAAATTTAAAAGCACAAGGAATGATTGTACC ACACAAAGTAATGTAAAACATTAA Human VEGF receptor 1 isoform 3 protein sequence (SEQ ID NO: 19) (sFlt1- 14) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQ HIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSA CGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKET ESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPV KLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRR RIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFK SVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVK AFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPD PALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARC DFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMAS TLVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFH VNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTM HYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARN VYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTL DCHANGVPEPQITWFKNNHKIQQEPELYTSTSPSSSSSSP LSSSSSSSSSSSS Human VEGF Receptor 1 Isoform 3 cDNA (SEQ ID NO: 20) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGC TGCTCAGCTGTCTGCTTCTCACAGGATCTAGTTCAGGTTC AAAATTAAAAGATCCTGAACTGAGTTTAAAAGGCACCCAG CACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCA GGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCC TGTGGAAGAAATGGCAAACAATTCTGCAGTACTTTAACCT TGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTG CAAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACA GAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACA CATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTT ACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCAC TTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGA CAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGC ATTTGTATAAGACAAACTATCTCACACATCGACAAACCAA TACAATCATAGATGTCCAAATAAGCACACCACGCCCAGTC AAATTACTTAGAGGCCATACTCTTGTCCTCAATTGTACTG CTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGA CGAATTGACCAAAGCAATTCCCATGCCAACATATTCTACA GTGTTCTTACTATTGACAAAATGCAGAACAAAGACAAAGG ACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAA TCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGT AGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGAAG GCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGT TACCTGCGACTGAGAAATCTGCTCGCTATTTGACTCGTGG CTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATG TGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAA ACCCCAGATTTACGAAAAGGCCGTGTCATCGTTTCCAGAC CCGGCTCTCTACCCACTGGGCAGCAGACAAATCCTGACTT GTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGT GACTTTTGTTCCAATAATGAAGAGTCCTTTATCCTGGATG CTGACAGCAACATGGGAAACAGAATTGAGAGCATCACTCA GCGCATGGCAATAATAGAAGGAAAGAATAAGATGGCTAGC ACCTTGGTTGTGGCTGACTCTAGAATTTCTGGAATCTACA TTTGCATAGCTTCCAATAAAGTTGGGACTGTGGGAAGAAA CATAAGCTTTTATATCACAGATGTGCCAAATGGGTTTCAT GTTAACTTGGAAAAAATGCCGACGGAAGGAGAGGACCTGA AACTGTCTTGCACAGTTAACAAGTTCTTATACAGAGACGT TACTTGGATTTTACTGCGGACAGTTAATAACAGAACAATG CACTACAGTATTAGCAAGCAAAAAATGGCCATCACTAAGG AGCACTCCATCACTCTTAATCTTACCATCATGAATGTTTC CCTGCAAGATTCAGGCACCTATGCCTGCAGAGCCAGGAAT GTATACACAGGGGAAGAAATCCTCCAGAAGAAAGAAATTA CAATCAGAGATCAGGAAGCACCATACCTCCTGCGAAACCT CAGTGATCACACAGTGGCCATCAGCAGTTCCACCACTTTA GACTGTCATGCTAATGGTGTCCCCGAGCCTCAGATCACTT GGTTTAAAAACAACCACAAAATACAACAAGAGCCTGAACT GTATACATCAACGTCACCATCGTCATCGTCATCATCACCA TTGTCATCATCATCATCATCGTCATCATCATCATCATCAT AG Human VEGF Receptor 1 Isoform 4 Protein Sequence (SEQ ID NO: 21) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQ HIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSA CGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKET ESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRV TSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYK EIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPV KLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRR RIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFK SVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVK AFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDA GNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPD PALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARC DFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKLPP ANSSFMLPPTSFSSNYFHFLP Human VEGF Receptor 1 Isoform 4 cDNA (SEQ ID NO: 22) ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGC TGCTCAGCTGTCTGCTTCTCACAGGATCTAGTTCAGGTTC AAAATTAAAAGATCCTGAACTGAGTTTAAAAGGCACCCAG CACATCATGCAAGCAGGCCAGACACTGCATCTCCAATGCA GGGGGGAAGCAGCCCATAAATGGTCTTTGCCTGAAATGGT GAGTAAGGAAAGCGAAAGGCTGAGCATAACTAAATCTGCC TGTGGAAGAAATGGCAAACAATTCTGCAGTACTTTAACCT TGAACACAGCTCAAGCAAACCACACTGGCTTCTACAGCTG CAAATATCTAGCTGTACCTACTTCAAAGAAGAAGGAAACA GAATCTGCAATCTATATATTTATTAGTGATACAGGTAGAC CTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACA CATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTT ACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCAC TTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGA CAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAA GAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGC ATTTGTATAAGACAAACTATCTCACACATCGACAAACCAA TACAATCATAGATGTCCAAATAAGCACACCACGCCCAGTC AAATTACTTAGAGGCCATACTCTTGTCCTCAATTGTACTG CTACCACTCCCTTGAACACGAGAGTTCAAATGACCTGGAG TTACCCTGATGAAAAAAATAAGAGAGCTTCCGTAAGGCGA CGAATTGACCAAAGCAATTCCCATGCCAACATATTCTACA GTGTTCTTACTATTGACAAAATGCAGAACAAAGACAAAGG ACTTTATACTTGTCGTGTAAGGAGTGGACCATCATTCAAA TCTGTTAACACCTCAGTGCATATATATGATAAAGCATTCA TCACTGTGAAACATCGAAAACAGCAGGTGCTTGAAACCGT AGCTGGCAAGCGGTCTTACCGGCTCTCTATGAAAGTGAAG GCATTTCCCTCGCCGGAAGTTGTATGGTTAAAAGATGGGT TACCTGCGACTGAGAAATCTGCTCGCTATTTGACTCGTGG CTACTCGTTAATTATCAAGGACGTAACTGAAGAGGATGCA GGGAATTATACAATCTTGCTGAGCATAAAACAGTCAAATG TGTTTAAAAACCTCACTGCCACTCTAATTGTCAATGTGAA ACCCCAGATTTACGAAAAGGCCGTGTCATCGTTTCCAGAC CCGGCTCTCTACCCACTGGGCAGCAGACAAATCCTGACTT GTACCGCATATGGTATCCCTCAACCTACAATCAAGTGGTT CTGGCACCCCTGTAACCATAATCATTCCGAAGCAAGGTGT GACTTTTGTTCCAATAATGAAGAGTCCTTTATCCTGGATG CTGACAGCAACATGGGAAACAGAATTGAGAGCATCACTCA GCGCATGGCAATAATAGAAGGAAAGAATAAGCTTCCACCA GCTAACAGTTCTTTCATGTTGCCACCTACAAGCTTCTCTT CCAACTACTTCCATTTCCTTCCGTGA The extracellular region of wild-type human VEGFR-1 (SEQ ID NO: 23) (the 7 Ig-like domains are in bold and underlined)

Chemical formula

Chemical Structure

Chem.

[0120] In some examples, the soluble VEGF receptor may further comprise a stabilizing domain (e.g., an Fc domain or a portion of an Fc domain). For example, the stabilizing domain may be an IgG1 Fc domain (e.g., a human wild-type IgG1 Fc domain or a portion thereof) and may be. For example, the stabilizing domain may be an IgG2 Fc domain (e.g., a human wild-type IgG 2 Fc domain or a portion thereof). For example, the stabilizing domain may be an IgG3 It can be an Fc domain (e.g., a human wild-type IgG3 domain or a part thereof).

[0121] Non-limiting examples of the human wild-type IgG1 Fc domain, human wild-type IgG2 Fc domain, and human wild-type IgG3 Fc domain are shown below. Human wild-type IgG1 Fc domain (SEQ ID NO: 32) pcpapellgg psvflfppkp kdtlmisrtp evtcvvv dvs hedpevkfnw yvdgvevhna ktkpreeqyn sty rvvsvlt vlhqdwlngk eykckvsnka lpapiektis kakgqprepq vytlppsrde ltknqvsltc lvkgfy psdi avewesngqp ennykttppv ldsdgsffly sk ltvdksrw qqgnvfscsv mhealhnhyt qkslslspg k Human wild-type IgG2 Fc region (SEQ ID NO: 33) vecppcpapp vagpsvflfp pkpkdtlmis rtpevtc vvv dvshedpevq fnwyvdgvev hnaktkpree qfn stfrvvs vltvvhqdwl ngkeykckvs nkglpapiek tisktkgqpr epqvytlpps reemtknqvs ltclvk gfyp sdiavewesn gqpennyktt ppmldsdgsf fl yskltvdk srwqqgnvfs csvmhealhn hytqkslsl s pgk Human wild-type IgG3 Fc region (SEQ ID NO: 34) tcprcpapel lggpsvflfp pkpkdtlmis rtpevtc​ vvv dvshedpevq fkwyvdgvev hnaktkpree qfn stfrvvs vltvlhqdwl ngkeykckvs nkalpapiek tisktkgqpr epqvytlpps reemtknqvs ltclvk gfyp sdiavewess gqpennyktt ppmldsdgsf fl yskltvdk srwqqgnifs csvmhealhn rftqkslsl s pgk

[0122] In some embodiments, the soluble VEGF receptor is aflibercept (Eylea( registered trademark)). Aflibercept comprises portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1 (size approximately 115k Da). Aflibercept inhibits the activities of VEGF-A, VEGF-B, and PIGF. Aflibercept has a K for VEGF-A of 0.49 pM. See, for example, WO2017 / 2 D 18974. See also. The amino acids encoding aflibercept (SEQ ID NO: 12) SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVT LKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCE ATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKL VLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSG SEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFV RVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0123] In some embodiments of the soluble VEGF receptor, at least 80% identical (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) to SEQ ID NO: 12. one (e.g., at least 82%, at least 84%, at least 86%, at least 88 %, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%) identical sequence.

[0124] In some embodiments of the soluble VEGF receptor, it is SEQ ID NO: 12, or contains an extracellular domain containing its sequence, except that it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the sequence of SEQ ID NO: 12. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions in the sequence of SEQ ID NO: 12. or contains an extracellular domain containing its sequence.

[0125] Further examples of soluble VEGF receptors are described, for example, in Kendall et al., PNAS 90:10705-10709, 1993, Kendall et al., Biochem Biophys Res Commun 226:324-328, 1996, Failla et al., Int J Mol Sci 19(5):pii. AS 90:10705-10709,1993, Kendall et al., Bi ochem Biophys Res Commun 226:324-328,199 6, Failla et al., Int J Mol Sci 19(5):pii. E1306,2018, and Jung et al., PLoS One 7(9):e 44572.

[0126] Vector A recombinant AAV vector or “rAAV” typically comprises, at a minimum, a transgene or a portion thereof and regulatory sequences, and optionally 5’ and 3’ AAV inverted terminal repeats (ITR s). Such recombinant AAV vectors are packaged into a capsid and delivered to a selected target cell (e.g., a cochlear hair cell).

[0127] The AAV sequences of this vector typically include cis - acting 5’ and 3’ ITR sequences ( see, e.g., B.J. Carter, “Handbook of Parvoviruses ”, ed., P. Tijsser, CRC Press, pp. 155 - 168, 199 0). A typical AAV ITR sequence is about 145 nucleotides in length. In some embodiments, at least 75% (e.g., at least 8 0%, at least 85%, at least 90%, or at least 95%) of a typical ITR sequence is incorporated into the AAV vector . The ability to modify these ITR sequences is within the skill in the art (see, e.g., Sambrook et al., “Molecular Cloni ng. A Laboratory Manual”, 2d ed., Cold Spri ng Harbor Laboratory, New York, 1989, and K.F isher et al., J Virol. 70:520 - 532, 1996, etc.). In some embodiments, any of the coding sequences described herein is adjacent to the 5’ and 3’ AAV ITR sequences in the AAV vector. The AAV ITR sequences can be obtained from any known AAV, including currently identified AAV types ​ is possible.

[0128] The AAV vectors described herein may contain any of the regulatory elements described herein (e.g., one or more of a promoter, a polyadenylation (poly(A)) signal sequence, and an IRES).

[0129] In some embodiments, the vector(s) is an adenovirus (e.g., see Mitriev et al. (1998) J. Virol. 72:9706-9713 and Poulin et al., J. Virol 8:10074-10086, 2 010). In some embodiments, the vector(s) is a retrovirus (e.g., see Maier et al. (2010) Future Microbiol iol 5:1507-23).

[0130] The vectors provided herein can be of different sizes. The selection of a vector for use in any of the compositions, kits, and methods described herein can depend on the size of the vector.

[0131] In some embodiments, the vector(s) can have a total nucleotide number of up to 10 kb. In some embodiments, the viral vector(s) can be about 1 kb to about 2 k b, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb , about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 k b, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 k b, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 k b to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or it may have a total number of nucleotides in the range of about 9 kb to about 10 kb.

[0132] In some embodiments, the vector(s) is / are a lentivirus and can have a total number of nucleotides of up to 8 kb. In some examples, the lentivirus(es) can have about 1 k b to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 k b to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 k b to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 k b to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 k b to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 k b to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 k b to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb of total number of nucleotides.

[0133] In some embodiments, the vector(s) is an adenovirus and can have a total nucleotide number of up to 8 kb. In some embodiments, the adenovirus(es) can be from about 1 kb to about 2 kb, from about 1 kb to about 3 kb, from about 1 kb to about 4 kb, from about 1 kb to about 5 kb, from about 1 kb to about 6 kb, from about 1 kb to about 7 kb, from about 1 kb to about 8 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb, from about 2 kb to about 6 kb, from about 2 kb to about 7 kb, from about 2 kb to about 8 kb, from about 3 kb to about 4 kb, from about 3 kb to about 5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 7 kb, from about 3 kb to about 8 kb, from about 4 kb to about 5 kb, from about 4 kb to about 6 kb, from about 4 kb to about 7 kb, from about 4 kb to about 8 kb, from about 5 kb to about 6 kb, from about 5 kb to about 7 kb, from about 5 kb to about 8 kb, from about 6 kb to about 7 kb, from about 6 kb to about 8 kb, or from about 7 kb to about 8 kb in terms of total nucleotide number.

[0134] In some embodiments, the vector(s) is an adeno-associated virus (AAV vector ), and can contain a total nucleotide number of up to 5 kb. In some embodiments, the AAV vector(s) can be from about 1 kb to about 2 kb, from about 1 kb to about 3 kb, from about 1 kb to about 4 kb , from about 1 kb to about 5 kb, from about 2 kb to about 3 kb, from about 2 kb to about 4 kb, from about 2 kb to about 5 kb , from about 3 kb to about 4 kb, from about 3 kb to about 5 kb, or from about 4 kb to about 5 kb in terms of total nucleotide number.

[0135] Any of the vectors disclosed herein can be introduced into mammalian cells (e.g., inner ear cells, inner hair cells of the cochlea) using various different methods known in the art. Non-limiting examples of methods for introducing nucleic acids into mammalian cells include lipofection ​ n, transfection (e.g., calcium phosphate transfection, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. n, transfection using highly branched organic compounds, transfection using cationic polymers, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. n, dendrimer-based transfection, optical transfection, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. n, particle-based transfection (e.g., nanoparticle transfection), or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. n, or transfection using liposomes (e.g., cationic liposomes)), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. ), microinjection, electroporation, cell squeezing, sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. sonoporation, protoplast fusion, imparfaction, hydrodynamic delivery, gene gun, magnetofection, virus transfection, and nucleofection. virus transfection, and nucleofection.

[0136] Any of the vectors described herein may further comprise a control sequence selected from the group consisting of a control sequence, e.g., a transcription start sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) signal, and a Kozak consensus sequence. Non-limiting examples of these control sequences are described herein. Any of the vectors described herein may further comprise a control sequence selected from the group consisting of a transcription start sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) signal, and a Kozak consensus sequence. Non-limiting examples of these control sequences are described herein. Any of the vectors described herein may further comprise a control sequence selected from the group consisting of a transcription start sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) signal, and a Kozak consensus sequence. Non-limiting examples of these control sequences are described herein. Any of the vectors described herein may further comprise a control sequence selected from the group consisting of a transcription start sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (polyA) signal, and a Kozak consensus sequence. Non-limiting examples of these control sequences are described herein. In some embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. In some embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Promoter

[0137] The term "promoter" means a DNA sequence recognized by an enzyme / protein in mammalian cells necessary to initiate transcription of a particular gene. A promoter typically binds, for example, RNA polymerase and / or any associated factors and initiates transcription. The term "promoter" means a DNA sequence recognized by an enzyme / protein in mammalian cells necessary to initiate transcription of a particular gene. A promoter typically binds, for example, RNA polymerase and / or any associated factors and initiates transcription. The term "promoter" means a DNA sequence recognized by an enzyme / protein in mammalian cells necessary to initiate transcription of a particular gene. A promoter typically binds, for example, RNA polymerase and / or any associated factors and initiates transcription. Promoters refer to the nucleotide sequence at which a gene is introduced. Non-limiting examples of promoters are described herein. Further examples of promoters are known in the art.

[0138] In some embodiments, an antibody (e.g., an antibody that specifically binds VEGF or A vector (e.g., an adeno-associated virus (AAV) vector) encoding an antigen-binding antibody fragment. The promoter may include a promoter and / or an enhancer. Vectors encoding the fragments can be prepared using vectors described herein or known in the art. The gene may include either a promoter and / or an enhancer.

[0139] In some embodiments, the promoter is an inducible promoter, a constitutive promoter , mammalian cell promoters, viral promoters, chimeric promoters, engineered promoters A promoter, a tissue specific promoter, or any other type known in the art. In some embodiments, the promoter is a mammalian RNA polymerase. In some embodiments, the promoter is an RNA polymerase II promoter, such as an RNA polymerase II promoter. In terms of form, the promoter may be an H1 promoter, a human U6 promoter, or a mouse U6 promoter. RNA polymerase promoters, including but not limited to the porcine U6 promoter, The promoter generally drives transcription in inner hair cells. In some instances, the promoter will be a promoter capable of promoting the expression of the cochlea. It is a cochlear-directed promoter.

[0140] A variety of promoters are known in the art that can be used herein. Non-limiting examples of promoters that can be used include human EF1a, human cytomegalo virus (CMV) (U.S. Patent No. 5,168,062), human ubiquitin C (UBC) , mouse phosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40), β-globin, β-actin, α-fetoprotein, γ-globin , β-interferon, γ-glutamyltransferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR (e.g., human T cell leukemia virus H TLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor , adenovirus 5 E2, stromelysin, mouse MX gene, glucose-regulated protein (GRP78 and GRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2κ b, HSP70, prolipherin, tumor necrosis factor, thyroid-stimulating hormone α gene, immunoglobulin light chain, T cell receptor, HLA DQα and DQβ, i nterleukin-2 receptor, MHC class II, MHC class II HLA-DRα, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponi n I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus, and ten gaza leukemia virus (GALV) promoters. Further Examples are known in the art. For example, see Lodish, Molecular Cel l Biology, Freeman and Company, New York 2 007. In some embodiments, the promoter is the CMV immediate early prom oter. In some embodiments, the promoter is the CAG promoter or the C AG / CBA promoter.

[0141] The term "constitutive" promoter, when operably linked to a nucleic acid encoding a protein (e.g., an antibody or an antigen-binding antibody fragment), refers to a nucleotide sequence that causes RNA to be transcribed from that nucleic acid in mammalian cells under most or all physiological conditions.

[0142] Examples of constitutive promoters include the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshar t et al, Cell 41:521-530, 1985), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the pho sphoglycerol kinase (PGK) promoter, and the EF1-alpha promoter (Invitrogen), but are not limited thereto.

[0143] Inducible promoters allow for regulation of gene expression and can be regulated by an exogenously supplied compound, an environmental factor such as temperature, or the presence of a specific physiological state (e.g., acute phase, a specific differentiation state of a cell, or only in replicating cells). Inducible promoters and induction systems include, but are not limited to, Invitrogen, Clontech, and Ariad . It is available from a variety of commercial sources and is not limited to. Further examples of inducible promoters are known in the art. Examples are known in the art.

[0144] Examples of inducible promoters regulated by exogenously supplied compounds include zinc inducible sheep metallothionein (MT) promoter, dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system (W O98 / 10088), ecdyson insect promoter (No et al, Proc. N atl. Acad. Sci. U.S.A. 93:3346-3351, 1996), tet racycline repressible system (Gossen et al, Proc. Natl. Acad. S ci. U.S.A. 89:5547-5551, 1992), tetracycline inducible system (Gossen et al, Science 268:1766-1769, 1995 、Harvey et al, Curr. Opin. Chem. Biol. 2:512- 518, 1998 also see), RU486 inducible system (Wang et al, Nat . Biotech. 15:239-243, 1997) and Wang et al, Ge ne Ther. 4:432-441, 1997), and rapamycin inducible system (Mag ari et al. J. Clin. Invest. 100:2865-2872, 19 97).

[0145] The term "tissue-specific" promoter refers to a promoter that is active only in a particular cell type and / or tissue (e.g., transcription of a particular gene occurs only in cells that express a transcription regulatory protein that binds to the tissue-specific promoter). Examples are known in the art. bind).

[0146] In some embodiments, the regulatory sequences confer tissue-specific gene expression capabilities. In certain instances, the tissue-specific regulatory sequences bind to tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0147] Exemplary tissue-specific promoters include, but are not limited to, the liver-specific thyroxine-binding globulin (T BG) promoter, the insulin promoter, the glucagon promoter, the somatostatin promoter, the pancreatic polypeptide (PPY) promoter, the synapsin-1 (Syn) promoter, the creatine kinase (MCK) promoter, the mammalian desmin (DES) promoter, the alpha-myosin heavy chain (a-MHC) promoter, and the cardiac troponin T (cTnT) promoter. Further exemplary promoters include the beta-actin promoter, the hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002-1009, 1996), the alpha-fetoprotein (AFP) promoter (Arbuthnot et al., Hum. Gene Ther. 7:1503-1514, 1996), the bone osteocalcin promoter (Stein et al., Mol. Biol. Re p. 24:185-196, 1997), the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res. 11:654-664, 1996) , the CD2 promoter (Hansal et al., J. Immunol. 161:1 063-1068, 1998), the immunoglobulin heavy chain promoter, the T cell receptor alpha chain promoter, and Neuronal promoters such as the α-chain promoter and the neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol. 13:503-515, 1993), the neurofilament light chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. U.S. A. 88:5611-5615, 1991), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron 15:373-384, 19 95) can be mentioned.

[0148] In some embodiments, the tissue-specific promoter is a snail-specific promoter . In some embodiments, the tissue-specific promoter is a snail hair cell-specific promoter . Non-limiting examples of snail hair cell-specific promoters include the ATOH1 promoter, the POU4F3 promoter, the LHX3 promoter, the MYO7A promoter, the MYO6 promoter, the α9ACHR promoter, and the α10ACHR promoter can be mentioned, but are not limited thereto. In some embodiments, the promoter is a snail hair cell-specific promoter such as the PRESTIN promoter or the ONCOMOD promoter. See, for example, Zheng et al., Nature 405:149- 155, 2000, Tian et al. Dev. Dyn. 231:199-203, 2004, and Ryan et al., Adv. Otorhinolaryngol. 66:99-115, 2009.

[0149] Enhancer ​In some cases, a vector (e.g., an AAV vector) may contain an enhancer sequence. The term "enhancer" refers to a nucleotide sequence that can increase the transcriptional level of a nucleic acid encoding a protein of interest (e.g., an antibody that specifically binds to VEGF or an antigen-binding antibody fragment thereof, or a soluble VEGF receptor). An enhancer sequence (50 - 1500 base pairs in length) generally increases the transcriptional level by providing additional binding sites for transcription-related proteins (e.g., transcription factors). In some embodiments, the enhancer sequence is found within an intron sequence. Unlike a promoter sequence, an enhancer sequence can act at a greater distance from the transcription start site (e.g., as compared to a promoter). Non-limiting examples of enhancers include the RSV enhancer, the CMV enhancer, and the SV40 enhancer.

[0150] Poly(A) signal sequence In some embodiments, any of the vectors provided herein (e.g., an AAV vector) may contain a polyadenylation (poly(A)) signal sequence. Most newly synthesized eukaryotic mRNAs have a poly(A) tail added during a complex process that includes cleavage of the primary transcript and a coupled polyadenylation reaction driven by the poly(A) signal sequence at their 3' ends (e.g., see Proudfoot et al., Cell 108 :501 - 512,2002). The poly(A) tail confers mRNA stability and mobility (Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland (Publishing, 1994). In some embodiments, the poly(A) signal sequence is positioned 3' to a nucleic acid sequence encoding an antibody heavy chain, an antibody light chain, an antigen-binding antibody fragment, or a soluble VEGF receptor.

[0151] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenyl moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) adenine nucleotide sequence added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a polyadenylation (or poly(A)) signal, which is a specific sequence. The poly(A) tail and the proteins bound to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA transport from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription termination, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0152] As used herein, "poly(A) signal sequence" or "polyadenylation signal"​​​​​​​​​​​ The "nal sequence" is a sequence that induces endonuclease cleavage of mRNA and a series of cleaved adenosines and addition to the 3' end of the mRNA.

[0153] Bovine growth hormone (bgh) (Woychik et al., Proc. Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984, U.S. Pat. No. 5,122,458), mouse-β-globin, mouse-α-globin (Orki n et al., EMBO J. 4(2):453-456, 1985, Thein et al., Blood 71(2):313-319, 1988), human collagen , polyomavirus (Batt et al., Mol. Cell Biol. 15( 9):4783-4790, 1995), herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (US2006 / 0040 354), human growth hormone (hGH) (Szymanski et al., Mol. Therapy 15(7):1340-1347, 2007), SV40 late and early poly(A) sites such as the SV40 poly(A) site consisting of a group (Schek et al., M ol. Cell Biol. 12(12):5386-5393, 1992) There are several poly(A) signal sequences that can be used, including those derived from .

[0154] The poly(A) signal sequence can be AATAAA. The AATAAA sequence is ATTA AA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AA TATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AA Having homology with AATAAA and capable of signaling polyadenylation, including TAAG It may be replaced with other hexanucleotide sequences that can do so (see, for example, WO06 / 12414). ).

[0155] In some embodiments, the poly(A) signal sequence is a synthetic polyadenylation site obtainable (see, for example, the Promega pCl-neo expression vector based on Levitt el al, Genes Dev. 3(7):1019 - 1025, 1989). In some embodiments, the poly(A) signal sequence is the polyadenylation signal of soluble neuropilin-1 ( sNRP) (AAATAAAATACGAAATG, SEQ ID NO 11) (see, for example, WO05 / 073384). Further examples of poly(A) signal sequences are known in the art.

[0156] Internal ribosome entry site (IRES) within the sequence In some embodiments, vectors encoding antibodies (e.g., antibody heavy chains and antibody light chains), antigen-binding antibody fragments , or soluble VEGF receptors (e.g., adeno-associated virus ( AAV) vectors) may contain an internal ribosome entry site (IRES) within the polynucleotide sequence . The IRES sequence is used to produce more than one polypeptide from a single gene transcript . The IRES forms a complex secondary structure that initiates translation from any position having an mRNA immediately downstream of where the IRES is located (see, for example, Pelletie r and Sonenberg, Mol. Cell. Biol. 8(3):1103 - 1112, 1988).​​

[0157] For example, foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV), human rhinovirus Human immunodeficiency virus (HRV), Cricket paralysis virus, Human immunodeficiency virus (HIV), Hepatitis A Hepatitis A virus (HAV), Hepatitis C virus (HCV), and Poliovirus (PV) derived There are several IRES sequences known to those of skill in the art, including, for example, Alberts ,Molecular Biology of the Cell,Garland S science, 2002, and Hellen et al., Genes Dev.15 (13):1593-612, 2001.

[0158] In some embodiments, the IRES sequence incorporated into the AAV vector is an IRES sequence derived from a foot and mouth disease virus. The foot-and-mouth disease virus 2A sequence is involved in the cleavage of the polyprotein. It is a small peptide (approximately 18 amino acids long) that has been shown to mediate D et al., EMBO 4:928-933, 1994, Mattion et al. al., J. Virology 70:8124-8127, 1996, Furler et al., Gene Therapy 8:864-873, 2001, and Ha lpin et al.,Plant Journal 4:453-459,1999 The cleavage activity of the 2A sequence has been shown to be essential for the synthesis of plasmids and gene therapy vectors (AAV and retrovirus vectors). This has been previously demonstrated in artificial systems containing erythropoietin (Ryan et al., EMBO 4:928-933,1994, Mattion et al., J.Virolog y 70:8124-8127, 1996, Furler et al., Gene T. Therapy 8:864-873, 2001, and Halpin et al., Pl ant Journal 4:453-459, 1999, de Felipe et al., Gene Therapy 6:198-208, 1999, de Felip e et al., Human Gene Therapy 11:1921-1931 , 2000, and Klump et al., Gene Therapy 8:811- 817, 2001).

[0159] Reporter sequence Any of the AAVs provided herein may optionally include a sequence that encodes a reporter protein (a "reporter sequence"). Non-limiting examples of reporter sequences include beta-lactamase, beta-galactosidase (LacZ), alkaline phosph atase, thymidine kinase, green fluorescent protein (GFP), red fluorescent protein, m Cherry fluorescent protein, yellow fluorescent protein, chloramphenicol acetyltrans ferase (CAT), and DNA sequences encoding luciferase. Further examples of reporter sequences are known in the art. When associated with regulatory elements that drive their expression, reporter sequences can provide signals detectable by conventional means including enzyme assays, X-ray assays, colorimetric assays, fluorescence assays, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immuno logical assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). In some embodiments, the reporter sequence is the LacZ gene and mammalian cells (e.g., (RIA), and immunohistochemistry) to provide a signal detectable by conventional means including enzyme assays, X-ray assays, colorimetric assays, fluorescence assays, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry). can provide a signal detectable by conventional means including enzyme assays, X-ray assays, colorimetric assays, fluorescence assays, or other spectroscopic assays, fluorescence-activated cell sorting (FACS) assays, immunological assays (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0160] In some embodiments, the reporter sequence is the LacZ gene and mammalian cells (e.g., For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein. For example, the presence of a vector carrying the LacZ gene in spiral ganglion hair cells is detected by an assay for beta-galactosidase activity. When the reporter is a fluorescent protein (e.g., green fluorescent protein) or luciferase, the presence of a vector carrying the fluorescent protein or luciferase in mammalian cells (e.g., spiral ganglion hair cells) can be measured by fluorescence techniques (e.g., fluorescence microscopy or FACS) or by light production in a luminometer (e.g., a spectrofluorometer or an IVIS imaging device). In some embodiments, the reporter sequence can be used to verify the tissue-specific targeting ability and tissue-specific promoter regulatory activity of any of the vectors described herein.

[0161] Adjacent region: Untranslated region (UTR) In some embodiments, any of the adeno-associated virus (AAV) vectors can include an untranslated region such as a 5' UTR or a 3' UTR. In some embodiments, any of the adeno-associated virus (AAV) vectors can include an untranslated region such as a 5' UTR or a 3' UTR.

[0162] The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor. The untranslated region (UTR) of a gene is copied but not translated. The 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon. The 3' UTR begins immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence for the regulatory role played by UTRs in terms of nucleic acid molecule stability and translation. The regulatory features of UTRs can be incorporated into any of the vectors, compositions, kits, or methods described herein to enhance the expression of an antibody (e.g., an antibody that specifically binds to VEGF), an antigen-binding antibody fragment (e.g., an antigen-binding fragment that specifically binds to VEGF), or a soluble VEGF receptor.

[0163] Natural 5’UTRs contain sequences involved in translation initiation. They possess characteristics similar to those of the Kozak sequence, which is generally known to be involved in the process by which ribosomes initiate translation of many genes. The Kozak sequence has the consensus sequence CCR(A / G)CCAUGG, where R is a purine (A or G) three bases upstream of the start codon (AUG), and another "G" follows the start codon. 5’UTRs are also known to form secondary structures involved in elongation factor binding.

[0164] In some embodiments, the 5’UTR is included in any of the vectors described herein. Non-limiting examples of 5’UTRs that can be used to enhance the expression of nucleic acid molecules such as mRNAs include those derived from the following genes: albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, and factor VII.

[0165] In some embodiments, the 5’UTR derived from mRNA replicated by cells in snails can be included in any of the vectors, compositions, kits, and methods described herein.

[0166] 3’UTRs are known to have stretches of adenosine and uridine (in RNA form) or thymidine (in DNA form) embedded in them. These AU-rich characteristics are found particularly in genes with high turnover rates. Based on their sequence features and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995, Chen et al.). ​​​​​​​​​​​​​​​ , Mol. Cell Biol. 15:2010 - 2018, 1995), Class I A RE contains several dispersed copies of the AUUUA motif in the U - rich region. For example , c - Myc and MyoD mRNAs contain Class I AREs. Class II A RE has two or more overlapping UUAUUUA(U / A)(U / A) nonamers. GM - C SF and TNF - alpha mRNAs are examples that contain Class II AREs. Class III AREs are less well - defined. These U - rich regions do not contain the AUUUA motif . Two well - studied examples of this class are c - Jun and myogenin m RNA.

[0167] While most proteins that bind to AREs are known to destabilize the messenger , members of the ELAV family, most notably HuR, have been demonstrated to increase mRNA stability. HuR binds to all three classes of AREs. By engineering HuR - specific binding sites into the 3’UTR of a nucleic acid molecule, HuR binding , and thus message stabilization in vivo, will be effected.

[0168] In some embodiments, the introduction, removal, or modification of 3’UTR AREs is used to regulate the stability of the mRNA encoding a protein of interest (e.g., any antibody described herein, any antigen - binding antibody fragment described herein, or any soluble VEGF receptor described herein) . In other embodiments, the ARE is removed or mutated to increase intracellular stability and thus the protein of interest. Translation and production of a protein (e.g., any antibody described herein, any antigen-binding antibody fragment, or any soluble VEGF receptor described herein) can be increased.

[0169] In other embodiments, non-ARE sequences can be incorporated into the 5' or 3' UTR. Some embodiments, a portion of an intron or intron sequence can be incorporated into the flanking region of a polynucleotide in any of the vectors, compositions, kits, and methods provided herein. Incorporation of the intron sequence can increase protein production as well as the mRNA level.

[0170] Fc mutations that decrease the half-life of an antibody, antigen-binding antibody fragment, or soluble VEGF receptor in a mammal Any of the antibodies, antigen-binding antibody fragments, or soluble VEGF receptors described herein either does not contain at least one of one or more amino acid substitutions in the Fc region, or alternatively can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions in the Fc region that decrease the half-life of the antibody, antigen-binding antibody fragment, or soluble VEGF receptor in a mammal as compared to the half-life of the same antibody, antigen-binding antibody fragment, or soluble VEGF receptor. Methods for determining the half-life of an antibody, antigen-binding antibody fragment, or soluble VEGF receptor in a mammal

[0171] are well known in the art. Non-limiting examples of point mutations in Fc mutations that can decrease the half-life of an ,It is described in MAbs 5(6):896-903, 2013.

[0172] Method Into the inner ear of a mammal, (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the signal peptides described herein) and an antibody light chain variable domain operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., scFv) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the soluble VEGF receptors described herein) in a therapeutically effective amount. Also provided herein is a method for increasing the level of an antibody or antigen-binding antibody fragment in the inner ear of a mammal that requires an increase in the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide (e.g., any of the exemplary antibody light chain variable domains described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., scFv) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the soluble VEGF receptors described herein) in a therapeutically effective amount. A method is also provided herein that includes introducing into the inner ear of a mammal a therapeutically effective amount of an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the soluble VEGF receptors described herein). In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide (e.g., any of the exemplary antibody light chain variable domains described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., scFv) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the soluble VEGF receptors described herein) in a therapeutically effective amount. Into the inner ear of a mammal, (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the signal peptides described herein) and an antibody light chain variable domain operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., scFv) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the soluble VEGF receptors described herein) in a therapeutically effective amount. In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide In the inner ear of a mammal that requires an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear, a method for increasing the level of the antibody or antigen-binding antibody fragment in the inner ear, the method comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide (e.g., any of the antibody heavy chain variable domains described herein) and an antibody light chain variable domain operably linked to a signal peptide A light chain variable domain (e.g., any of the antibody light chain variable domains described herein) A polypeptide comprising, or (b) an antigen-binding antibody fragment (e.g., any of the exemplary signal peptides described herein) operably linked to a signal peptide such as an scFv) (e.g., any of the exemplary antigen-binding antibody fragments described herein ), a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide is introduced, which introduction results in, for example, an increase in the level of an antibody or antigen-binding antibody fragment in the inner ear of a mammal compared to the level of the antibody or antigen-binding antibody fragment in the inner ear of the mammal before administration (e.g., an increase of 1% to 400% (or any of the partial ranges within this range described herein), or at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 6 0%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 1100%, at least 120 0%, at least 1300%, at least 1400%, at least 1500%, at least 1600%, at least 1700%, at least 1800%, at least 1900%, or at least 2000% increase), and methods are also provided herein. ​​​

[0173] A method for increasing the level of soluble VEGF receptor, comprising introducing into the inner ear of a mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein said introduction results in an increase in the level of soluble VEGF receptor in the inner ear of the mammal (e.g., an increase of 1% to 400% (or any of the sub-ranges within this range described herein), or at least 1%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 950%, at least 1000%, at least 1100%, at least 1200%, at least 1300%, at least 1400%, at least 1500%, at least 1600%, at least 1700%, at least 1800%, at least 1900%, or at least 2000% increase) compared to the level of soluble VEGF receptor in the inner ear of the mammal before administration. Methods are also provided herein.

[0174] ​​​​​​​​​​​​​​​​​ A method for treating inner ear disorders in mammals in need of treatment for inner ear disorders, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising (a) a polypeptide comprising an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. For example, any of the antibody heavy chain variable domains described herein And a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. For example, any of the signal peptides described herein And a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. Introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising (a) a polypeptide comprising an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. For example, any of the signal peptides described herein And a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. Or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. For example, any of the signal peptides described herein And a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. Introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising (a) a polypeptide comprising an antibody heavy chain variable domain (e.g., any of the antibody heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the exemplary antigen-binding antibody fragments described herein) linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGR receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), whereby treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. Thereby, treatment of the inner ear disorder in the mammal is effected. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. In some embodiments, treatment of the inner ear disorder results in a reduction (e.g., a 1% to 100% reduction, or any of the sub-ranges within this range described herein) in the severity, frequency, or number of symptoms of the inner ear disorder in the mammal after introduction as compared to before introduction. For example, a 1% to 100% reduction, or any of the sub-ranges within this range described herein In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. In some embodiments, treatment of any inner ear disorder results in an improvement in the hearing of the mammal (e.g., one or more measures of hearing) after introduction as compared to before introduction. Improvement of the measurement standard (for example, an improvement of 1% to 400%, or any of the sub-ranges described herein) is achieved. is brought about.

[0175] In some embodiments of any of these methods, an antibody or antigen-binding antibody fragment, or a soluble VEGF receptor specifically binds to a vascular endothelial growth factor (VEGF) (for example, one or more of VEG F-A, VEGF-B, VEGF-C, and VEGF-D, such as one or more of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D). In some embodiments of any of these methods, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding an antibody or antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. In some embodiments of any of these methods, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of these methods, the mammal is human. In some embodiments of any of these methods, the mammal (e.g., human) is identified as having an inner ear disorder. In some embodiments of any of these methods, the mammal (e.g., human) has previously been diagnosed as having an inner ear disorder. In some embodiments of any of these methods, the vector comprises a nucleic acid sequence encoding a polypeptide comprising an antibody heavy chain and an antibody light chain. In some embodiments of any of these methods, the vector comprises a nucleic acid sequence encoding an antigen-binding antibody fragment ​​​​​​​ comprises. In some embodiments of any of these methods, the vector encodes a nucleic acid sequence for a soluble VEGF receptor operably linked to a signal peptide.

[0176] A method of reducing VEGF activity (e.g., one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, such as human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D) in the inner ear of a mammal in need of reducing VEGF activity, the method comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising (a) a polypeptide comprising an anti-body heavy chain variable domain (e.g., any of the anti-body heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an anti-body light chain variable domain (e.g., any of the anti-body light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity one or more of) and reducing VEGF activity in the inner ear of a mammal in need thereof, the method comprising administering to the inner ear of the mammal (a) a polypeptide comprising an anti-body heavy chain variable domain (e.g., any of the anti-body heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an anti-body light chain variable domain (e.g., any of the anti-body light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity one or more) of human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF-D), the method comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising (a) a polypeptide comprising an anti-body heavy chain variable domain (e.g., any of the anti-body heavy chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), and a polypeptide comprising an anti-body light chain variable domain (e.g., any of the anti-body light chain variable domains described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity e.g., any of the signal peptides described herein) operably linked to an anti- body heavy chain variable domain (e.g., any of the anti-body heavy chain variable domains described herein ), and (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity e.g., any of the signal peptides described herein) operably linked to an anti-body light chain variable domain (e.g., any of the anti-body light chain variable domains described herein ), (b) a polypeptide comprising an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity e.g., any of the signal peptides described herein) operably linked to an antigen-binding antibody fragment (e.g., Fab or scFv) (e.g., any of the antigen-binding antibody fragments described herein) ), or (c) a nucleotide sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein), wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity e.g., any of the signal peptides described herein) operably linked to a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein ), and introducing a therapeutically effective amount of an AAV vector comprising the nucleotide sequence, wherein the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity and the polypeptide of (a) specifically binds to VEGF and reduces VEGF activity or (b) a polypeptide that specifically binds to VEGF and inhibits VEGF activity. or (c) a soluble VEGF receptor comprising an antigen-binding antibody fragment that reduces the affinity of the soluble VEGF receptor to one or more of the soluble VEGF receptors. Specifically binds to one or more VEGF proteins and reduces the activity of one or more VEGF proteins. The introduction of the VEGF reduces, for example, the VEGF activity in the mammal compared to that in the mammal before the introduction. VEGF activity (e.g., VEGF-A, VEGF-B, VEGF- C, and one or more of VEGF-D, e.g., one or more of human VEGF-A, human V Reduction (e.g., 1% to 20% activity of human EGF-B, human VEGF-C, and human VEGF-D) a 100% reduction, or any subrange of this range described herein) Also provided herein is a method for reducing VEGF activity in a mammal. Detection may be performed using any of the exemplary methods described herein.

[0177] Acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II (NF2) in the mammalian inner ear The present invention relates to a method for treating an inflammatory bowel disorder, comprising administering to the inner ear of a mammal (a) a signal peptide (e.g., operably linked to a signal peptide (any of the signal peptides described in the specification) A polypeptide comprising a domain (e.g., any of the antibody heavy chain variable domains described herein). and signal peptides (e.g., the signal peptides described herein). operably linked to an antibody light chain variable domain (e.g., an antibody light chain variable domain as described herein) (b) a polypeptide comprising a signal peptide ( For example, any of the signal peptides described herein. Antigen-binding antibody fragments (e.g., Fab or scFv) (e.g., the antigen-binding fragments described herein) or (c) a polypeptide comprising a signal peptide (e.g., operably linked to a signal peptide (e.g., any of the signal peptides described herein). A soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) The method includes introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding The polypeptide of (a) is a VEGF (e.g., VEGF-A, VEGF-B, VEGF FC, and one or more of VEGF-D, such as human VEGF-A, human VEGF VEGF-B, human VEGF-C, and human VEGF-D) The polypeptide of (b) encodes an antibody that reduces VEGF activity, or For example, one or more of VEGF-A, VEGF-B, VEGF-C, and VEGF-D; For example, human VEGF-A, human VEGF-B, human VEGF-C, and human VEGF- D) and reduce VEGF activity. or (c) a soluble VEGF receptor that is capable of binding to VEGF-A, VEGF-B, or VEGF-V. EGF-C, and one or more of VEGF-D (e.g., human VEGF-A, human VEGF-C, human VEGF-D, VEGF-B, human VEGF-C, and human VEGF-D) and, by this introduction, acoustic neuromas, vestibular schwannomas, or nerve fiber tumors in the inner ear of mammals. Also provided herein are methods that result in the treatment of NF2. As described, one or more of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II Successful treatment of the above may, for example, result in a decrease in the level of the inflammatory response in the mammal compared to before the introduction step, respectively. The number, severity, and / or frequency of one or more symptoms of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II (e.g., a reduction of 1% to 100%, or any of the sub-ranges within this range described herein) can be detected by observing it.

[0178] In some embodiments of any of these methods, the vector comprises a nucleic acid sequence encoding a polypeptide encoding an antibody heavy chain variable domain (e.g., any of the antibody heavy chains described herein) and an antibody light chain variable domain (e.g., any of the antibody light chain variable domains described herein). In some embodiments of any of these methods, the vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment (e.g., any of the antigen-binding antibody fragments described herein). In some embodiments of any of these methods, the vector comprises a nucleic acid sequence encoding a soluble VEGF receptor (e.g., any of the soluble VEGF receptors described herein) operably linked to a signal peptide (e.g., any of the signal peptides described herein). In some embodiments of any of these methods, the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding an antibody or an antigen-binding antibody fragment. In some embodiments, the AAV vector comprises a promoter, and the promoter is selected from the group consisting of an inducible promoter, a constitutive promoter, or a tissue-specific promoter. In some embodiments, the AAV vector further comprises a polyadenylation signal sequence. In some embodiments of any of the methods, the mammal is a human. These In some embodiments of any of the methods, the mammal (e.g., human) has an inner ear disorder. In some embodiments of any of these methods , the mammal (e.g., human) has been previously diagnosed with an inner ear disorder. In some embodiments of any of these methods, the mammal (e.g., human) has been identified or diagnosed as having drug-induced hearing loss. In some embodiments of any of these methods, the mammal (e.g., human) has been identified or diagnosed as having age-related

[0179] In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc region that contains one or more point mutations that

[0180] decrease the half-life of the antibody or antigen-binding antibody fragment in vivo. In some embodiments of any of these methods, two or more doses of any of the adeno-associated virus (AAV) vectors described herein are introduced or administered to the inner ear of the mammal or subject. Some embodiments of these methods include introducing or administering a first dose of an adeno-associated virus (AAV) vector to the inner ear of the mammal or subject, evaluating the auditory function of the mammal or subject after introduction or administration of the first dose, and

[0181] In some embodiments of any of the methods described herein, adeno-associated virus (AAV) vectors can be formulated for intracochlear administration. In some embodiments of any of the methods described herein herein, the adeno-associated virus (AAV) vectors described herein can be administered by intracochlear or topical administration. In some embodiments of any of the methods described herein herein, the adeno-associated virus (AAV) vectors can be administered using a medical device (e.g., any of the exemplary medical devices described herein).

[0182] In some embodiments, intracochlear administration can be performed using any of the methods described herein or known in the art. For example, adeno-associated virus (AAV) vectors can be administered or introduced into the cochlea using the following surgical procedures. First , the external auditory canal is cleaned using visualization with a 0-degree / 2.5-mm rigid endoscope, and a round blade is used to sharply outline the contour of the approximately 5-mm external auditory canal tympanic flap. Then, the external auditory canal tympanic flap is lifted and entered into the middle ear from the posterior. The chorda tympani nerve is identified and divided, and a curette is used to remove the scutal bone of the epitympanic wall and expose the round window membrane. To enhance the apical distribution of the administered or introduced adeno-associated virus (AAV) vectors, an otologic laser is used to create a small 2-mm fenestra in the oval window to allow for perilymph replacement during injection of the adeno-associated virus (AAV) vectors through the round window membrane. Then, a microinfusion device is prepared and brought to the surgical field. This device is maneuvered towards the round window and the tip is placed within the bony protrusion of the round window, and a micro-needle (s) can be used to allow for perilymph replacement during injection of the adeno-associated virus (AAV) vectors through the round window membrane. After that, a microinfusion device is prepared and brought to the surgical field. This device is maneuvered towards the round window and the tip is placed within the bony protrusion of the round window, and a micro-needle(s) is placed within the bony protrusion of the round window, and a micro-needle(s) Permits penetration of the membrane. Engage and interlock the foot pedal to enable a measured and constant injection of an adeno-associated virus (AAV) vector. Then, withdraw this device and seal the round window and the cribriform plate with a gel foam patch.

[0183] In some embodiments of any of the methods described herein, the subject or mammal is a rodent, non-human primate, or human. In some embodiments of any of the methods described herein, the subject or mammal is an adult, adolescent, juvenile, child, infant, newborn, or neonate. In some embodiments of any of the methods described herein, the subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-7 0, 70-80, 80-90, 90-100, 100-110, 10-30, 10-40 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10 -110, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 3 0-90, 30-100, 40-60, 40-70, 40-80, 40-90, 40-1 00, 50-70, 50-80, 50-90, 50-100, 60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110 or 90-110 years old. In some embodiments of any of the methods described herein, In some embodiments, the subject or mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months old.

[0184] In some embodiments of any of the methods described herein, the subject or mammal has, or is at risk of developing, hearing loss (e.g., drug-induced hearing loss).

[0185] In some embodiments of any of the methods described herein, the subject or mammal has previously been identified as having a mutation in the VEGF gene.

[0186] In some embodiments, successful treatment of hearing loss (e.g., drug-induced hearing loss) can be determined in a subject using any of the conventional auditory function tests known in the art. Non-limiting examples of auditory function tests are various types of audiometry assays (e.g., pure tone tests, speech tests, middle ear tests, auditory brainstem responses, and otoacoustic emissions).

[0187] Methods for introducing any of the adeno-associated virus (AAV) vectors described herein into mammalian cells are known in the art (e.g., by lipofection or by use of a viral vector, such as any of the viral vectors described herein).

[0188] Pharmaceutical Compositions and Kits In some embodiments, any of the compositions described herein may further comprise one or more agents that facilitate entry of a nucleic acid or any of the vectors described herein into mammalian cells (e.g., liposomes or cationic lipids).

[0189] In some embodiments, any of the vectors described herein may be formulated using natural and / or synthetic polymers. Non-limiting examples of polymers that may be included in any of the compositions described herein include DYNAMIC POLYCONJU GATE (registered trademark) (Arrowhead Research Corp., Pasa dena, Calif.), formulations manufactured by Mirus Bio (Madison, Wis.) and Ro che Madison (Madison, Wis.), PhaseRX polymer formulations (e.g., SMARTT POLYMER TECHNOLOGY (registered trademark) ( PhaseRX, Seattle, Wash.), but not limited thereto), DM RI / DOPE, poloxamers, VAXFECTIN (registered trademark) adjuvant manufactured by Vical (San Diego, Calif.), chitosan, cyclodextrin manufactured by Calando Pharm aceuticals (Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers, RONDEL( trademark) (RNAi / oligonucleotide nanoparticle delivery) polymers (Arrowhead Research Corporation, Pasadena, Calif.), and pH-responsive coblock polymers (e.g., those manufactured by PhaseRX (Seattle, Wash. ), but not limited thereto), but are not limited to these. Many of these polymers have demonstrated efficacy in the delivery of oligonucleotides to mammalian cells in vivo (e.g., deFougerolles , Human Gene Ther. 19:125-132, 2008, Rozema , et al., Nature 461:745-748, 2009, and Zimmermann , et al., Nature 461:754-758, 2009). et al., Proc. Natl. Acad. Sci. U.S.A. 104:1298 2 - 12887, 2007, Rozema et al., Proc. Natl. Aca d. Sci. U.S.A. 104:12982 - 12887, 2007, Hu - Lies kovan et al., Cancer Res. 65:8984 - 8982, 200 5, Heidel et al., Proc. Natl. Acad. Sci. U.S.A . 104:5715 - 5721, 2007 (see also).

[0190] Any of the compositions described herein can be, for example, a pharmaceutical composition. The pharmaceutical composition can include any of the compositions described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include one or more buffer solutions (e.g., neutral buffered saline, phosphate - buffered saline, etc.), one or more carbohydrates (e.g., glucose, mannose, sucrose, and dextran), mannitol, one or more proteins, polypeptides, or amino acids (e.g., glycine), one or more antioxidants, one or more chelating agents (e.g., EDTA or glutathione), and / or one or more preservatives.

[0191] In some embodiments, the composition includes a pharmaceutically acceptable carrier (e.g., phosphate - buffered saline, saline, or bacteriostatic water). When formulated, the solution is administered in a therapeutically effective amount in a manner compatible with the dosage formulation. The formulation can be readily administered in various dosage forms such as, for example, injectable solutions, injectable gels, drug - releasing capsules, etc.

[0192] As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into any of the compounds described herein.

[0193] In some embodiments, a single dose of any of the compositions described herein is, for example, in a buffered solution, at least 1 ng, at least 2 ng, at least 4 ng, about 6 n g, about 8 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40 ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, at least 90 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, at least 500 ng, at least 1 μg , at least 2 μg, at least 4 μg, at least 6 μg, at least 8 μg, at least 10 μg, at least 12 μg, at least 14 μg, at least 16 μg, at least 18 μg, at least 20 μg, at least 22 μg, at least 24 μg, at least 26 μg, at least 28 μg, at least 30 μg, at least 32 μg, at least 34 μg, at least 36 μg, at least 38 μg, at least 40 μg, at least 42 μg, at least 44 μg, at least 46 μg, at least 48 μg, at least 50 μg, at least 52 μg, at least 54 μg, at least 56 μg, at least 58 μg, at least 60 μg, at least 62 μg, at least 64 μg, at least 66 μg, at least 68 μg, at least 70 μg, at least 72 μg, at least at least 74 μg, at least 76 μg, at least 78 μg, at least 80 μg, at least 82 μg, at least 84 μg, at least 86 μg, at least 88 μg, at least 90 μg, at least 92 μg, at least 94 μg, at least 96 μg, at least 98 μg, at least 100 μg, at least 102 μg, at least 104 μg, at least 106 μg, at least 108 μg, at least 110 μg, at least 11 2 μg, at least 114 μg, at least 116 μg, at least 118 μg, at least 120 μg, at least 122 μg, at least 124 μg, at least 126 μg 、 at least 128 μg, at least 130 μg, at least 132 μg, at least 1 34 μg, at least 136 μg, at least 138 μg, at least 140 μg, at least 142 μg, at least 144 μg, at least 146 μg, at least 148 μ g, at least 150 μg, at least 152 μg, at least 154 μg, at least 156 μg, at least 158 μg, at least 160 μg, at least 162 μg, at least 164 μg, at least 166 μg, at least 168 μg, at least 170 μg, at least 172 μg, at least 174 μg, at least 176 μg, at least 178 μg, at least 180 μg, at least 182 μg, at least 184 μg, at least 186 μg, at least 188 μg, at least 190 μg, at least 19 2 μg, at least 194 μg, at least 196 μg, at least 198 μg, or contain at least two different vectors with a total amount of at least 200 μg.

[0194] The compositions provided herein can be formulated, for example, to be compatible with their intended route of administration. Non-limiting examples of the intended route of administration include topical administration (e.g., intracochlear administration).

[0195] In some embodiments, the therapeutic composition is formulated to include lipid nanoparticles. In some embodiments, the therapeutic composition is formulated to include polymer nanoparticles . In some embodiments, the therapeutic composition is formulated to include minicircle DNA . In some embodiments, the therapeutic composition is formulated to include CELiD DNA . In some embodiments, the therapeutic composition is formulated to include synthetic perilymph . Exemplary synthetic perilymph includes 20 - 200 mM NaCl, 1 - 5 mM KCl , 0.1 - 10 mM CaCl2, 1 - 10 mM glucose, 2 - 50 mM HEPES and has a pH of about 6 to about 9.

[0196] Kits are also provided that include any of the compositions described herein. In some embodiments , the kit can include a solid composition (e.g., a lyophilized composition including at least two different vectors described herein) and a liquid for solubilizing the lyophilized composition. In some embodiments, the kit can include a pre-filled syringe containing any of the compositions described herein. In some embodiments, the kit can include a vial containing any of the compositions described herein (e.g., an aqueous composition, e.g., formulated as an aqueous pharmaceutical composition).

[0197] In some embodiments, the kit can include a vial containing any of the compositions described herein (e.g., an aqueous composition, e.g., formulated as an aqueous pharmaceutical composition).

[0198] ​In some embodiments, the kit may include instructions for performing any of the methods described herein. It may include instructions for use.

[0199] Devices and Surgical Methods A therapeutic delivery system for treating hearing loss (e.g., acoustic neuroma / vestibular schwannoma and associated hearing loss) is provided herein. In one aspect, the therapeutic delivery system includes: i) a medical device capable of creating one or more incisions in the round window membrane of the inner ear of a human subject, which requires creating one or more incisions in the round window membrane of the inner ear, and ii) an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of microneedles. It includes an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of microneedles. It includes an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of microneedles. It includes an effective dose of a composition (e.g., any of the compositions described herein). In some embodiments, the medical device includes a plurality of microneedles. In some embodiments, the medical device includes a plurality of microneedles. In some embodiments, the medical device includes a plurality of microneedles.

[0200] Surgical methods for treating hearing loss (e.g., acoustic neuroma / vestibular schwannoma and associated hearing loss) are also provided herein. In some embodiments, these methods include introducing a first incision into the cochlea of a human subject at a first incision point and administering a therapeutically effective amount of any of the compositions provided herein into the cochlea. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision. In some embodiments, these methods include introducing a first incision into the cochlea of a human subject at a first incision point and administering a therapeutically effective amount of any of the compositions provided herein into the cochlea. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision. In some embodiments, these methods include introducing a first incision into the cochlea of a human subject at a first incision point and administering a therapeutically effective amount of any of the compositions provided herein into the cochlea. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision. In some embodiments, these methods include introducing a first incision into the cochlea of a human subject at a first incision point and administering a therapeutically effective amount of any of the compositions provided herein into the cochlea. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision. In some embodiments, the composition is administered to the subject at the first incision point. In some embodiments, the composition is administered to the subject within or through the first incision. In some embodiments, the composition is administered to the subject within or through the first incision.

[0201] In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the oval window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the round window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the oval window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the round window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the oval window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the round window membrane of the cochlea. In some embodiments of any of the methods described herein, any of the compositions described herein is administered to the subject within or through the round window membrane of the cochlea. In some embodiments of any of the methods described herein, the composition is administered intracellularly within the round window membrane. The administration is performed using a medical device capable of creating multiple incisions. In some embodiments, the medical device comprises a plurality of microneedles. The therapeutic device includes a plurality of microneedles including a generally circular first side, each microneedle having a first side. The needle has a diameter of at least about 10 microns. The device includes a base and / or reservoir capable of holding the composition. In embodiments, the medical device comprises a plurality of individual lumen through which the composition can be delivered. In some embodiments, the medical device comprises at least a hollow microneedle. A means for generating a partial vacuum is included.

[0202] The present invention will now be described in further detail with reference to the following experimental examples. These examples are Provided for illustrative purposes only and not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way. Rather, any and all of the techniques that become apparent as a result of the teachings provided herein. It should be construed to encompass variations.

[0203] Without further elaboration, those skilled in the art can, using the preceding description and the illustrative examples below, can be used to make and utilize the compositions of the invention and to practice the claimed methods. It is believed that the following examples are intended to specifically point out various aspects of the present invention and in no way constitute a limitation on the present disclosure. They should not be construed as limiting the remainder. EXAMPLES

[0204] Example 1. Construction of viral vectors Four different recombinant AAV vectors were generated and are shown in FIGS. 1A - D.

[0205] The vector of FIG. 1A is a 4474 bp exemplary AAV vector (SEQ ID NO: 35) containing the following sub - arrays proceeding in the 5' to 3' direction. CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTGCGGCCGCACGCGT (5' ITR, SEQ ID NO: 36), GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTG CGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAG CGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGC CCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGG GACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCT TAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGG CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC GCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGG GCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGC TGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGG GGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTA ACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGG CCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGG TGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCG TCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG GACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA CCATGTTCATGCCTTCTTCTTTTTCCTACAG (CBA sequence, sequence number 37), CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC ( spacer, sequence number 38), ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTC TGGCCCTGGTCACCAATTCT (IL-2 secretion signal sequence, sequence number 39 ), GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAAC CTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTA CACCTTCACCAACTACGGCATGAACTGGGTCCGACAGGCC CCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCT ACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTAC CTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGCGCCAAGTATCCCCACTACTACGGCAGCAGCCA CTGGTACTTTGACGTGTGGGGACAGGGCACACTGGTCACA GTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGC TCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTG ACCGTGTCCTGGAATTCTGGCGCTCTGACAAGCGGCGTGC ACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTC TCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCA ACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGA CAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTG CTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTA AGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTG CGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAG TTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCA AGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAG AGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTG AACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCC TGCCTGCTCCTATCGAGAAAACCATCAGCAAGGCCAAGGG CCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGC CGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCC TCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATG GGAGAGCAATGGCCAGCCAGAGAACAACTACAAGACAACC CCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACA GCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAA CGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCTCTGAGCCTGTCTCCTGGCAAG( The sequence encoding the heavy chain of bevacizumab, SEQ ID NO: 40), CGGAAGAGAAGA (linker sequence, SEQ ID NO: 41), GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCG ACGTGGAAGAGAACCCCGGACCT (T2A sequence, SEQ ID NO: 42), ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCC TGGCTCTCGTGACCAACAGC (IL-2 secretion signal sequence, SEQ ID NO: 43 )、 GACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCT CTGTGGGAGACAGAGTGACCATCACCTGTAGCGCCAGCCA GGACATCTCCAACTACCTGAACTGGTATCAGCAAAAGCCC GGCAAGGCCCCTAAGGTGCTGATCTACTTCACAAGCAGCC TGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCT GAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCG TGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAA GCGGACTGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCT AGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCA GTGGAAAGTGGACAATGCCCTGCAGAGCGGCAACAGCCAA GAGAGCGTGACAGAGCAGGACTCCAAGGATAGCACCTATA GCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGA GAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAAT GTTAA (sequence encoding the light chain of bevacizumab, SEQ ID NO: 44), GAGCTCGCTGATCAGCCTCGA (linker sequence, SEQ ID NO: 45), CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA GGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG (Bovine growth hormone polyA tail sequence, SEQ ID NO: 46), sequence, SEQ ID NO: 46), AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT (Linker sequence, SEQ ID NO: 47), and sequence, SEQ ID NO: 47), and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGCTGCCTGCAGG (3’ ITR, SEQ ID NO: 48).

[0206] The IL-2 signal sequences encoded by SEQ ID NOs: 39 and 43 are MYRM QLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50) is. SEQ ID NO: 40 encodes the heavy chain of bevacizumab (SEQ ID NO: 6). SEQ ID NO 4 encodes the light chain of bevacizumab (SEQ ID NO: 5). The last three nucleotides of SEQ ID NO: 44 are a stop codon. are a stop codon.

[0207] The vector of Figure 1B contains a 3814bp sub-sequence that proceeds in the 5' to 3' direction It is an exemplary AAV vector (SEQ ID NO: 51). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTGCGGCCGCACGCGT (3’ ITR, SEQ ID NO: 36), GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTG CGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAG CGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGC CCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGG GACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCT TAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGG CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC GCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGG GCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGC TGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGG GGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTA ACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGG CCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGG TGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCG TCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG GACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA CCATGTTCATGCCTTCTTCTTTTTCCTACAG (CBA sequence, sequence number 37), CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC ( linker sequence, sequence number 38), ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTC TGGCCCTGGTCACCAATTCT (IL-2 secretion signal sequence, sequence number 39 ), GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAAC CTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTA CGACTTCACCCACTACGGCATGAACTGGGTCCGACAGGCC CCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCT ACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTAC CTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCA CTGGTACTTTGACGTGTGGGACAGGGCACACTGGTCACA GTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGC TCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTG ACCGTGTCCTGGAATTCTGGCGCTCTGACAAGCGGCGTGC ACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTC TCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCA ACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGA CAAGACCCACACCGGCAAG (the sequence encoding the heavy chain of ranibizumab, sequence Number 52), CGGAAGAGAAGA (linker sequence, SEQ ID NO: 41), GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCG ACGTGGAAGAGAACCCCGGACCT (T2A sequence, SEQ ID NO: 42), ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCC TGGCTCTCGTGACCAACAGC (IL-2 signal secretion sequence, SEQ ID NO: 43 ), GACATCCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCT CTGTGGGAGACAGAGTGACCATCACCTGTAGCGCCAGCCA GGACATCTCCAACTACCTGAACTGGTATCAGCAAAAGCCC GGCAAGGCCCCTAAGGTGCTGATCTACTTCACAAGCAGCC TGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCT GAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCG TGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAA GCGGACTGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCT AGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCA GTGGAAAGTGGACAATGCCCTGCAGAGCGGCAACAGCCAA GAGAGCGTGACAGAGCAGGACTCCAAGGATAGCACCTATA GCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGA GAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAAT GTTAA (sequence encoding the light chain of ranibizumab, SEQ ID NO: 53). GAGCTCGCTGATCAGCCTCGA (linker sequence, SEQ ID NO: 45), CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA GGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG (Bovine growth hormone polyA tail sequence, SEQ ID NO: 46), and and AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT (Linker, SEQ ID NO: 47), AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 48).

[0208] The IL-2 signal sequences encoded by SEQ ID NOs: 39 and 43 are MYRM QLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50) is. SEQ ID NO: 52 is the heavy chain of ranibizumab (EVQLVESGGGLVQPGGSL RLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEP TYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAK YPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSS YPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTGK, SEQ ID NO: 54) encodes. SEQ ID NO: 53 is bev The light chain of trastuzumab (SEQ ID NO: 7) is encoded. The last three nucleotides of SEQ ID NO: 53 are a stop codon.

[0209] Figure 1C is an exemplary 4573 bp A containing the following subarray proceeding in the 5' to 3' direction AV vector (SEQ ID NO: 55). CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTGCGGCCGCACGCGT (5' ITR, SEQ ID NO: 36), GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTG CGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAG CGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGC CCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGG GACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCT TAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGG CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC GCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGG GCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGC TGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGG GGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTA ACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGG CCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGG TGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCG TCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG GACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA CCATGTTCATGCCTTCTTCTTTTTCCTACAG (CBA sequence, sequence number 37), CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC ( linker sequence, sequence number 38), ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTC TGGCCCTGGTCACCAATTCT (IL-2 secretion signal sequence, sequence number 39 ) GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAAC CTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTA CGACTTCACCCACTACGGCATGAACTGGGTCCGACAGGCC CCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCT ACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAG ATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTAC CTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGT ACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCA CTGGTACTTTGACGTGTGGGGACAGGGCACACTGGTCACA GTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCAC TGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGC TCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTG ACCGTGTCCTGGAATTCTGGCGCTCTGACAAGCGGCGTGC ACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTC TCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGC ACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCA ACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGA CAAGACCCACACCGGCAAG (the sequence encoding the heavy chain of ranibizumab, sequence number 52), CGGAAGAGAAGA (linker sequence, SEQ ID NO: 41), GGCTCTGGCGAAGGCAGAGGCAGCCTGCTTACATGTGGCG ACGTGGAAGAGAACCCCGGACCT (T2A sequence, SEQ ID NO: 42), ATGTATAGAATGCAGCTCCTGTCCTGCATTGCCCTGAGCC TGGCTCTCGTGACCAACAGC (IL-2 signal secretion sequence, SEQ ID NO: 43 ), GACATCCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCT CTGTGGGAGACAGAGTGACCATCACCTGTAGCGCCAGCCA GGACATCTCCAACTACCTGAACTGGTATCAGCAAAAGCCC GGCAAGGCCCCTAAGGTGCTGATCTACTTCACAAGCAGCC TGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAG CGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCT GAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCG TGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAA GCGGACTGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCT AGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGT GCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCA GTGGAAAGTGGACAATGCCCTGCAGAGCGGCAACAGCCAA GAGAGCGTGACAGAGCAGGACTCCAAGGATAGCACCTATA GCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGA GAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGC CTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAAT GT (sequence encoding the light chain of ranibizumab, SEQ ID NO: 56), GGCTCCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTG ACGTCGAGGAGAATCCTGGCCCA (linker sequence, SEQ ID NO: 57), ATGGAGAGCGACGAGAGCGGCCTGCCCGCCATGGAGATCG AGTGCCGCATCACCGGCACCCTGAACGGCGTGGAGTTCGA GCTGGTGGGCGGCGGAGAGGGCACCCCCGAGCAGGGCCGC ATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCT TCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTT CTACCACTTCGGCACCTACCCCAGCGGCTACGAGAACCCC TTCCTGCACGCCATCAACAACGGCGGCTACACCAACACCC GCATCGAGAAGTACGAGGACGGCGGCGTGCTGCACGTGAG CTTCAGCTACCGCTACGAGGCCGGCCGCGTGATCGGCGAC TTCAAGGTGATGGGCACCGGCTTCCCCGAGGACAGCGTGA TCTTCACCGACAAGATCATCCGCAGCAACGCCACCGTGGA GCACCTGCACCCCATGGGCGATAACGATCTGGATGGCAGC TTCACCCGCACCTTCAGCCTGCGCGACGGCGGCTACTACA GCTCCGTGGTGGACAGCCACATGCACTTCAAGAGCGCCAT CCACCCCAGCATCCTGCAGAACGGGGGCCCCATGTTCGCC TTCCGCCGCGTGGAGGAGGATCACAGCAACACCGAGCTGG GCATCGTGGAGTACCAGCACGCCTTCAAGACCCCGGATGC AGATGCCGGTGAAGAATAA (sequence encoding TurboGFP, SEQ ID NO: 58), GAGCTCGCTGATCAGCCTCGA (linker sequence, SEQ ID NO: 45), CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA GGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG (bovine growth hormone polyA tail sequence, SEQ ID NO: 46), AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT (linker sequence, SEQ ID NO: 47), and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGCTGCCTGCAGG (3’ ITR, SEQ ID NO: 48).

[0210] The IL-2 signal sequences encoded by each of SEQ ID NOs: 39 and 43 are MYRM It is QLLSCIALSLALVTNS (SEQ ID NO: 49). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50). The T2A sequence encoded by SEQ ID NO: 42 is GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 50). SEQ ID NO: 52 encodes the heavy chain of ranibizumab (SEQ ID NO: 54). SEQ ID NO: 56 encodes the light chain of bevacizumab (SEQ ID NO: 7). SEQ ID NO: 58 encodes Turbo GFP (MESDESGLPAMEIECRITGTLNGVEFELVGGGEGTP EQGRMTNKMKSTKGALTFSPYLLSHVMGYGFYHFGTYPSG YENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRYEAGR VIGDFKVMGTGFPEDSVIFTDKIIRSNATVEHLHPMGDND LDGSFTRTFSLRDGGYYSSVVDSHMHFKSAIHPSILQNGG PMFAFRRVEEDHSNTELGIVEYQHAFKTPDADAGEE, SEQ ID NO : 59). The last three nucleotides of SEQ ID NO: 58 are a stop codon .

[0211] Figure 1D is an exemplary AAV vector (SEQ ID NO: 60) of 3631 bp containing the following sub-sequence proceeding in the 5' to 3' direction . CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGC CCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAG CGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCTGCGGCCGCACGCGT (5' ITR, SEQ ID NO: 36), GACATTGATTATTGACTAGTTATTAATAGTAATCAATTAC GGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAA TGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCC AATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCG GGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTG CGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCC TTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAG CGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGC CCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGG GACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT AATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCT TAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGG CTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCC GCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGG GCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGG GGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGC TGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGG GGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTA ACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGG CCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCG GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGG TGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTC GGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGT CGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG TGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCT AGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGG AAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCG TCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGG GACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA CCATGTTCATGCCTTCTTCTTTTTCCTACAG (CBA sequence, sequence number 37), CTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACC ( spacer, sequence number 38), ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTC TGGCCCTGGTCACCAATTCT (IL-2 secretion signal sequence, sequence number 39 ), AGCGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGA TCCCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGT CATCCCCTGCAGAGTGACAAGCCCCAACATCACCGTGACT CTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCA AGAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAG CAACGCCACCTACAAAGAGATCGGCCTGCTGACCTGTGAA GCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGA CACACAGACAGACCAACACCATCATCGACGTGGTGCTGAG CCCTAGCCACGGCATTGAACTGTCTGTGGGCGAGAAGCTG GTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGGCA TCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCA CAAGAAACTGGTCAACCGGGACCTGAAAACCCAGAGCGGC AGCGAGATGAAGAAATTCCTGAGCACCCTGACCATCGACG GCGTGACCAGATCTGACCAGGGCCTGTACACATGTGCCGC CAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGTG CGGGTGCACGAGAAGGACAAGACCCACACCTGTCCTCCAT GTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCT GTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGA ACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACG AGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGT GGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAG TACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGC TGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAA GGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACC ATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTT ACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCA GGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCC GATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGA ACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGG CTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGC AGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGC ACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAG CCTGTCTCCTGGATAA (the sequence encoding aflibercept, SEQ ID NO: 61 )、 GAGCTCGCTGATCAGCCTCGA (linker sequence, SEQ ID NO: 45), CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACT GTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCA GGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT GCTGGGGATGCGGTGGGCTCTATGG (bovine growth hormone polyA tail sequence SEQ ID NO: 46), AAGCTTGAATTCAGCTGACGTGCCTCGGACCGCT (linker sequence SEQ ID NO: 47), and AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCG CGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCC CGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAGCTGCCTGCAGG (3’ ITR, SEQ ID NO: 48).

[0212] The IL-2 signal sequence encoded by SEQ ID NO: 39 is MYRMQLLSCIA It is LSLALVTNS (Accession No. 49). Accession No. 61 encodes aflibercept (Accession No. 12). The last three nucleotides of Accession No. 61 are a stop codon .

[0213] To determine protein expression driven by the AAV vectors shown in FIGS. 1A-1C, HEK293FT cells were seeded overnight at 7×10 cells / 4 well (400 μL / well) in the wells of a 24-well plate. The HEK293FT cells were transfected at approximately 800 ng with the AAV vectors shown in FIGS. 1A-1D using Jetprime Polypus reagent (used to generate the data in lanes 2-5 and 10-13 of FIG. 2). The HEK293FT cells were seeded at 4×10 cells / well (50 μL / well) in the wells of a 96-well plate in the presence of 2 μM etoposide (used to generate the data in lanes 6-8 and 14-16 of FIG. 2) for 6 hours. The AAV vector shown in FIG. 1A was added to the medium at a multiplicity of infection (MOI) of 7.5×10 4

[0214] 4 5 5 . The supernatant was collected from the wells at 72 hours after treatment and loaded onto a 4-12% Bis-Tris protein gel under reducing conditions (lanes 2-8 of FIG. 2) and non-reducing conditions (lanes 10-16 of FIG. 2). An anti-ranibizumab antibody that detects the Fab region was used as the primary antibody, and anti-human IgG was used as the secondary antibody. As shown in FIG. 2, the heavy and light chain ranibizumab were detected in lanes 3 and 6-8, and intact ranibizumab (heterodimer) was detected in lanes 11 and 14-16.

[0214]

[0214]

[0215] Example 2. Binding Activity of Anti-Human VEGF Monoclonal Antibody HEK293FT cells after transfection with the AAV vector shown in Figure 1A A set of experiments was conducted to determine the binding activity of bevacizumab produced in Recombinant human VEGF was used as a binder in buffer or conditioned medium (Figure 3A and Figure 3B, respectively) in a surface plasmon resonance instrument (mouse anti-human VEGF monoclonal antibody (anti-hVEGF MmAb, R&D, MAB293-100) for the first set of control experiments to calibrate the instrument. A second set of control experiments was conducted to determine the human VEGF binding activity of the control conditioned medium and conditioned medium (Figure 4A and Figure 4B, respectively) of HEK29 3TF cells after transfection with the AAV vector shown in Figure 1A.

[0216] The sample (bevacizumab in the medium or conditioned medium derived from HEK293T F cells transfected with the AAV vector shown in Figure 1A) was prepared by diluting it 1:10 in 1× kinetic buffer (Fortebio, 18-1105) in a 384-well sample plate The anti-hVEGF MmAb (R&D, MAB293-100) was diluted as a positive control at a concentration of 10 μg / mL. The capture agent, recombinant human VEGF (R&D, 293 -VE-010), was serially diluted from 200 nM to 3.125 nM at a dilution ratio of 1:2.

[0217] The binding affinity of the conditioned medium sample and the mouse anti-human VEGF antibody (R&D) sample was measured in 1× kinetic buffer in an Octet (registered trademark) HTX biosensor instrument. The binding characteristics and K DValues were generated by Data Analysis, an Octet® analysis software and HT10.0. As shown in FIGS. 3A - B, the K of the anti - hVEGF D MmAb in buffer was less than 1.0×10 -12 M, and the anti - hVEGF M mAb in conditioned medium was less than 1.0×10 -12 M. The conditioned medium itself had no binding affinity and the intensity was very low (only background signal) (FIG. 4A). In contrast, the conditioned medium containing bevacizumab produced by HEK293TF cells transfected with the AAV vector shown in FIG. 1A had high binding affinity but low intensity (FIG. 4B, less than 1.0×10 M K ). FIG. 4C shows a table of the loaded samples, as well as their respective K -12 , K D error, equilibrium association constant (k D ), and dissociation constant (k D ), and k a error. dis dis FIG. 36 shows .

[0218] In summary, the anti - hVEGF mouse antibody (R & D) showed high binding affinity (K D was lower than the measurable range of 1 .0×10 -12 M). The bevacizumab - conditioned medium sample showed high binding affinity (K D was lower than the measurable range). K values could not be estimated from the binding data of the control - conditioned medium sample. D

[0219] In summary, these data demonstrate that the AAV vectors provided herein can result in the expression and secretion of anti - VEGF antibodies, and using these, anti - VEGF antibodies can be delivered to the inner ear of mammals ​​​It shows that the body can be expressed.

[0220] Other embodiments The present invention has been described in conjunction with its detailed description, but the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. It should be understood that it is intended. Other aspects, advantages, and modifications are within the scope of the following claims. within.

[0221] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control, as well as the headings of the sections, and any description of materials, methods, and examples is illustrative only and not intended to be limiting. In certain embodiments, for example, the following items are provided. (Item 1) into the inner ear of a mammal (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 introducing a therapeutically effective amount of an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding the same. (Item 2) A method for increasing the level of an antibody or antigen-binding antibody fragment in the inner ear of a mammal in need thereof, comprising introducing into the inner ear of the mammal (a) a polypeptide comprising an antibody heavy chain variable domain operably linked to a signal peptide ​​​​​​​​A polypeptide comprising an antibody light chain variable domain operably linked to a chid and a signal peptide chid, or (b) introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide, wherein said introduction results in an increase in the level of said antibody or said antigen-binding antibody fragment in said inner ear of said mammal, said method. The method according to item 1 or 2, wherein said antibody or said antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). The method according to item 3, wherein said antibody or said antigen-binding antibody fragment reduces VEGF activity. (Item 3) The method according to item 1 or 2, wherein said antibody or said antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). The method according to item 1 or 2, wherein said antibody or said antigen-binding antibody fragment specifically binds to vascular endothelial growth factor (VEGF). (Item 4) The method according to item 3, wherein said antibody or said antigen-binding antibody fragment reduces VEGF activity. . (Item 5) The method according to any one of items 1 to 4, wherein said AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding said antibody or said antigen-binding antibody fragment. The method according to any one of items 1 to 4, wherein said AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding said antibody or said antigen-binding antibody fragment. The method according to any one of items 1 to 4, wherein said AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to a sequence encoding said antibody or said antigen-binding antibody fragment. (Item 6) The method according to item 5, wherein said AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. The method according to item 5, wherein said AAV vector comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. (Item 7) The method according to any one of items 1 to 6, wherein said AAV vector further comprises a polyadenylation signal sequence. The method according to any one of items 1 to 6, wherein said AAV vector further comprises a polyadenylation signal sequence. (Item 8) The method according to any one of items 1 to 7, wherein said mammal is a human. (Item 9) The method according to any one of items 1 to 8, wherein said mammal has been identified as having an inner ear disorder. The method according to any one of items 1 to 8, wherein said mammal has been identified as having an inner ear disorder. (Item 10) The method according to any one of items 1 to 8, wherein the mammal is diagnosed as having an inner ear disorder. The method. (Item 11) The AAV vector further 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, according to any one of items 1 to 10. The method according to any one of items 1 to 10, wherein the AAV vector further comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal. The method. (Item 12) The AAV vector further comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal, according to any one of items 1 to 10. The method according to any one of items 1 to 10, wherein the AAV vector further comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal. (Item 13) A method for treating inner ear disorder in a mammal in need of treatment for inner ear disorder, comprising , introducing into the inner ear of the mammal (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 therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide, wherein the introduction results in treatment of the inner ear disorder in the mammal. (Item 14) The method according to item 13, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. (Item 15) The method according to item 13, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. (Item 15) The method according to item 13, wherein the AAV vector further comprises one or both of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. The method according to item 13, wherein the AAV vector further comprises one or both of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. The method according to item 13, wherein the AAV vector further comprises one or both of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. (Item 15) The method according to item 13, wherein the AAV vector further comprises one or both of an inducible promoter, a constitutive promoter, and a tissue-specific promoter. The method according to item 14, comprising a promoter selected from the group consisting of a promoter. (Item 16) The method according to any one of items 13 to 15, wherein the AAV vector further comprises a polyadenylation signal sequence. (Item 17) The method according to any one of items 13 to 16, wherein the mammal is a human. (Item 18) The method according to any one of items 13 to 17, wherein the mammal is identified as having an inner ear disorder. (Item 19) The method according to any one of items 13 to 17, wherein the mammal is diagnosed as having an inner ear disorder. (Item 20) The method according to any one of items 13 to 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. (Item 21) The method according to any one of items 13 to 19, wherein the AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal. (Item 22) A method for reducing VEGF activity in the inner ear of a mammal in need of reducing VEGF activity in the inner ear, comprising: administering to the inner ear of the mammal: either (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 encoding a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide.​​​​​​​​​ introducing a therapeutically effective amount of an AAV vector comprising the nucleotide sequence, wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity, and the introduction results in a reduction of VEGF activity in the inner ear of the mammal, said method. (Item 23) (Item 23) The method according to item 22, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. The method according to item 22, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. The method according to item 22, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the antibody or the antigen-binding antibody fragment. (Item 24) The method according to item 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. The method according to item 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. (Item 25) The method according to any one of items 22 to 24, wherein the AAV vector further comprises a polyadenylation signal sequence. The method according to any one of items 22 to 24, wherein the AAV vector further comprises a polyadenylation signal sequence. (Item 26) The method according to any one of items 22 to 25, wherein the mammal is a human. (Item 27) The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. (Item 28) The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. (Item 29) The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having neurofibromatosis type 2. The method according to any one of items 22 to 26, wherein the mammal is identified or diagnosed as having neurofibromatosis type 2. (Item 30) The AAV vector comprises 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 and a nucleic acid sequence encoding the polypeptides, according to any one of items 22 to 29 . (Item 31) (Item 31) The AAV vector comprises a nucleic acid sequence encoding a polypeptide comprising an antigen-binding antibody fragment operably linked to a signal peptide and is according to any one of items 22 to 29 . (Item 32) A method for treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in the inner ear of a mammal , comprising introducing into the inner ear of the mammal (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 therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a polypeptide comprising an antigen-binding antibody fragment linked to a signal peptide , wherein the polypeptide of (a) encodes an antibody that specifically binds to VEGF and reduces VEGF activity, and the polypeptide of (b) encodes an antigen-binding antibody fragment that specifically binds to VEGF and reduces VEGF activity , and the introduction results in treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively . (Item 33) (Item 33) The AAV vector is operable with the sequence encoding the antibody or the antigen-binding antibody fragment . (Item 33) The AAV vector is operable with the sequence encoding the antibody or the antigen-binding antibody fragment Item further comprising one or both of a promoter and a Kozak sequence that may be linked The method according to item 32 (Item 34) The method according to item 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 (Item 35) The method according to any one of items 32 to 34, wherein the AAV vector further comprises a polyadenylation signal sequence (Item 36) The method according to any one of items 32 to 35, wherein the mammal is a human (Item 37) The method according to any one of items 32 to 36, wherein the mammal is identified or diagnosed as having a vestibular schwannoma (Item 38) The method according to any one of items 32 to 36, wherein the mammal is identified or diagnosed as having a vestibular schwannoma (Item 39) The method according to any one of items 32 to 36, wherein the mammal is identified or diagnosed as having neurofibromatosis type 2 (Item 40) The method according to any one of items 32 to 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 (Item 41) The method according to any one of items 32 to 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 (Item 42)​​​​​​​​​​ wherein the antibody comprises an Fc region comprising one or more amino acid substitutions that decrease the half-life of the antibody in a mammal compared to a control antibody, or wherein the antigen-binding antibody fragment has a decreased in vivo half-life compared to a control antigen-binding antibody fragment, The method according to any one of items 1 to 41. (Item 43) Introducing a therapeutically effective amount of an adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide into the inner ear of a mammal. (Item 44) A method comprising introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide into the inner ear of a mammal in need of an increase in the level of soluble vascular endothelial growth factor (VEGF) receptor in the inner ear. (Item 45) wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-1 (VEGFR-1). A method for increasing the level of soluble VEGF receptor in the inner ear of a mammal, comprising introducing, into the inner ear of the mammal, a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide, wherein the introduction results in an increase in the level of the soluble VEGF receptor in the inner ear of the mammal. (Item 46) wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1. The method according to item 43 or 44, wherein the portion of the extracellular region of VEGFR-1 comprises a flanking sequence derived from wild-type human VEGFR-1. (Item 47) The method according to item 45, wherein the portion of the extracellular region of VEGFR-1 comprises a flanking sequence derived from wild-type human VEGFR-1. (Item 48) wherein the portion of the extracellular region of VEGFR-1 comprises a flanking sequence derived from wild-type human VEGFR-1. The method according to item 45, (Item 49) wherein the portion of the extracellular region of VEGFR-1 comprises a flanking sequence derived from wild-type human VEGFR-1. The method according to item 46, comprising one or more immunoglobulin-like domains in the extracellular region. Method. (Item 48) The method according to item 45, wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to the flanking sequence derived from wild-type human VEGFR-1. (Item 49) The method according to item 43 or 44, wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-2 (VEGFR-2). (Item 50) The method according to item 49, wherein the portion of the extracellular region of VEGFR-2 comprises the flanking sequence derived from wild-type human VEGFR-2. (Item 51) The method according to item 50, wherein the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-2. (Item 52) The method according to item 49, wherein the portion of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to the flanking sequence derived from wild-type human VEGFR-2. (Item 53) The method according to item 43 or 44, wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1 and a portion of the extracellular region of VEGFR-2. (Item 54) The portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-1, and the portion of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region derived from wild-type human VEGFR-2. The method according to item 53. Method. (Item 55) The method according to item 54, wherein the soluble VEGF receptor is aflibercept. (Item 56) The method according to item 43 or 44, wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-3 (VEGFR-3). (Item 57) (Item 57) The method according to item 56, wherein the portion of the extracellular region of VEGFR-3 comprises an adjacent sequence derived from wild-type human VEGFR-3. (Item 58) (Item 58) The method according to item 57, wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the aforementioned extracellular region derived from wild-type human VEGFR-3. (Item 59) (Item 59) (Item 59) The method according to item 56, wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to the adjacent sequence derived from wild-type human VEGFR-3. (Item 60) (Item 60) The method according to any one of items 43 to 59, wherein the soluble VEGF receptor comprises an Fc domain. (Item 61) (Item 61) The method according to item 60, wherein the Fc domain is an IgG1 Fc domain. (Item 62) The method according to item 61, wherein the IgG1 Fc domain is a human wild-type IgG1 Fc domain. (Item 63) (Item 63) The method according to any one of items 43 to 62, wherein the soluble VEGF receptor reduces the ability of VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3. (Item 64) (Item 64) (Item 64) The AAV vector is operably linked to a sequence encoding the soluble VEGF receptor. The method according to any one of items 43 to 63, further comprising one or both of the promoter and the Kozak sequence to be used. The method according to any one of the preceding items. (Item 65) The method according to item 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. (Item 66) The method according to any one of items 43 to 65, wherein the AAV vector further comprises a polyadenylation signal sequence. (Item 67) The method according to any one of items 43 to 66, wherein the mammal is a human. (Item 68) The method according to any one of items 43 to 67, wherein the mammal is identified as having an inner ear disorder. (Item 69) The method according to any one of items 43 to 67, wherein the mammal is diagnosed as having an inner ear disorder. (Item 70) A method for treating an inner ear disorder in a mammal in need of treatment for the inner ear disorder, comprising introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide, wherein the introduction results in treatment of the inner ear disorder in the mammal. The method according to item 70, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. (Item 72) The method according to item 70, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. (Item 72) The method according to item 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. (Item 73) The method according to any one of items 70 to 72, wherein the AAV vector further comprises a polyadenylation signal sequence. (Item 74) The method according to any one of items 70 to 73, wherein the mammal is a human. (Item 75) The method according to any one of items 70 to 74, wherein the mammal is identified as having an inner ear disorder. (Item 76) The method according to any one of items 70 to 74, wherein the mammal is diagnosed as having an inner ear disorder. (Item 77) A method for reducing VEGF activity in the inner ear of a mammal in need of reducing VEGF activity in the inner ear, comprising: introducing into the inner ear of the mammal a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide; wherein the introduction results in a reduction of the VEGF activity in the inner ear of the mammal. (Item 78) The method according to item 77, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. (Item 79) The method according to item 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. ​​​​​​​​​​​ (Item 80) The method according to any one of Items 77 to 79, wherein the AAV vector further comprises a polyadenylation signal sequence. (Item 81) The method according to any one of Items 77 to 80, wherein the mammal is a human. (Item 82) The method according to any one of Items 77 to 81, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. (Item 83) The method according to any one of Items 77 to 81, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. (Item 84) The method according to any one of Items 77 to 81, wherein the mammal is identified or diagnosed as having neurofibromatosis type 2. (Item 85) A method for treating acoustic neuroma, vestibular schwannoma, or neurofibromatosis type 2 in the inner ear of a mammal, comprising: introducing a therapeutically effective amount of an AAV vector comprising a nucleotide sequence encoding a nucleotide sequence encoding a soluble vascular endothelial growth factor (VEGF) receptor operably linked to a signal peptide into the inner ear of the mammal, wherein the introduction results in the treatment of acoustic neuroma, vestibular schwannoma, or neurofibromatosis type II in the inner ear of the mammal, respectively. The method according to Item 85, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. (Item 86) The method according to Item 85, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. The method according to Item 85, wherein the AAV vector further comprises one or both of a promoter and a Kozak sequence operably linked to the sequence encoding the soluble VEGF receptor. (Item 87) The AAV vector is an inducible promoter, a constitutive promoter, and a tissue-specific promoter​​​​​​ The method according to item 86, comprising a promoter selected from the group consisting of a Romoter. (Item 88) The method according to any one of items 85 to 87, wherein the AAV vector further comprises a polyadenylation signal sequence. The method according to any one of items 85 to 87. (Item 89) The method according to any one of items 85 to 88, wherein the mammal is a human. (Item 90) The method according to any one of items 85 to 89, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. The method according to any one of items 85 to 89. (Item 91) The method according to any one of items 85 to 89, wherein the mammal is identified or diagnosed as having a vestibular schwannoma. The method according to any one of items 85 to 89. (Item 92) The method according to any one of items 85 to 89, wherein the mammal is identified or diagnosed as having neurofibromatosis type 2. The method according to any one of items 85 to 89. (Item 93) The method according to any one of items 70 to 92, wherein the soluble VEGF receptor comprises a portion of the extracellular region of VEGF receptor-1 (VEGFR-1). The method according to any one of items 70 to 92. (Item 94) The method according to item 93, wherein the portion of the extracellular region of VEGFR-1 comprises a contiguous sequence derived from wild-type human VEGFR-1. The method according to item 93. (Item 95) The method according to item 94, wherein the portion of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-1. The method according to item 94. The method according to item 94. (Item 96) The method according to item 93, wherein the portion of the extracellular region of VEGFR-1 comprises a sequence that is at least 90% identical to the contiguous sequence derived from wild-type human VEGFR-1. The method according to item 93. (Item 97) The soluble VEGF receptor comprises an extracellular region of VEGF receptor-2 (VEGFR-2) The method according to any one of items 70 to 92, which comprises a part of the extracellular region of VEGFR-2. (Item 98) The method according to item 97, wherein the part of the extracellular region of VEGFR-2 comprises a contiguous sequence derived from wild-type human VEGFR-2. (Item 99) (Item 99) The method according to item 98, wherein the part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. The method according to item 98, wherein the part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. The method according to item 98, wherein the part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. (Item 100) The method according to item 97, wherein the part of the extracellular region of VEGFR-2 comprises a sequence that is at least 90% identical to the contiguous sequence derived from wild-type human VEGFR-2. (Item 101) (Item 101) The soluble VEGF receptor comprises a part of the extracellular region of VEGFR-1 and a part of the extracellular region of VEGFR-2. The method according to any one of items 70 to 92, which comprises a part of the extracellular region of VEGFR-1 and a part of the extracellular region of VEGFR-2. (Item 102) The part of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-1. The part of the extracellular region of VEGFR-1 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-1. The part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. The method according to item 101, wherein the part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. The method according to item 101, wherein the part of the extracellular region of VEGFR-2 comprises one or more immunoglobulin-like domains in the extracellular region of wild-type human VEGFR-2. (Item 103) The method according to item 102, wherein the soluble VEGF receptor is aflibercept. (Item 104) The soluble VEGF receptor comprises a part of the extracellular region of VEGF receptor-3 (VEGFR-3). The method according to any one of items 70 to 92, which comprises a part of the extracellular region of VEGFR-3. (Item 105) The method according to item 104, wherein the portion of the extracellular region of VEGFR-3 comprises a flanking sequence derived from wild-type human VEGFR-3 (Item 106) The method according to item 105, wherein the portion of the extracellular region of VEGFR-3 comprises one or more immunoglobulin-like domains in the aforementioned extracellular region derived from wild-type human VEGFR-3 (Item 107) The method according to item 104, wherein the portion of the extracellular region of VEGFR-3 comprises a sequence that is at least 90% identical to a flanking sequence derived from wild-type human VEGFR-3 (Item 108) The method according to any one of items 70 to 107, wherein the soluble VEGF receptor comprises an Fc domain (Item 109) The method according to item 108, wherein the Fc domain is an IgG1 Fc domain (Item 110) The method according to item 109, wherein the IgG1 Fc domain is a human wild-type IgG1 Fc domain (Item 111) The method according to any one of items 70 to 110, wherein the soluble VEGF receptor reduces the ability of VEGF to bind to one or more of VEGFR-1, VEGFR-2, and VEGFR-3 (Item 112) The method according to any one of items 43 to 111, wherein the AAV vector further comprises a secretion sequence ​​​​​​​​​

Claims

1. An adeno-associated virus (AAV) vector comprising a nucleotide sequence, wherein the nucleotide sequence comprises a first coding sequence encoding a first polypeptide comprising a variable region of an antibody heavy chain operably linked to a first signal peptide, and a second coding sequence encoding a second polypeptide comprising a variable region of an antibody light chain operably linked to a second signal peptide, wherein the nucleotide sequence comprises a 5' to 3' sequence encoding SEQ ID NO: 39, SEQ ID NO: 52, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or SEQ ID NO: 53, and the polypeptide specifically binds to one or more mammalian VEGF proteins, and the AAV vector is formulated for administration into the cochlea. An AAV vector.

2. The AAV vector according to claim 1, wherein the polypeptide reduces VEGF activity.

3. The AAV vector according to claim 1 or 2, wherein the nucleotide sequence further comprises one or both of a promoter and a Kozak sequence operably linked to the nucleotide sequence.

4. The AAV vector according to claim 3, wherein the nucleotide sequence further comprises a promoter selected from the group consisting of an inducible promoter, a constitutive promoter, and a tissue-specific promoter.

5. The AAV vector according to claim 4, wherein the promoter is a constitutive promoter.

6. The AAV vector according to claim 5, wherein the promoter is a CAG promoter.

7. The AAV vector according to any one of claims 1 to 6, wherein the nucleotide sequence further comprises a polyadenylation signal sequence.

8. The AAV vector according to any one of claims 1 to 7, wherein the nucleotide sequence further comprises two inverted terminal repeats (ITRs) adjacent to the first coding sequence and the second coding sequence.

9. One of the two ITRs has the nucleotide sequence set forth in SEQ ID NO: 36; and / or One of the two ITRs has the nucleotide sequence set forth in SEQ ID NO:

48. The AAV vector according to claim 8.

10. The AAV vector according to any one of claims 1 to 9, wherein the polypeptide comprises a variable domain of an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:

54.

11. The AAV vector according to any one of claims 1 to 9, wherein the polypeptide comprises a variable domain of an antibody light chain comprising the amino acid sequence of SEQ ID NO:

7. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The AAV vector according to any one of claims 1 to 10.

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