Anti-VEGF antibody constructs and related methods for treating vestibular schwannoma-associated conditions

Abstract

The present disclosure provides a construct comprising a coding sequence operably linked to a promoter, the coding sequence encoding a vascular endothelial growth factor (VEGF) binding agent or a portion thereof. In some embodiments, the construct is an AAV construct. In some embodiments, the AAV construct is part of an AAV particle. The compositions comprising the constructs and AAV particles described herein can be useful for treating hearing loss, for example, hearing loss associated with vestibular schwannoma.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 305,923, filed February 2, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Hearing loss can be conductive (originating from the ear canal or middle ear), sensorineural (originating from the inner ear or auditory nerve), or mixed. Sensorineural hearing loss includes hearing loss caused by dysfunction of cells (e.g., hair cells) in the mammalian inner ear. Non-limiting causes of sensorineural hearing loss include noise exposure, head trauma, viral infection, autoimmune inner ear disease, hereditary hearing loss, aging, inner ear malformations, Meniere's disease, otosclerosis, and tumors. As described herein, another cause of hearing loss can be vestibular schwannoma (VS), which is a tumor that develops, for example, on the nerve that leads from the inner ear to the brain. Summary of the Invention

[0003] The present disclosure provides the recognition that administering an anti-VEGF protein (e.g., ranibizumab, bevacizumab, and / or aflibercept) to a subject can be useful in treating conditions, diseases, or disorders associated with neovascularization. The present disclosure further recognizes that administering an anti-VEGF protein is not always easy. For example, administration of an anti-VEGF protein should be achieved in a manner that provides an appropriate level of the anti-VEGF protein locally in cells and tissues associated with neovascularization.

[0004] The present disclosure provides that administration of anti-VEGF constructs capable of expressing anti-VEGF proteins (e.g., ranibizumab, bevacizumab, and / or aflibercept) can be useful in treating conditions, diseases, or disorders associated with neovascularization. In particular, recombinant AAV (rAAV) constructs encoding anti-VEGF proteins (e.g., ranibizumab, bevacizumab, and / or aflibercept) can be particularly useful in treating conditions, diseases, or disorders associated with neovascularization of the ear and eye, particularly when used with rAAVAnc80 capsids to form rAAVAnc80-anti-VEGF particles.

[0005] Among other things, the present disclosure provides constructs comprising a coding sequence operably linked to a promoter, the coding sequence encoding a vascular endothelial growth factor (VEGF) binding agent or portion thereof (also collectively referred to herein as an anti-VEGF protein).

[0006] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, or a tissue-specific promoter. In some embodiments, the promoter is a CAG promoter, a CBA promoter, a CMV promoter, or a CB7 promoter. In some embodiments, the promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 49 or 50, SEQ ID NO: 64, and / or SEQ ID NO: 65.

[0007] In some embodiments, the coding sequence is or comprises a primate coding sequence, in some embodiments, the coding sequence is or comprises a human coding sequence, in some embodiments, the coding sequence is or comprises an engineered coding sequence.

[0008] In some embodiments, the VEGF-binding agent, or portion thereof, is a primate VEGF-binding agent. In some embodiments, the VEGF-binding agent is or comprises a human VEGF-binding agent. In some embodiments, the VEGF-binding agent is or comprises a humanized VEGF-binding agent.

[0009] In some embodiments, the VEGF-binding agent can bind to at least one VEGF protein. In some embodiments, the at least one VEGF protein is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or a combination thereof. In some embodiments, the at least one VEGF protein is VEGF-A.

[0010] In some embodiments, the VEGF-binding agent comprises at least one polypeptide. In some embodiments, the VEGF-binding agent is or comprises an antibody or a fragment thereof. In some embodiments, the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd fragment, an Fd' fragment, a complementarity-determining region (CDR), a single-chain Fv, or an Fc domain. In some embodiments, the VEGF-binding agent is or comprises an immunoglobulin heavy chain, an immunoglobulin light chain, or a combination thereof.

[0011] In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 16. In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 20. In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 16 and a polypeptide comprising the amino sequence set forth in SEQ ID NO: 20.

[0012] In some embodiments, the VEGF-binding agent is or comprises ranibizumab.

[0013] In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 13. In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 13 and the nucleic acid sequence set forth in SEQ ID NO: 19.

[0014] In some embodiments, the coding sequence is or comprises the nucleic acid sequence set forth in SEQ ID NO:103.

[0015] In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 24. In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 25. In some embodiments, the VEGF-binding agent comprises a polypeptide comprising the amino sequence set forth in SEQ ID NO: 24 and a polypeptide comprising the amino sequence set forth in SEQ ID NO: 25.

[0016] In some embodiments, the VEGF-binding agent is or comprises bevacizumab.

[0017] In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 108. In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 109. In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO: 108 and the nucleic acid sequence set forth in SEQ ID NO: 109.

[0018] In some embodiments, the coding sequence is or comprises the nucleic acid sequence set forth in SEQ ID NO:22.

[0019] In some embodiments, the coding sequence comprises one or more nucleic acid sequences each encoding a signal peptide, hi some embodiments, at least one nucleic acid sequence encodes an interleukin 2 (IL2) signal peptide.

[0020] In some embodiments, the coding sequence comprises one or more sequences that encode a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a thosea asigna virus 2A (T2A) peptide.

[0021] In some embodiments, the VEGF-binding agent comprises an Fc domain. In some embodiments, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO:111.

[0022] In some embodiments, the coding sequence comprises the nucleic acid sequence set forth in SEQ ID NO:110.

[0023] In some embodiments, the VEGF-binding agent comprises one or more extracellular domains of a VEGF receptor. In some embodiments, the one or more extracellular domains of a VEGF receptor comprise an extracellular domain comprising the amino acid sequence set forth in SEQ ID NO:112.

[0024] In some embodiments, the VEGF-binding agent comprises two extracellular domains of a VEGF receptor.

[0025] In some embodiments, the coding sequence comprises one or more nucleic acid sequences each encoding a signal peptide, hi some embodiments, at least one nucleic acid sequence encodes an IL2 signal peptide.

[0026] In some embodiments, the construct comprises two AAV inverted terminal repeats (ITRs). In some embodiments, the two AAV ITRs flank the coding sequence and the promoter.

[0027] In some embodiments, the two AAV ITRs are or are derived from AAV2 ITRs.

[0028] In some embodiments, the two AAV ITRs comprise a 5' ITR comprising the nucleic acid sequence set forth in SEQ ID NO:45 or 47 and a 3' ITR comprising the nucleic acid sequence set forth in SEQ ID NO:46 or 48.

[0029] In some embodiments, the construct comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 90, 91, 92, 93, 94, 106, or 107.

[0030] In some embodiments, the construct comprises a nucleic acid sequence set forth in either SEQ ID NO:95 or 96.

[0031] In some embodiments, the constructs described herein are for use in treating an otic disorder characterized by one or more symptoms associated with neovascularization and / or the otic disorder. In some embodiments, the use of the constructs described herein is provided for the manufacture of a medicament for treating an otic disorder characterized by one or more symptoms associated with neovascularization and / or the otic disorder. In some embodiments, the one or more symptoms associated with the otic disorder include hearing loss, hair cell degeneration, altered biochemical environment of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear aperture obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, or death.

[0032] The present disclosure further provides AAV particles comprising the constructs described herein.

[0033] In some embodiments, the rAAV particles comprise an rAAV capsid that is or is derived from an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rh10, AAV-rh39, AAV-rh43, or AAV Anc80 capsid. In some embodiments, the rAAV capsid is an rAAV Anc80 capsid. In some embodiments, the rAAV Anc80 capsid is an rAAV Anc80L65 capsid.

[0034] In some embodiments, the AAV particles described herein are for use in treating an otic disorder characterized by one or more symptoms associated with neovascularization and / or the otic disorder. In some embodiments, the use of the AAV particles described herein is for the manufacture of a medicament for treating an otic disorder characterized by one or more symptoms associated with neovascularization and / or the otic disorder. In some embodiments, the one or more symptoms associated with the otic disorder include hearing loss, hair cell degeneration, altered biochemical environment of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear opening obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, or death.

[0035] The present disclosure provides compositions comprising the constructs described herein and / or the AAV particles described herein.

[0036] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0037] In some embodiments, the pharmaceutical compositions described herein comprise disodium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, or at least 8 mM disodium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise up to 15 mM, up to 14 mM, up to 13 mM, up to 12 mM, up to 11 mM, or up to 10 mM disodium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise 5-12 mM, 6-10 mM, or 7-9 mM disodium phosphate.

[0038] In some embodiments, the pharmaceutical compositions described herein comprise monopotassium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1.0 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, or at least 1.5 mM monopotassium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise up to 2.0 mM, up to 1.9 mM, up to 1.8 mM, up to 1.7 mM, up to 1.6 mM, or up to 1.5 mM monopotassium phosphate. In some embodiments, the pharmaceutical compositions described herein comprise 0.5-2.0 mM, or 1.0-2.0 mM monopotassium phosphate.

[0039] In some embodiments, the pharmaceutical compositions described herein comprise potassium chloride. In some embodiments, the pharmaceutical compositions described herein comprise at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2.0 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, at least 2.5 mM, at least 2.6 mM, or at least 2.7 mM potassium chloride. In some embodiments, the pharmaceutical compositions described herein comprise up to 3.5 mM, up to 3.4 mM, up to 3.3 mM, up to 3.2 mM, up to 3.1 mM, up to 3.0 mM, up to 2.9 mM, or up to 2.8 mM potassium chloride. In some embodiments, the pharmaceutical compositions described herein comprise 2.0-3.5 mM, or 2.5-3.0 mM potassium chloride.

[0040] In some embodiments, the pharmaceutical compositions described herein comprise sodium chloride. In some embodiments, the pharmaceutical compositions described herein comprise at least 50 mM, at least 75 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, or at least 170 mM sodium chloride. In some embodiments, the pharmaceutical compositions described herein comprise up to 250 mM, up to 240 mM, up to 230 mM, up to 220 mM, up to 210 mM, up to 200 mM, up to 190 mM, or up to 180 mM sodium chloride. In some embodiments, the pharmaceutical compositions described herein comprise 150-250 mM, or 150-200 mM sodium chloride.

[0041] In some embodiments, the pharmaceutical compositions described herein comprise poloxamer 188. In some embodiments, the pharmaceutical compositions described herein comprise at least 0.0005%, at least 0.0006%, at least 0.0007%, at least 0.0008%, at least 0.0009%, or at least 0.001% poloxamer 188 (vol / volume %). In some embodiments, the pharmaceutical compositions described herein comprise up to 0.002%, up to 0.0019%, up to 0.0018%, up to 0.0017%, up to 0.0016%, up to 0.0015%, up to 0.0014%, up to 0.0013%, up to 0.0012%, up to 0.0011%, or up to 0.001% poloxamer 188 (vol / volume %). In some embodiments, the pharmaceutical compositions described herein contain 0.0005-0.002%, or 0.0005-0.0015% poloxamer 188 (vol / vol %).

[0042] In some embodiments, the pharmaceutical compositions described herein comprise (% v / v) about 8.10 mM disodium phosphate, about 1.5 mM monopotassium phosphate, about 2.7 mM potassium chloride, about 172 mM sodium chloride, and about 0.001% poloxamer 188. In some embodiments, the pharmaceutical compositions described herein comprise (% v / v) about 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and 0.001% poloxamer 188.

[0043] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising the AAV particles described herein, e.g., rAAV-anti-VEGF particles, comprises about 1 x 10 11 vg / mL ~ approx. 1×10 15 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising an AAV particle described herein, e.g., an rAAV-anti-VEGF particle, is administered at a dose (e.g., amount) of 2.5×10 vg / mL. 12 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising an AAV particle described herein, e.g., an rAAV-anti-VEGF particle, is administered at a dose (e.g., amount) of 5×10 vg / mL + / −10%. 12 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising the AAV particles described herein, e.g., rAAV-anti-VEGF particles, is administered at a dose (e.g., amount) of 1 x 10 vg / mL + / - 10%. 13 In some embodiments, the pharmaceutical compositions described herein comprise about 8.10 mM disodium phosphate, about 1.5 mM monopotassium phosphate, about 2.7 mM potassium chloride, about 172 mM sodium chloride, and about 0.001% poloxamer 188 (vol / vol %). In some embodiments, the pharmaceutical compositions described herein comprise 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and 0.001% poloxamer 188 (vol / vol %).

[0044] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising the AAV particles described herein, e.g., rAAV-anti-VEGF particles, comprises about 1 x 10 10 ~Approx. 1×10 13 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising an AAV particle described herein, e.g., an rAAV-anti-VEGF particle, is administered at a dose of about 2.3 x 10 vg / cochlea. 11 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising an AAV particle described herein, e.g., an rAAV-anti-VEGF particle, is administered at a dose of about 4.5 x 10 vg / cochlea. 11 In some embodiments, a composition (e.g., a pharmaceutical composition) comprising an AAV particle described herein, e.g., an rAAV-anti-VEGF particle, is administered at a dose of about 9×10 vg / cochlea. 11 vg / cochlea. In some embodiments, the pharmaceutical compositions described herein comprise about 8.10 mM disodium phosphate, about 1.5 mM monopotassium phosphate, about 2.7 mM potassium chloride, about 172 mM sodium chloride, and about 0.001% poloxamer 188 (vol / vol %). In some embodiments, the pharmaceutical compositions described herein comprise 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and 0.001% poloxamer 188 (vol / vol %).

[0045] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising AAV particles described herein, e.g., rAAV-anti-VEGF particles, is administered to a subject in a volume of about 0.01 mL to 0.1 mL. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising AAV particles described herein, e.g., rAAV-anti-VEGF particles, is administered to a subject in a volume of about 0.09 mL. In some embodiments, a pharmaceutical composition described herein comprises about 8.10 mM disodium phosphate, about 1.5 mM monopotassium phosphate, about 2.7 mM potassium chloride, about 172 mM sodium chloride, and about 0.001% poloxamer 188 (vol / vol%). In some embodiments, a pharmaceutical composition described herein comprises 8.10 mM disodium phosphate, 1.5 mM monopotassium phosphate, 2.7 mM potassium chloride, 172 mM sodium chloride, and 0.001% poloxamer 188 (vol / vol%).

[0046] In some embodiments, the compositions described herein are for use in treating an otic disorder, e.g., an otic disorder in a mammal, wherein the otic disorder is characterized by neovascularization and / or one or more symptoms associated with the otic disorder. In some embodiments, the use of a construct described herein is provided for the manufacture of a medicament for treating an otic disorder, e.g., an otic disorder in a mammal, wherein the otic disorder is characterized by neovascularization and / or one or more symptoms associated with the otic disorder. In some embodiments, the one or more symptoms associated with the otic disorder include hearing loss, hair cell degeneration, altered biochemical environment of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear opening obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, death, or a combination thereof.

[0047] In some embodiments, the compositions described herein are for use in treating an inner ear disorder, e.g., an inner ear disorder in a mammal. In some embodiments, the use of the constructs described herein is provided for the manufacture of a medicament for treating an inner ear disorder, e.g., an inner ear disorder in a mammal. In some embodiments, the inner ear disorder comprises a vestibular schwannoma or neurofibromatosis type II. In some embodiments, the inner ear disorder is or comprises a vestibular schwannoma. In some embodiments, the inner ear disorder is or comprises a neurofibromatosis type II. A vestibular schwannoma can also be referred to as an acoustic neuroma, and both terms are used interchangeably herein.

[0048] In some embodiments, the compositions described herein are for use in treating vestibular schwannoma, e.g., vestibular schwannoma in a mammal. In some embodiments, the use of the constructs described herein is provided for the manufacture of a medicament for treating vestibular schwannoma, e.g., vestibular schwannoma in a mammal.

[0049] In some embodiments of any of the methods or uses disclosed herein, the mammal is a human.

[0050] The present disclosure also provides cells, in some embodiments, comprising a construct described herein, an rAAV particle described herein, and / or a composition described herein.

[0051] In some embodiments, the cells are in vivo, ex vivo, or in vitro.

[0052] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is present in the ear of a subject.

[0053] In some embodiments, the cells are immortalized to generate stable cell lines.

[0054] The present disclosure provides systems, which include a construct described herein, an rAAV particle described herein, a composition described herein, and / or a cell described herein.

[0055] The present disclosure provides methods, in some embodiments, that include contacting a cell with a construct described herein, an rAAV particle described herein, and / or a composition described herein.

[0056] In some embodiments, the cell is a cell of a subject.

[0057] In some embodiments, the cell is an ear cell. In some embodiments, the cell is an inner ear cell. In some embodiments, the inner ear cell is an outer hair cell. In some embodiments, the inner ear cell is an inner hair cell. In some embodiments, the inner ear cell is a pillar cell. In some embodiments, the inner ear cell is a Hensen cell. In some embodiments, the inner ear cell is a Clausius cell.

[0058] In some embodiments, the inner ear cells are present in vitro or ex vivo.

[0059] In some embodiments, the methods include introducing a construct described herein, an rAAV particle described herein, and / or a composition described herein into the inner ear of a subject.

[0060] In some embodiments, the construct, rAAV particle, or composition is introduced into the cochlea of ​​a subject. In some embodiments, the construct, rAAV particle, or composition is introduced via round window membrane injection.

[0061] In some embodiments, administration of a construct, rAAV particle, or composition disclosed herein to the cochlea of ​​a subject results in expression of a transgene encoded by the construct, rAAV particle, or composition in the cochlea of ​​the subject. In some embodiments, the transgene encodes an anti-VEGF protein. In some embodiments, the anti-VEGF protein is expressed in one or more cells in the cochlea of ​​the subject. In some embodiments, the anti-VEGF protein is expressed in outer hair cells, inner hair cells, pillar cells, Hensen cells, Clausius cells, or a combination thereof. In some embodiments, expression of the anti-VEGF protein in one or more cochlear cells has minimal impact (e.g., no impact) on the survival and / or function of one or more cochlear cells.

[0062] In some embodiments, the method includes measuring the hearing level of the subject. In some embodiments, the hearing level is measured by performing an auditory brainstem response (ABR) test.

[0063] In some embodiments, the method includes comparing the subject's hearing level to a reference hearing level, hi some embodiments, the reference hearing level is a published or historical reference hearing level.

[0064] In some embodiments, the subject's hearing level is measured after a construct described herein, an rAAV particle described herein, and / or a composition described herein is introduced, and the baseline hearing level is the subject's hearing level measured before a construct described herein, an rAAV particle described herein, and / or a composition described herein is introduced.

[0065] In some embodiments, the methods include measuring the level of a vascular endothelial growth factor (VEGF) binding agent or a portion thereof in the subject.

[0066] In some embodiments, the level of the vascular endothelial growth factor (VEGF) binding agent, or a portion thereof, is measured in the inner ear of the subject. In some embodiments, the level of the vascular endothelial growth factor (VEGF) binding agent, or a portion thereof, is measured in the cochlea of ​​the subject.

[0067] In some embodiments, the methods include comparing the level of a vascular endothelial growth factor (VEGF) binding agent, or a portion thereof, in the subject to a reference level of the vascular endothelial growth factor (VEGF) binding agent, or a portion thereof.

[0068] In some embodiments, the reference level of a vascular endothelial growth factor (VEGF) binding agent, or portion thereof, is a published or historical reference level of a vascular endothelial growth factor (VEGF) binding agent, or portion thereof.

[0069] In some embodiments, the level of a vascular endothelial growth factor (VEGF)-binding agent or portion thereof in the subject is measured after a construct described herein, an AAV particle described herein, and / or a composition described herein is introduced, and the baseline level of the vascular endothelial growth factor (VEGF)-binding agent or portion thereof is the level of the vascular endothelial growth factor (VEGF)-binding agent or portion thereof in the subject measured before a construct described herein, an AAV particle described herein, and / or a composition described herein is introduced.

[0070] In some embodiments, the method includes measuring a tumor dimension or volume in the subject. In some embodiments, the dimension is the greatest diameter or length of the entire tumor.

[0071] In some embodiments, the method comprises comparing the subject's tumor size or volume to a reference tumor size or volume, respectively.

[0072] In some embodiments, the baseline tumor size or volume is a published or historical baseline tumor size or volume.

[0073] In some embodiments, the subject's tumor size or volume is measured after introduction of a construct described herein, an AAV particle described herein, and / or a composition described herein, and the baseline tumor size or volume is the subject's tumor size or volume measured before introduction of a construct described herein, an AAV particle described herein, and / or a composition described herein.

[0074] In some embodiments, the method is a method of treating hearing loss comprising administering a construct described herein, an rAAV particle described herein, and / or a composition described herein to a subject in need thereof.

[0075] In some embodiments, the subject has or is at risk for an otic disease characterized by neovascularization, hi some embodiments, the otic disease is or comprises vestibular schwannoma.

[0076] In some embodiments, the method is a method of treating an inner ear disorder comprising administering a construct described herein, an rAAV particle described herein, and / or a composition described herein to a subject in need thereof. In some embodiments, the inner ear disorder is vestibular schwannoma or neurofibromatosis type II.

[0077] In some embodiments of any of the methods or uses disclosed herein, the subject is a human.

[0078] In some embodiments, one or more symptoms associated with an otic disorder are alleviated or ameliorated following administration of a construct described herein, an AAV particle described herein, and / or a composition described herein, In some embodiments, the one or more symptoms include hearing loss, hair cell degeneration, altered biochemical milieu of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear orifice obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, or death.

[0079] In some embodiments, the method is a method of treating a vestibular schwannoma.

[0080] In some embodiments, the method is a method of modulating the level of VEGF.

[0081] In some embodiments, the method is a method of modulating the level of active VEGF.

[0082] In some embodiments, the method is a method of reducing the activity of VEGF.

[0083] The disclosure provides methods that include contacting a cell with a construct described herein and one or more constructs that include the AAV Rep gene, the AAV Cap gene, the AAV VA gene, the AAV E2a gene, and the AAV E4 gene.

[0084] In some embodiments, the cells are inner ear cells. In some embodiments, the inner ear cells are outer hair cells. In some embodiments, the inner ear cells are inner hair cells. In some embodiments, the inner ear cells are pillar cells. In some embodiments, the inner ear cells are Hensen cells. In some embodiments, the inner ear cells are Clausius cells. In some embodiments, the inner ear cells are present in the ear of a subject. In some embodiments, the inner ear cells are present in vitro or ex vivo.

[0085] The present disclosure provides a population of cells comprising one or more cells described herein, wherein the population is or comprises a stable cell line.

[0086] Although methods and materials are described herein for use in the present invention, other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control.

[0087] definition The scope of the present disclosure is defined by the claims appended hereto, and is not limited by the specific embodiments described herein. Those skilled in the art will recognize, upon reading this specification, various modifications that may be equivalent to such described embodiments or that may otherwise fall within the scope of the claims. Generally, terms used herein adhere to their art-understood meanings unless expressly indicated otherwise. Explicit definitions of certain terms are provided below, but the meaning of these and other terms in specific instances throughout the specification will be apparent to those skilled in the art from the context.

[0088] The use of ordinal terms such as "first," "second," "third," etc. in the claims to modify claim elements does not, by itself, imply a priority, precedence, or order of one element of the claim over another element, or a chronological order in which the actions of a method are performed, but rather is merely used as a label to distinguish one element of a claim having a particular name from another element having the same name (apart from the use of ordinal terms) and to distinguish between these claim elements.

[0089] As used herein, the articles "a" and "an" should be understood to include plural referents unless expressly stated to the contrary. A claim or description including "or" between one or more members of a group is considered to stand when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless stated to the contrary or otherwise apparent from the context. In some embodiments, exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. In some embodiments, two or more, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. The present disclosure should be understood to encompass all variations, combinations, and permutations of one or more limitations, elements, clauses, descriptive language, etc. from one or more of the enumerated claims, introduced into another claim (or any other claim, as appropriate) dependent on the same base claim, unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one of ordinary skill in the art. Where elements are presented as a list (e.g., in a Markush group or similar format), it is to be understood that each subgroup of the elements is also disclosed, and that any element(s) can be deleted from the group. In general, when an embodiment or aspect is referred to as "comprising" certain elements, features, etc., it is to be understood that the particular embodiment or aspect "consists of" or "consists essentially of" such elements, features, etc. For the sake of brevity, those embodiments have not in every instance been specifically described in as many words herein. It is also to be understood that any embodiment or aspect can be explicitly excluded from the claims, regardless of whether that specific exclusion is set forth herein.

[0090] Throughout this specification, when a polynucleotide or polypeptide is represented by a string of letters (e.g., in the case of a polynucleotide, A, C, G, T, representing adenosine, cytidine, guanosine, and thymidine, respectively), the polynucleotide or polypeptide is represented in left-to-right, 5' to 3' or N-terminal to C-terminal order.

[0091] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of an agent to the subject or system. In some embodiments, the agent is or is contained in a composition, and in some embodiments, the agent is produced through metabolism of the composition or one or more components thereof. Those of skill in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be systemic or local. In some embodiments, systemic administration may be intravenous. In some embodiments, administration may be local. Local administration may include delivery to the perilymph of the cochlea, for example, by injection through the round window membrane, or injection into the scala tympani, scala media, endolymph, perilymph, and / or endolymph after canalostomy. In some embodiments, administration may include only a single administration. In some embodiments, administration may include the application of a series of doses. In some embodiments, administration may include administration that is intermittent (e.g., multiple administrations separated in time) and / or periodic (e.g., individual administrations separated by a common period of time) administration. In some embodiments, administration may include continuous administration (e.g., perfusion) over at least a selected period of time.

[0092] Allele: As used herein, the term "allele" refers to one of two or more existing genetic variants at a particular polymorphic genomic locus.

[0093] Amelioration: As used herein, the term "amelioration" refers to the prevention, alleviation, or alleviation of a condition, or improvement of a condition, in a subject. Amelioration can include, but does not necessarily include, complete recovery or complete prevention of a disease, disorder, or condition.

[0094] Amino acid: As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain, for example, through the formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure, e.g., HN-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is an unnatural amino acid, in some embodiments, an amino acid is a D-amino acid, and in some embodiments, an amino acid is an L-amino acid. A "standard amino acid" refers to any of the 20 standard L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than the standard amino acids, whether synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including the carboxy-terminal amino acid and / or the amino-terminal amino acid in a polypeptide, can include structural modifications compared to the general structure described above. For example, in some embodiments, an amino acid can be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, carboxylic acid group, one or more protons, and / or hydroxyl group) compared to the general structure. In some embodiments, such modifications may, for example, alter the serum half-life of a polypeptide comprising the modified amino acid compared to one comprising otherwise identical, unmodified amino acids, hi some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid compared to one comprising otherwise identical, unmodified amino acids.

[0095] Antibody: As used herein, the term "antibody" refers to a polypeptide containing sufficient standard immunoglobulin sequence elements to specifically bind to a particular target antigen. As known in the art, intact antibodies, as produced in nature, are approximately 150 kD tetrameric agents composed of two identical heavy chain polypeptides (approximately 50 kD each) and two identical light chain polypeptides (approximately 25 kD each), which associate together in what is commonly referred to as a "Y-shaped" structure. Each heavy chain consists of at least four domains (each approximately 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y-shaped structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain consists of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer consists of two heavy-light chain dimers, with one disulfide bond linking the heavy and light chains, and two additional disulfide bonds connecting the heavy chain hinge regions to form the tetramer. Naturally produced antibodies are also typically glycosylated on the CH2 domain. Each domain of a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two β-sheets (e.g., a three-, four-, or five-stranded sheet) packed together in a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops known as "complementarity-determining regions" (CDR1, CDR2, and CDR3), and four more or less invariant "framework" regions (FR1, FR2, FR3, and FR4).When a native antibody folds, the FR regions form a β-sheet that provides the structural framework for the domain, and the CDR loop regions of both the heavy and light chains are organized in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells (including, e.g., effector cells that mediate cytotoxicity). As is known in the art, the affinity and / or other binding properties of the Fc region for Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized in accordance with the present invention comprise a glycosylated Fc domain, including Fc domains with modified or engineered glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that comprises a sufficient immunoglobulin domain sequence as found in a native antibody may be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, the antibody is polyclonal, and in some embodiments, the antibody is monoclonal. In some embodiments, the antibody has constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody," as used herein, can, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any art-known or developed construct or configuration for utilizing the structural and functional characteristics of alternative forms of antibodies.For example, in some embodiments, antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies, bi- or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, masked antibodies (e.g., Probodies®), Small Modular ImmunoPharmaceutical (SMIP™), single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies®, minibodies, BiTE®, ankyrin repeat proteins or DARPIN®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, the antibody can comprise a heavy and / or light chain variable domain. In some embodiments, the antibody may not comprise a constant domain. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have if produced naturally. In some embodiments, the antibody may comprise a covalent modification (e.g., the attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.).

[0096] Approximately or about: As used herein, the term "approximately" or "about" may apply to one or more values ​​of interest, including values ​​similar to a reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within ±10% (greater or less than) of the reference value, unless otherwise stated or otherwise apparent from the context (except where such number may exceed 100% of possible values). For example, in some embodiments, the term "approximately" or "about" may encompass a range of values ​​that is 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0097] Associated / Associated: As used herein, the term "associated / associated" refers to two events or entities being "associated" with one another when the presence, level, and / or form of one correlates with that of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they interact, directly or indirectly, such that they are in and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently linked by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0098] Biologically active: As used herein, the term "biologically active" refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed through detection of a direct or indirect product produced by the biological pathway or event of interest.

[0099] Characteristic portion: As used herein, the term "characteristic portion" broadly refers to a portion of a substance whose presence (or absence) correlates with the presence (or absence) of a particular characteristic, property, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion found in a given substance and related substances that share a common specific characteristic, property, or activity, but not in substances that do not share the common specific characteristic, property, or activity. In some embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is a portion comprising a contiguous stretch of amino acids or a collection of contiguous stretches of amino acids that together are characteristic of the protein or polypeptide. In some embodiments, each such contiguous stretch typically comprises at least 2, 5, 10, 15, 20, 50, or more amino acids. Generally, a characteristic portion of a substance (e.g., a protein, antibody, etc.) is a portion that shares at least one functional characteristic with the related intact substance, in addition to the sequence and / or structural identity described above. In some embodiments, a characteristic portion may be biologically active.

[0100] Characteristic sequence: As used herein, the term "characteristic sequence" is a sequence that is found in all members of a family of polypeptides or nucleic acids and can therefore be used by one of skill in the art to define members of the family.

[0101] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., of a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or non-member) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., sequentially linked monomers). In some embodiments, the characteristic sequence element comprises at least first and second stretches of consecutive monomers spaced apart by one or more spacer regions that may or may not vary in length between polymers having the common sequence element.

[0102] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more agents may be administered simultaneously. In some embodiments, two or more agents may be administered sequentially. In some embodiments, two or more agents may be administered in overlapping dosing regimens.

[0103] Equivalent: As used herein, the term "equivalent" refers to two or more agents, entities, circumstances, sets of conditions, subjects, populations, etc. that may not be identical to one another, but are sufficiently similar to permit their comparison, such that a person of ordinary skill in the art would understand that they could reasonably draw conclusions based on the observed differences or similarities. In some embodiments, a set of equivalent agents, entities, circumstances, sets of conditions, subjects, populations, etc. is characterized by multiple substantially identical characteristics and one or a few different characteristics. One of ordinary skill in the art will understand the degree of identity required, depending on the circumstances, for two or more such agents, entities, circumstances, sets of conditions, subjects, populations, etc. to be considered equivalent in any given situation. For example, one skilled in the art will recognize that sets of agents, entities, circumstances, sets of conditions, subjects, populations, etc. are equivalent to one another when they are characterized by a sufficient number and variety of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or by different sets of circumstances, stimuli, agents, entities, circumstances, sets of conditions, subjects, populations, etc. are caused by or indicate variation in those characteristics that vary.

[0104] Construct: As used herein, the term "construct" refers to a composition comprising a polynucleotide capable of carrying at least one heterologous polynucleotide. In some embodiments, a construct can be a plasmid, transposon, cosmid, artificial chromosome (e.g., human artificial chromosome (HAC), yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)), or viral construct, and any Gateway® plasmid. A construct can, for example, include sufficient cis-acting elements for expression; other elements for expression can be supplied by the host primate cell or in an in vitro expression system. A construct can include any genetic element (e.g., a plasmid, transposon, cosmid, artificial chromosome, or viral construct) capable of replication when linked to appropriate control elements. Thus, in some embodiments, a "construct" can include a cloning and / or expression construct and / or a viral construct (e.g., an adeno-associated virus (AAV) construct, an adenoviral construct, a lentiviral construct, or a retroviral construct).

[0105] Conservative: As used herein, the term "conservative" refers to cases that represent conservative amino acid substitutions, including the replacement of an amino acid residue with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the desired functional properties of a protein, such as, for example, the ability of a receptor to bind a ligand. Examples of groups of amino acids having side chains with similar chemical properties include: aliphatic side chains such as glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), and isoleucine (Ile, I); aliphatic-hydroxy side chains such as serine (Ser, S) and threonine (Thr, T); amide-containing side chains such as asparagine (Asn, N) and glutamine (Gln, Q); aromatic side chains such as phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W); basic side chains such as lysine (Lys, K), arginine (Arg, R), and histidine (His, H); acidic side chains such as aspartic acid (Asp, D) and glutamic acid (Glu, E); and sulfur-containing side chains such as cysteine ​​(Cys, C) and methionine (Met, M). Conservative amino acid substitutions include, for example, valine / leucine / isoleucine (Val / Leu / Ile, V / L / I), phenylalanine / tyrosine (Phe / Tyr, F / Y), lysine / arginine (Lys / Arg, K / R), alanine / valine (Ala / Val, A / V), glutamic acid / aspartic acid (Glu / Asp, E / D), and asparagine / glutamine (Asn / Gln, N / Q). In some embodiments, conservative amino acid substitutions can be substitutions of alanine for any naturally occurring residue in a protein, for example, as used in alanine scanning mutagenesis. In some embodiments, conservative substitutions are made to have a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet, GH et al., 1992, Science 256:1443-1445, which is incorporated herein by reference in its entirety. In some embodiments, the substitution is a moderately conservative substitution, where the substitution has a non-negative value in the PAM250 log-likelihood matrix.Those skilled in the art will understand that changes (e.g., substitutions, additions, deletions, etc.) in amino acids that are not conserved between the same proteins from different species are unlikely to affect the function of the protein, and therefore these amino acids should be selected for mutation. Amino acids that are conserved between the same proteins from different species should not be altered (e.g., deleted, added, substituted, etc.) because these mutations are likely to result in changes in the function of the protein. [Table 1]

[0106] Control: As used herein, the term "control" refers to its art-recognized meaning as a standard against which results are compared. Typically, controls are used to increase consistency in experiments by isolating variables in order to draw conclusions about them. In some embodiments, a control is a reaction or assay performed simultaneously with a test reaction or assay to provide a comparator. For example, in one experiment, the "test" (i.e., the variable being tested) is administered. In a second experiment, the "control" does not receive the variable being tested. In some embodiments, a control is a pre-existing control (e.g., of a test or assay already performed, or of a known amount or result). In some embodiments, a control is or includes a printed or otherwise kept record. In some embodiments, a control is a positive control. In some embodiments, a control is a negative control.

[0107] Determining, Measuring, Evaluation, Study, Assay, and Analysis: As used herein, the terms “determining,” “measuring,” “evaluating,” “studying,” “assay,” and “analyzing” can be used interchangeably to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Assays can be relative or absolute. For example, in some embodiments, “assaying for the presence of” can be determining the amount of something present and / or determining whether it is present or not.

[0108] Engineered: Generally, as used herein, the term "engineered" refers to aspects of manipulation by the hand of man. For example, a cell or organism is considered "engineered" if it has been manipulated to change its genetic information (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutations or by breeding protocols). As is common practice and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell are typically still considered "engineered," even though the actual manipulation was performed on the antecedent entity. In some embodiments, "engineered" includes "humanization" of a coding sequence. In some embodiments, "humanization" can include introducing human non-coding sequences, such as introns and regulatory elements, into a non-human sequence. In some embodiments, "humanization" can include codon-optimizing a nucleotide sequence for human use. In some embodiments, "humanization" can include replacing a portion of a polypeptide (e.g., a domain, such as a framework region or complementary domain region) or a nucleotide sequence (e.g., coded or non-coding) with a human polypeptide or nucleotide sequence.

[0109] Excipient: As used herein, the term "excipient" refers to an inert (e.g., non-therapeutic) substance that can be included in a pharmaceutical composition to, for example, provide or contribute a desired consistency or stabilizing effect. In some embodiments, suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0110] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product (e.g., a transcription product, e.g., mRNA, e.g., a polypeptide, etc.) from the nucleic acid sequence. In some embodiments, the gene product can be a transcription product. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence includes one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0111] Functional: As used herein, the term "functional" refers to something that exists in a form in which it exhibits a property and / or activity that characterizes it. For example, in some embodiments, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and / or activity that characterizes it. In some such embodiments, a functional biological molecule is characterized relative to another biological molecule that is non-functional in that the "non-functional" version does not exhibit the same or equivalent property and / or activity as the "functional" molecule. A biological molecule may have one function, two functions (i.e., bifunctional), or many functions (i.e., multifunctional).

[0112] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a gene product (e.g., an RNA product, e.g., a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene comprises non-coding sequence. In some particular embodiments, a gene may comprise both coding (e.g., exon) and non-coding (e.g., intron) sequence. In some embodiments, a gene may comprise one or more regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences that can, for example, control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.). As used herein, the term "gene" generally refers to a portion of a nucleic acid that encodes a polypeptide or fragment thereof; the term may optionally encompass regulatory sequences, as will be clear to one of skill in the art from the context. This definition is not intended to exclude the application of the term "gene" to non-protein-coding expression units, but is intended to clarify that in most cases, the term as used herein refers to a nucleic acid that encodes a polypeptide. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional; for example, a gene variant may encode a polypeptide that functions similarly or not at all compared to a wild-type gene. In some embodiments, a gene may encode a transcript that, in some embodiments, may be toxic above a threshold level. In some embodiments, a gene may encode a polypeptide, but the polypeptide may not be functional and / or may be toxic above a threshold level.

[0113] Hearing loss: As used herein, the term "hearing loss" may refer to the partial or complete inability of an organism to hear. In some embodiments, the hearing loss may be acquired. In some embodiments, the hearing loss may be hereditary. In some embodiments, the hearing loss may be genetic. In some embodiments, the hearing loss may be the result of disease or trauma (e.g., physical trauma, treatment with one or more agents that result in hearing loss, etc.). In some embodiments, the hearing loss may be due to one or more known genetic causes and / or syndromes. In some embodiments, the hearing loss may be of unknown etiology. In some embodiments, the hearing loss may or may not be alleviated by the use of hearing aids or other treatments.

[0114] Heterologous: As used herein, the term "heterologous" may be used in reference to one or more regions of a particular molecule compared to another region and / or another molecule. For example, in some embodiments, heterologous polypeptide domains refer to polypeptide domains that do not naturally occur together (e.g., within the same polypeptide). For example, in a man-made fusion protein, a polypeptide domain from one polypeptide may be fused to a polypeptide domain from a different polypeptide. In such a fusion protein, the two polypeptide domains would be considered "heterologous" to each other because they do not naturally occur together.

[0115] Identity: As used herein, the term "identity" refers to the overall relatedness between polymers, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polymers are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of the aligned sequences for comparison is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence, and then nucleotides at corresponding positions are compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the two molecules (i.e., the first and second) are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17, incorporated herein by reference in its entirety), which is incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0116] Improve, increase, enhance, inhibit, or reduce: As used herein, "improve," "increase," "enhance," "inhibit," "reduce," or their grammatical equivalents refer to a value compared to a baseline or other reference measurement. In some embodiments, the value is statistically significantly different from the baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., a single individual) under otherwise equivalent conditions in the absence (e.g., before and / or after) of a particular agent or treatment, or in the presence of an appropriate equivalent reference agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment. In some embodiments, a suitable reference standard is a negative reference standard; in some embodiments, a suitable reference standard is a positive reference standard.

[0117] Nucleic Acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA. In some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to that of naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or entirely single-stranded, and in some embodiments, the nucleic acid is partially or entirely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or is complementary to a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0118] Operably linked: As used herein, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is attached such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest; in some embodiments, the control element acts in trans or otherwise on the functional element of interest. In some embodiments, "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some embodiments, for example, functional linkage can include transcriptional regulation. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, in the same reading frame.

[0119] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including, for example, those adapted for administration as an injectable solution, e.g., an aqueous or non-aqueous solution or suspension, or ear drops designed to be administered into the ear canal. In some embodiments, the pharmaceutical composition may be formulated for administration by injection into either a specific organ or compartment, e.g., direct injection into the ear, or systemic injection, e.g., intravenous injection. In some embodiments, the formulation may be or include a drench (aqueous or non-aqueous solution or suspension), tablet, bolus, powder, granule, paste, capsule, powder, etc. In some embodiments, the active agent may be or include an isolated, purified, or pure compound.

[0120] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable," which may be used in reference to, for example, a carrier, diluent, or excipient used to formulate a pharmaceutical composition disclosed herein, means the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0121] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0122] Polyadenylation: As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylated portion or modified variants thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. In some embodiments, the 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) (SEQ ID NO: 117) added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(A) tail can be added to transcripts containing a specific sequence, a polyadenylation signal or "poly(A) sequence." The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation can affect the termination of transcription, the export of mRNA from the nucleus, and translation. Typically, polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur in the cytoplasm later. After transcription is terminated, the mRNA strand can be cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site can be characterized by the presence of the base sequence AAUAAA near the cleavage site. After mRNA cleavage, adenosine residues can be added to the free 3' end of the cleavage site. As used herein, a "poly(A) sequence" is a sequence that triggers endonuclease cleavage of mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0123] Polypeptide: As used herein, the term "polypeptide" refers to any polymeric chain of residues (e.g., amino acids) typically linked by peptide bonds. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or created by the action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide may comprise one or more pendant groups or other modifications, e.g., one or more amino acid side chain modifications or attachments, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, PEGylation, etc. (including combinations thereof). In some embodiments, a polypeptide may comprise L-amino acids, D-amino acids, or both, and may include any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0124] Polynucleotide: As used herein, the term "polynucleotide" refers to any polymeric strand of nucleic acid. In some embodiments, a polynucleotide is or comprises RNA. In some embodiments, a polynucleotide is or comprises DNA. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleic acid analogs. In some embodiments, polynucleotide analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. Alternatively or additionally, in some embodiments, a polynucleotide has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, a polynucleotide is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). In some embodiments, a polynucleotide is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof). In some embodiments, polynucleotides comprise one or more modified sugars (eg, 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids.In some embodiments, the polynucleotide has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. In some embodiments, the polynucleotide comprises one or more introns. In some embodiments, the polynucleotide is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, a polynucleotide is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the polynucleotide is partially or entirely single-stranded, and in some embodiments, the polynucleotide is partially or entirely double-stranded. In some embodiments, the polynucleotide has a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes it. In some embodiments, the polynucleotide has enzymatic activity.

[0125] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). A protein may include moieties other than amino acids (e.g., may be a glycoprotein, proteoglycan, etc.) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be the entire polypeptide chain (with or without a signal sequence) produced by a cell, or a characteristic portion thereof. Those of skill in the art will understand that a protein may in some cases include multiple polypeptide chains, for example, those linked by one or more disulfide bonds or those associated by other means.

[0126] Recombinant: As used herein, the term "recombinant" refers to a polypeptide that is designed, engineered, prepared, expressed, made, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression construct transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise engineered to express one or more genes or genetic components that encode and / or directly express the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide prepared, expressed, made, or isolated by any other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directly expresses the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements arise, for example, from mutagenesis (e.g., in vivo or in vitro) of known sequence elements from natural or synthetic sources, for example, in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0127] Reference: As used herein, the term "reference" refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is an existing reference or control, optionally embodied in a tangible medium. Typically, as will be understood by those of skill in the art, a reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. One of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular potential reference or control. In some embodiments, the reference is a negative control reference. In some embodiments, the reference is a positive control reference.

[0128] Regulatory element: As used herein, the term "regulatory element" or "regulatory sequence" refers to a non-coding region of DNA that regulates in some way the expression of one or more specific genes. In some embodiments, such genes are juxtaposed to or "near" a given regulatory element. In some embodiments, such genes are located at a considerable distance from a given regulatory element. In some embodiments, a regulatory element impairs or promotes transcription of one or more genes. In some embodiments, a regulatory element can be located in cis with respect to the gene being regulated. In some embodiments, a control element can be located in trans with respect to the gene being regulated. For example, in some embodiments, a regulatory sequence refers to a nucleic acid sequence that regulates the expression of a gene product operably linked to the regulatory sequence. In some such embodiments, this sequence may be an enhancer sequence or other regulatory element that regulates expression of the gene product.

[0129] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest may be or include a cell or organism, such as a microorganism (e.g., a virus), a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or cellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or irrigation (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage or irrigation). In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components and / or adding one or more agents), for example, filtration using a semipermeable membrane.Such a "processed sample" may include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of specific components, etc.

[0130] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including, in some embodiments, prenatal human forms). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is a person with one or more characteristics that characterize a susceptibility to, or risk for, a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or to whom diagnosis and / or treatment is and / or has been administered.

[0131] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a desired characteristic or property. Those skilled in the art will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion, or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to express the potential lack of completeness inherent in many biological and chemical phenomena.

[0132] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of therapy that partially or completely alleviates, relieves, eliminates, reverses, reduces, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be treatment of a subject who does not exhibit signs of the associated disease, disorder, and / or condition and / or who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of a subject who exhibits one or more established symptoms of the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who has been diagnosed with the associated disease, disorder, and / or condition. In some embodiments, treatment may be treatment of a subject who is known to have one or more susceptibility factors that statistically correlate with an increased risk of developing a given disease, disorder, and / or condition.

[0133] Tumor: As used herein, the term "tumor" refers to an abnormal growth of cells or tissue. In some embodiments, a tumor can include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic. In some embodiments, a tumor is associated with or is a manifestation of cancer. In some embodiments, a tumor can be a dispersed tumor or a liquid tumor. In some embodiments, a tumor can be a solid tumor.

[0134] Variant: As used herein, the term "variant" refers to a version of something, e.g., a gene sequence that differs in some way from another version. To determine whether something is a variant, typically a reference version is selected, and the variant differs relative to that reference version. In some embodiments, a variant can have the same or a different (e.g., increased or decreased) level of activity or functionality as the wild-type sequence. For example, in some embodiments, a variant can have improved functionality compared to the wild-type sequence, e.g., if it is codon-optimized to resist degradation by an inhibitory nucleic acid, e.g., miRNA. Such variants are referred to herein as gain-of-function variants. In some embodiments, a variant has a reduced or eliminated activity or functionality, or an altered activity that has a negative consequence (e.g., increased electrical activity resulting in chronic depolarization leading to cell death). Such variants are referred to herein as loss-of-function variants. For example, in some embodiments, a gene sequence is a wild-type sequence that encodes a functional protein and is present in the majority of members of a species whose genome contains the gene. In some such embodiments, a gain-of-function variant can be a gene sequence that contains one or more nucleotide differences relative to the wild-type gene sequence. In some embodiments, a gain-of-function variant is a codon-optimized sequence that encodes a transcript or polypeptide that may have improved properties (e.g., less susceptible to degradation, e.g., less susceptible to miRNA-mediated degradation) than the corresponding wild-type (e.g., non-codon-optimized) version. In some embodiments, a loss-of-function variant has one or more changes that result in a transcript or polypeptide that is defective in some way (e.g., reduced function, non-functional) compared to the wild-type transcript and / or polypeptide.

[0135] VEGF inhibitor: As used herein, the term "VEGF inhibitor" is used interchangeably with the term "anti-VEGF protein." [Brief explanation of the drawings]

[0136] [Figure 1] FIG. 1 is a schematic diagram of representative anatomy of the human ear, including common areas where vestibular schwannomas (VS) arise. [Figure 2] Figure 1 shows a graphical representation of the location of VSs (<5 mm wide) within the internal auditory canal. In this analysis, the majority of small internal auditory canal VSs (those located entirely within the internal auditory canal) were located close to the floor of the internal auditory canal (e.g., within a few millimeters of the base of the cochlea) based on measurements from MRI scans (Koen 2020, incorporated herein by reference in its entirety). When the origin of these 38 tumors was localized to the outer, middle, or inner third of the length of the internal auditory canal, 60% occurred within the outer third (adjacent to the inner ear) (Koen 2020, incorporated herein by reference in its entirety); when the distribution of these tumors was analyzed as a function of percent internal auditory canal length (normalized for the moderate interindividual variability in internal auditory canal length [mm]), approximately 85% of the area under the curve was within the outer half of the internal auditory canal (adjacent to the inner ear) (Koen 2020, incorporated herein by reference in its entirety). [Figure 3] Schematic diagram of the inner ear showing perilymph fluid communication between the left vestibular system and the right cochlea (scala tympani, scala vestibuli). Perilymph is shown in light purple, as shown in A-B of U.S. Provisional Patent Application No. 63 / 152,832 (the entire contents of which are incorporated herein by reference). A is a schematic diagram of a spiral cochlea. The number of cochlear turns shown is representative of the mouse inner ear. B is a schematic diagram showing a cross section of the cochlea. In this schematic, the scala tympani and scala vestibuli are filled with perilymph, and the scala media is filled with endolymph (Talaei 2019, incorporated herein by reference in its entirety). [Figure 4]Schematic diagrams of the administration methods described herein. A includes an image of a delivery device described herein (Appendix A, incorporated herein by reference in its entirety). The illustrated delivery device is for administering infusion fluid into the cochlea through the round window membrane and includes a stopper to guide insertion depth. The stopper is shown in green in Figure 4A of U.S. Provisional Patent Application No. 63 / 152,832, the entire contents of which are incorporated herein by reference. B includes an image showing the expected flow of infusion fluid from the delivery device within the oval window, from the scala tympani to the scala vestibuli (via a communication at the helicotrema at the cochlear apex) and out of the cochlea through a vent located at the stapes footplate (Talei 2019, incorporated herein by reference in its entirety). [Figure 5] A is a schematic diagram of a simplified endogenous AAV construct. B is a schematic diagram of a simplified recombinant AAV (rAAV) construct. [Figure 6A] 1A-1C are a series of schematic diagrams of exemplary rAAV constructs described herein. An exemplary rAAV-anti-VEGF construct includes, among other things, sequences encoding an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain separated by a sequence encoding a self-cleaving peptide. Such constructs are referred to herein as "VH / VL constructs" or "rAAV-VH / VL constructs." Exemplary rAAV-VH / VL constructs include rAAV-ranibizumab and rAAV-ranibizumab-PC, which encode ranibizumab. [Figure 6B]1A-1C are a series of schematic diagrams of exemplary rAAV constructs described herein. An exemplary rAAV-anti-VEGF construct includes, among other things, an immunoglobulin heavy chain variable domain, an optional immunoglobulin heavy chain constant domain, an immunoglobulin light chain variable domain, an optional immunoglobulin light chain variable domain, and a sequence encoding green fluorescent protein (GFP). Each of these components may be separated by a sequence encoding a self-cleaving peptide. Such a construct may be referred to as an "ABGFP construct" or "rAAV-ABGFP construct." Exemplary rAAV-ABGFPs include rAAV-ranibizumab-GFP and rAAV-bevacizumab-GFP, which are rAAV-ABGFPs encoding ranibizumab or bevacizumab, respectively. [Figure 6C] 1A-1C are a series of schematic diagrams of exemplary rAAV constructs described herein. An exemplary rAAV-anti-VEGF construct includes, among other things, sequences encoding an immunoglobulin heavy chain (comprising an immunoglobulin heavy chain variable domain and an immunoglobulin heavy chain constant domain) and an immunoglobulin light chain (comprising an immunoglobulin light chain variable domain and an immunoglobulin light chain constant domain), the chains separated by a sequence encoding a self-cleaving peptide. Such constructs are referred to herein as "AB constructs" or "rAAV-AB constructs." Exemplary rAAV-ABs include rAAV-bevacizumab and rAAV-bevacizumab-PC, which are rAAV-ABs encoding bevacizumab. [Figure 6D]

[0023] Figure 1 is a series of schematic diagrams of exemplary rAAV constructs described herein. These exemplary rAAV constructs include, among other things, sequences encoding VEGF receptor extracellular domain 1, VEGF receptor extracellular domain 2, and a portion of human immunoglobulin gamma (IgG) Fc. Such constructs may be referred to as "VEGF TRAP constructs" or "rAAV-TRAP constructs." Exemplary rAAV-TRAPs include rAAV-aflibercept and rAAV-aflibercept-PC, which are rAAV-TRAPs encoding aflibercept. [Figure 7]Figure 1 includes a Western blot showing HEK cell expression of different anti-VEGF proteins, ranibizumab and bevacizumab, using transfection or transduction with exemplary rAAV-anti-VEGF constructs described herein. Lanes are labeled along the top of the figure, with predicted protein sizes labeled on the left side of the figure. Lanes 2-8 contain reduced protein, and lanes 10-16 contain non-reduced protein. Lane 1: Prestained PageRuler™ protein ladder. Lane 2: Untransfected / negative control. Lane 3: Transfection with rAAV-bevacizumab-PC construct. Lane 4: Transfection with rAAV-ranibizumab-GFP construct. Lane 5: Transfection with rAAV-ranibizumab-PC construct. Lane 6: Transduction with rAAVAnc80-bevacizumab-PC particles at a multiplicity of infection (MOI) of 7.5 x 10. Lane 7: Transduction with rAAVAnc80-bevacizumab-PC particles at an MOI of 2.2 x 10. Lane 8: Transduction with rAAVAnc80-bevacizumab-PC particles at an MOI of 5.5 x 10. Lane 9: Prestained PageRuler™ protein ladder. Lane 10: Untransfected / negative control. Lane 11: Transfection with rAAV-bevacizumab-PC construct. Lane 12: Transfection with rAAV-ranibizumab-GFP construct. Lane 13: Transfection with rAAV-ranibizumab-PC construct. Lane 14: Transduction with rAAVAnc80-bevacizumab-PC particles at an MOI of 7.5 x 10. Lane 15: Transduction with rAAVAnc80-bevacizumab-PC particles at an MOI of 2.2 x 10. Lane 16: Transduction with rAAVAnc80-bevacizumab-PC particles at an MOI of 5.5 x 10. [Figure 8A]

[0023] Figure 1 is a series of graphs showing the affinity of certain anti-VEGF proteins described herein as measured on an Octet® HTX biosensor instrument using Octet® analysis software, Data Analysis HT10.0.

[0024] Figure 2 is a graph showing the affinity of a control mouse anti-human VEGF monoclonal antibody (anti-hVEGF MmAb) in buffer with recombinant human VEGF as the binder. In this assay, anti-hVEGF MmAb was prepared in CM at 100 μg / mL and then diluted to a final concentration of 10 μg / mL in 1× kinetics buffer. [Figure 8B]

[0023] Figure 1 is a series of graphs showing the affinity of certain anti-VEGF proteins described herein as measured by an Octet® HTX biosensor instrument using Octet® analysis software, Data Analysis HT 10.0.

[0024] Figure 2 is a graph showing the affinity of secreted proteins in culture medium from HEK cells transfected with the rAAV-ranibizumab-PC construct corresponding to SEQ ID NO: 90, using recombinant human VEGF as a binder. [Figure 8C] 1 is a series of graphs showing the affinity of certain anti-VEGF proteins described herein as measured by an Octet® HTX biosensor instrument using Octet® analysis software, Data Analysis HT 10.0. 2 is a graph showing the affinity of secreted proteins in culture medium from HEK cells transfected with the rAAV-bevacizumab-PC construct corresponding to SEQ ID NO: 93, using recombinant human VEGF as a binder. [Figure 8D] 1 is a series of graphs showing the affinity of certain anti-VEGF proteins described herein as measured by an Octet® HTX biosensor instrument using Octet® analysis software, Data Analysis HT 10.0. 2 is a graph showing the affinity of secreted proteins in control culture medium (CM) from HEK cells that were not transfected with recombinant human VEGF as a binder. [Figure 9]Graphical representation of the phylogeny and ancestral sequence reconstruction of the AAV evolutionary lineage. The dendrogram models the evolutionary path of AAV, with the early speciation of AAV4 and 5 serotypes paralleled by a single node, Anc80. Open circles with solid lines represent evolutionary intermediates reconstructed by ancestral sequence reconstruction. Open circles with dotted lines represent libraries of probabilistic sequence space around AAV Anc80 variants. Subclades have been collapsed for clarity (Zinn 2015, incorporated herein by reference in its entirety). [Figure 10] Schematic diagram of structural modeling of the AAVAnc80 capsid surface. Structural mapping of amino acid changes comparing AAV2 (left) and AAV8 (right) in the VP1 trimer visualizing the exterior (top) and interior (bottom) of the virion. AAVAnc80 has several different residues, and Anc80Lib has several ambiguous, and therefore dimorphic, residues (Zinn 2015, incorporated herein by reference in its entirety). As shown in Figure 10 of U.S. Provisional Patent Application No. 63 / 152,832 (incorporated herein by reference in its entirety), different residues are shown in blue, and ambiguous, dimorphic residues are shown in red. [Figure 11] Representative fluorescence images depict in vivo cochlear transduction of naturally occurring AAV serotypes and AAVAnc80 variants via round window membrane delivery in neonatal mice. Mice (P1) were injected with different AAV capsids (AAV1, AAV2, AAV8, AAV6 [not shown], and AAVAnc80) containing a construct encoding enhanced GFP (eGFP). Phalloidin labels actin, as shown in red in Figure 11 of U.S. Provisional Patent Application No. 63 / 152,832 (the entire contents of which are incorporated herein by reference). Quantification of eGFP-positive inner hair cells (IHCs) and outer hair cells (OHCs) demonstrated approximately 90-100% transduction efficiency from base to apex after delivery of rAAVAnc80 containing a construct encoding enhanced GFP (rAAVAnc80-eGFP) (Landegger 2017, incorporated herein by reference in its entirety). [Figure 12]Representative fluorescence images depict in vivo vestibular transduction of rAAVAnc80 particles via round window membrane delivery in neonatal mice. Mice (P1) were injected with AAVAnc80-eGFP and phalloidin-stained actin. eGFP is shown in green, and phalloidin is shown in red in Figure 12 of U.S. Provisional Patent Application No. 63 / 152,832, the contents of which are incorporated herein by reference in their entirety. Transduction was observed in both type I and type II hair cells of the utricle (Panel (A)) and cells of the semicircular canal ridge (Panel (B)) (Landegger 2017, incorporated herein by reference in its entirety). [Figure 13] Representative fluorescence images depicting in vivo cochlear transduction of rAAVAnc80 particles via posterior semicircular canal delivery in adult mice. Seven-week-old mice were injected with rAAVAnc80-eGFP particles. Panel (A) contains a low-magnification cross-section of the midmodiolus of the injected cochlea, showing eGFP signal in the IHCs (labeled (I) in panel A), OHCs (labeled (O) in panel B), the spiral lamina (labeled (SL) in panel B), Reissner's membrane (labeled (RM) in panel B), and the spiral ganglion (labeled (SG) in panel B). Panels (B1) and (B2) contain high-magnification views of the organ of Corti in the apical (panel (B1)) and intermediate (panel (B2)) regions of the cochlea. Quantification of eGFP-positive cells showed that approximately 100% of the IHCs were transduced, whereas OHC transduction decreased from the apex to the base. Panel (C) is a low-magnification view showing that eGFP signal was detected in a subset of cells (neurons and satellite glial cells) in the spiral ganglion (Suzuki 2017, incorporated herein by reference in its entirety). A color image of the panel provided in this figure is shown in Figure 13 of U.S. Provisional Patent Application No. 63 / 152,832, incorporated herein by reference in its entirety. [Figure 14]Representative fluorescence images depicting in vivo vestibular transduction of rAAVAnc80-eGFP via posterior semicircular canal delivery in adult mice. Seven-week-old mice were injected with rAAVAnc80-eGFP. Panels (A1) and (A2) contain low-magnification cross-sections of the vestibule, demonstrating eGFP signal in both the utricle and saccule. Panels (B) and (C) contain high-magnification cross-sections of vestibular end organs (panel (B): utricle; panel (C): crista ampulla) demonstrating eGFP expression in supporting cells and hair cells. Open arrows indicate examples of transduced supporting cells (hair cells not shown) (Suzuki 2017, incorporated herein by reference in its entirety). A color image of the panel provided in this figure is shown in Figure 14 of U.S. Provisional Patent Application No. 63 / 152,832, incorporated herein by reference in its entirety. [Figure 15] Representative fluorescence images are included to demonstrate in vivo cochlear and vestibular transduction of naturally occurring AAV2 serotypes compared with the rAAVAnc80 variant via round window membrane delivery with auditory canal fenestration in adult mice. Mice (4 weeks old) were injected with different AAV particles encoding eGFP (AAV2 and rAAVAnc80 are shown; AAV1, AAV8, and AAV9 are not shown). Compared to AAV2, rAAVAnc80-mediated transduction demonstrated comparable IHC and OHC transduction rates (Panels (A1) and (A2)), but more widespread transduction in spiral ganglion cells (Panels (B1) and (B2)) and sacculus hair cells (Panels (C1) and (C2): entire; Panels (D1) and (D2): partial) (Omichi 2020, incorporated herein by reference in its entirety). A color image of the panel provided in this figure is shown in Figure 15 of US Provisional Patent Application No. 63 / 152,832, the entire contents of which are incorporated herein by reference. [Figure 16]Figure 1 shows a graphical representation of RNA expression in cochlear explants and secreted protein expression in cochlear explant medium after transduction of cochlear explants from WT neonatal (P2) mice with rAAVAnc80 particles containing the anti-VEGF protein disclosed herein (rAAVAnc80-anti-VEGF). Cells from WT mouse cochlear explants were transduced with rAAVAnc80-bevacizumab-PC particles (construct set forth in SEQ ID NO: 93) or rAAVAnc80-ranibizumab-PC particles (construct set forth in SEQ ID NO: 90) to drive the expression and secretion of mRNA encoding the anti-VEGF protein. (A) depicts RNA expression analysis, showing the expression of mRNA encoding ranibizumab and bevacizumab in cells from explants that received rAAVAnc80-ranibizumab-PC or rAAVAnc80-bevacizumab-PC, respectively. No expression was detected in explants that received vehicle. Results are shown as mean + SD. (B) Quantification of ranibizumab detected in the culture medium of explants fed various concentrations (1.4E10, 2.8E10, or 4.2E10 vg) of rAAVAnc80-ranibizumab-PC particles using the mesoscale discovery (MSD) method. Ranibizumab was detected in the culture medium of explants fed rAAVAnc80-ranibizumab-PC particles, but not in explants fed the vehicle. Open circles indicate ranibizumab concentrations in individual samples (n = 4 / group), and bars indicate the mean. [Figure 17]Representative low-magnification fluorescent staining images from the first study (referred to herein as "Study 1") are included, showing the inner ear of a CBA / CaJ mouse transduced with rAAVAnc80-anti-VEGF particles described herein. The images are representative cochlear photomicrographs of the middle turn of a microdissected cochlea after intracochlear administration of either rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea, rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea, or vehicle control. To detect anti-VEGF protein expression, the sensory epithelium was immunostained with primary antibodies against the hair cell marker phalloidin (also showing faint nonspecific labeling of the spiral lamina margin in these photomicrographs) and ranibizumab ("anti-Fab staining"). As shown in Figure 17 of U.S. Provisional Patent Application No. 63 / 152,832 (the entire contents of which are incorporated herein by reference), phalloidin is shown in red and ranibizumab in green. A human anti-ranibizumab antibody was used to detect the Fab segment of the protein shared by ranibizumab and bevacizumab. Clear labeling was detected by IHC and in supporting cells outside of OHCs. Background staining was detected in the nerve fiber region of the cochlea (e.g., nerve fiber labeling was evident in both particle- and vehicle-injected cochleae), preventing reliable expression assessment in this specific region. [Figure 18]Representative fluorescent staining images from Study 1 are included, depicting the inner ears of CBA / CaJ mice transduced with rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea or rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea, compared with uninjected and / or vehicle-injected controls. IHCs and OHCs were immunostained with anti-myosin VIIa antibody and imaged at 8, 16, and 32 kHz using a published cochleogram (Viberg and Canlon, 2004, incorporated herein by reference in its entirety). Images of the control uninjected ear were obtained from the ear contralateral to the control vehicle-injected ear. Scale bar = 20 μm. A color image of the panel provided in this figure is shown in Figure 18 of US Provisional Patent Application No. 63 / 152,832, the entire contents of which are incorporated herein by reference. [Figure 19] Representative confocal images from Study 1 are included, depicting transduced hair cells and neurons from a population of CBA / CaJ mice transduced with rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90). Panels (A) and (C) depict transduced cells immunostained with anti-Fab antibody. Panels (B) and (D) depict neuronal processes immunostained with anti-neurofilament 200. Both the vehicle-injected sample (Panels (A) and (B)) and the rAAVAnc80-ranibizumab-PC particle-injected sample (Panels (C) and (D)) have transduced neuronal processes in the medial sulcus region, but not in the organ of Corti. Only the rAAVAnc80-ranibizumab-PC particle-injected sample has transduced inner and outer hair cells. Scale bar = 20 μm. A color image of the panel provided in this figure is shown in Figure 19 of US Provisional Patent Application No. 63 / 152,832, the entire contents of which are incorporated herein by reference. [Figure 20]

[0049] Figure 18 is a graphical representation from Study 1, showing histograms of IHC and OHC counts for CBA / CaJ mice transduced with rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea or rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea, compared to uninjected and / or vehicle-injected controls. A representative population of the data set utilized in this analysis is shown in Figure 18. A represents OHC counts for uninjected controls, vehicle-injected controls, and test articles. B represents IHC counts for uninjected controls, vehicle-injected controls, and test articles. Both A and B counts were quantified and graphed as a function of treatment group and frequency range. N for each group was either 9 or 10, and data are presented as the mean + / - standard error of the mean (SEM). Quantification of the control non-injected ear was performed from the ear contralateral to the control vehicle-injected ear. *p<0.05, **p<0.01, and ***p<0.001 compared to the non-injected ear, excluding brackets. p-values ​​were determined by two-way ANOVA followed by post-hoc Tukey's test. [Figure 21]Figure 18 shows graphical representations of hair cell (HC) and non-hair cell (non-HC) transduction frequencies. A representative population of the data set utilized in this analysis is shown in Figure 18. A represents hair cell (HC) counts for uninjected controls, vehicle-injected controls, and test articles. B represents non-hair cell (non-HC) counts for uninjected controls, vehicle-injected controls, and test articles. Data are from Study 1 and are representative of populations of CBA / CaJ mice transduced with rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea or rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea. Quantitative values ​​of transduced (Fab+) hair cells (A) and non-hair cells (B) are graphed as a function of treatment group and frequency range, combining data from both genders. N for each group was either 9 or 10, and data are presented as the mean + / - standard error of the mean (SEM). Quantitative values ​​for the control non-injected ear were obtained from the ear contralateral to the control vehicle-injected ear. Statistical comparisons were not performed due to variability between samples. [Figure 22] Representative high-magnification images from Study 2 are included, showing fluorescent staining of cochlear transduction with rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10vg / cochlea. Photomicrographs (63x) of cochleae from three regions of the injected cochlea are shown, demonstrating anti-ranibizumab (mAb) labeling. Each column represents a maximum projection of a confocal image stack acquired from an injected mouse, and each row represents the apical (8 kHz), mid-region (16 kHz), and basal (32 kHz) frequency regions of the cochlea. The primary cell type positive for immunostaining is listed to the right of each row. Significant background staining was detected in the nerve fiber regions of the cochlea (e.g., nerve fiber labeling was evident in both injected and non-injected cochleae [non-injected cochleae not shown]), preventing reliable expression assessment in this specific region. A color image of the panel provided in this figure, with anti-ranibizumab labeled in green, is shown in Figure 22 of U.S. Provisional Patent Application No. 63 / 152,832, the entire contents of which are incorporated herein by reference. [Figure 23] 1 is a graphical representation of the detection and quantification of secreted anti-VEGF protein in serum from Study 2, following intracochlear delivery of rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea. Higher concentrations of anti-VEGF protein were detected using electrochemiluminescence in the serum of mice injected with rAAVAnc80-ranibizumab-PC particles compared to the serum of mice injected with vehicle. Open circles indicate anti-VEGF protein concentrations in individual samples (vehicle, n=7; ranibizumab, n=9), and bars indicate the mean. [Figure 24] Figure 1 is a graphical representation of the detection and quantification of secreted anti-VEGF protein in mouse cerebrospinal fluid (CSF) from Study 1 after intracochlear delivery of rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea or rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea. Anti-VEGF protein (ranibizumab or bevacizumab) was detected using MSD in the CSF of mice administered rAAVAnc80-anti-VEGF particles but not in the CSF of mice administered vehicle. Open circles indicate anti-VEGF protein concentrations in individual samples (vehicle, n = 10; ranibizumab, n = 9; bevacizumab, n = 4), and bars represent the mean. [Figure 25]

[0033] Figure 11 is a graphical representation of auditory brainstem response (ABR) thresholds in Study 1 mice before and after intracochlear administration of rAAVAnc80-ranibizumab-PC (construct set forth in SEQ ID NO: 90) particles at 1.4E10 vg / cochlea or rAAVAnc80-bevacizumab-PC (construct set forth in SEQ ID NO: 93) particles at 1.2E10 vg / cochlea. Mean ABR thresholds were elevated in all groups after intracochlear surgery, including vehicle-injected and rAAVAnc80 particle-injected mice. Mean ABR thresholds were elevated in rAAVAnc80 particle-injected and vehicle-injected ears compared to baseline ABRs measured before surgery. Error bars represent standard deviation. [Figure 26]Figure 1 shows graphical representations of distortion product otoacoustic emissions (DPOAEs) and ABR thresholds for Study 2 mice after intracochlear delivery of rAAVAnc80-ranibizumab-PC particles (construct set forth in SEQ ID NO: 90). Cochlear and hearing function (A: DPOAEs, B: ABRs) showed normal mean thresholds in mice administered exemplary rAAVAnc80-ranibizumab-PC particles compared to uninjected mice. * indicates that one mouse administered rAAVAnc80-ranibizumab-PC particles died during functional testing; therefore, only DPOAEs and chirp-evoked ABRs were measured in this animal prior to death; therefore, N=2 for ABRs at 8, 16, and 32 kHz. Error bars represent standard deviation. [Figure 27]Figure 1 provides a schematic illustration and graphical model showing anti-VEGF protein concentration (modeled using measured concentrations of NHPs fed rAAVAnc80-ranibizumab particles (construct set forth in SEQ ID NO: 91)) relative to distance to VS. (A) Schematic of the computational modeling approach: Three-dimensional diffusion of a given anti-VEGF protein source within a 90 μL sphere is depicted relative to the distance (mm) from the surface of the sphere, e.g., the scala tympani / fundus border. Distances from the fundus to the medial and lateral borders of the tumor (<5 mm wide) were estimated from data obtained in Koen 2020 (most recently Figure 2), which is incorporated herein by reference in its entirety. (B) Represents a conservative modeling approach showing that perilymphatic anti-VEGF protein concentration decreases with diffusion distance but remains within the reported biologically active range (region between 10-100% on the Y-axis and 0-11 mm on the X-axis) near the tumor within the internal auditory canal. The estimated anti-VEGF protein concentrations vary depending on the choice of diffusion coefficient and are represented as a range (shaded area with solid dots) based on the three reported diffusion coefficients. A color image of B, including the shading, is provided in Figure 27B of U.S. Provisional Patent Application No. 63 / 152,832 (the entire contents of which are incorporated herein by reference). The biologically active range, and predicted therapeutically relevant range, is estimated to be approximately 28 ng / mL; the concentration required to inhibit 50% of VEGF-A biological activity in an in vitro cell proliferation assay is 11-27 ng / mL, as described in Genentech 2017 (the entire contents of which are incorporated herein by reference). [Figure 28]Inner hair cell (IHC) transduction events are shown for seven NHPs (six unilateral, one bilateral) that received AAVAnc80-eGFP intracochlearly (RWM) with stapedial base plate aerators and two NHPs (bilateral) that received AAVAnc80-eGFP intracochlearly (RWM) without stapedial base plate aerators. Cochleae were analyzed for eGFP expression in IHCs after a 3-week survival period. In macaque IHCs, approximately 80%–100% transduction efficiency was achieved at high doses (seen in M6–M9 of NHPs), while an apical-to-basal gradient of eGFP expression was observed at lower doses (seen in M1–M3 of NHPs). In animals operated without stapedial base plate aerators, only sporadic transduction was observed in the apical 75% of the cochlea. [Figure 29] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 30] FIG. 1 illustrates a side view of a curved needle subassembly according to aspects of the present disclosure. [Figure 31] 1 shows a perspective view of a device for delivering fluid to the inner ear according to an aspect of the present disclosure. [Figure 32] FIG. 10 shows a perspective view of a curved needle subassembly coupled to a distal end of a device, according to an aspect of the present disclosure. [Figure 33] FIG. 1 is a schematic diagram of the experimental setup used in Example 12. [Figure 34] 34A-34C are a series of graphical representations showing mean ABR threshold shifts after intracochlear delivery of rAAVAnc80-ranibizumab particles in NHPs. ABR threshold shifts (compared to baseline ABRs obtained in the same ear before intracochlear injection) are shown for 1, 2, 3, and 6 months after intracochlear injection of either vehicle (A) or rAAVAnc80-ranibizumab particles at doses of 6.0E10 vg / cochlea (B) or 3.0E11 vg / cochlea (FIG. 34C). Group means (±SD) at each time point reflect bilateral measurements for each NHP in the study. [Figure 35] FIG. 1 is a schematic diagram of the experimental setup used in Example 14. [Figure 36]1 is a graph showing anti-VEGF concentrations in the perilymph of NHPs for each dose of AAV-anti-VEGF. Each dose group had 3 NHPs, and the vehicle group had 2 NHPs. [Figure 37] Figure 2 shows a computational model of estimated anti-VEGF concentrations (y-axis) at various distances from the floor of the internal auditory canal (IAC). The reported biologically active threshold concentrations are similar to those shown in Figure 27. The red-shaded areas represent locations where early-stage vestibular schwannomas (VS) have been reported (Koen et al., 2020). The colored lines indicate the estimated amount of anti-VEGF protein from each of the modeled doses. All doses of AAV anti-VEGF are predicted to produce amounts of anti-VEGF protein above the biologically active threshold in locations where VS tumors are typically found. [Figure 38] FIG. 1 is a schematic diagram of the experimental setup used in Example 15. [Figure 39] 1 shows a method for quantifying protein expression in cochlear micrographs. [Figure 40] Representative images showing cochlear anti-VEGF protein expression at three frequency locations (0.25 kHz, 4 kHz, and 22.6 kHz) at all doses tested. A shows anti-VEGF protein expression at 1 month. B shows anti-VEGF protein expression at 6 months. C shows anti-VEGF, phalloidin, and Hoechst staining in specialized hair cells, including inner hair cells, pillar cells, outer hair cells, Hensen cells, and Clausius cells. D is a high-resolution magnification of a portion of the image in C. [Figure 41] 1 is a graph showing anti-VEGF expression in NHP cochleae at 63x photomicrographs. Data shows anti-VEGF expression at 1, 3, and 6 months in animals treated with either vehicle control or 1x, 2x, and 4x doses of AAV anti-VEGF. For each group of three bars on the graph, the first bar represents data at 1 month, the second bar represents data at 3 months, and the last bar represents data at 6 months. Anti-VEGF protein expression relative to vehicle control is shown, summed across all cochlear frequency locations. [Figure 42] Graphs depicting overall hair cell survival in NHP cochleae across all study groups at three frequency locations: 0.25 kHz, 4 kHz, and 22.6 kHz. Animals were treated with either vehicle control or 1x, 2x, or 4x doses of AAV anti-VEGF. For each frequency location in A and B, the first, fourth, seventh, and tenth bars represent data at 1 month; the second, fifth, eighth, and eleventh bars represent data at 3 months; and the third, sixth, ninth, and twelfth bars represent data at 6 months. A shows inner hair cell survival, and B shows outer hair cell survival. DETAILED DESCRIPTION OF THE INVENTION

[0137] In certain embodiments, the present disclosure relates to rAAV-anti-VEGF particles intended for the treatment of subjects with otic diseases associated with neovascularization. In certain embodiments, such otic diseases associated with neovascularization are vestibular schwannomas (VS), or benign tumors that form in the cells surrounding the vestibulocochlear nerve within the internal auditory canal. In certain embodiments, common symptoms associated with early VS include hearing loss, tinnitus, and dizziness, and there is great concern that continued tumor growth can lead to compression of the brainstem, leading to more serious morbidity and, in rare cases, death.

[0138] In certain embodiments, the present disclosure relates to rAAV-anti-VEGF particles intended for treating a subject with an inner ear disorder, such as those described herein. In some embodiments, the inner ear disorder comprises vestibular schwannoma or neurofibromatosis type II. In some embodiments, the inner ear disorder is or comprises vestibular schwannoma. In some embodiments, the inner ear disorder is or comprises neurofibromatosis type II.

[0139] In some embodiments, the present disclosure relates to rAAV-anti-VEGF particles intended for the treatment of subjects with vestibular schwannomas.

[0140] In certain cases, treatment with vascular endothelial growth factor (VEGF) inhibitors offers the potential to attenuate aggressive VS tumors rather than using invasive alternatives such as surgical resection and / or radiation therapy, which are the current standard of care. In certain embodiments, clinical data supports the use of systemically administered VEGF inhibitors in patients with VS tumors in terms of efficacy, but long-term systemic administration of VEGF inhibitors carries significant safety concerns. In certain embodiments, rAAV-anti-VEGF is designed and intended to treat individuals with VS by gene transfer to the inner ear to promote local expression and secretion of anti-VEGF proteins. In certain embodiments, the goal is to provide local exposure of therapeutic VEGF inhibitors (e.g., anti-VEGF proteins, e.g., bevacizumab, ranibizumab, and / or aflibercept) at the site of VS, thereby limiting systemic exposure and minimizing potential side effects associated with systemic administration.

[0141] In certain embodiments, cochlear and vestibular cells of the inner ear are transduced with rAAV-anti-VEGF and secrete the anti-VEGF protein into the perilymph, a cochlear fluid that is diffusively continuous with the interstitium and perineural space of the vestibulocochlear nerve where the VS tumor is located. In certain embodiments, the lack of a barrier to diffusion along the internal auditory canal provides a potential route for therapeutic anti-VEGF proteins expressed in the perilymph to reach their intended VS targets in the neural interstitium. In certain embodiments, transduced cells in the modiolus are positioned such that the desired anti-VEGF protein is secreted directly into the interstitial fluid of the nerve.

[0142] In certain embodiments, increasing doses of a composition described herein (e.g., comprising rAAV-anti-VEGF) are administered via unilateral intracochlear injection to an individual (e.g., a mammal, e.g., a human patient in need thereof) with unilateral (e.g., sporadic) progressive VS. In certain embodiments, the growth rate of these tumors is variable, and some VS tumors do not progress. In certain embodiments, individuals (e.g., mammals, e.g., humans) may be limited to individuals with tumors that show clear evidence of progression, excluding individuals with evidence of stable tumors on serial imaging evaluations. In certain embodiments, the growth rate of these tumors is variable, and some VS tumors do not progress. In certain embodiments, individuals (e.g., a mammal, e.g., a human patient in need thereof) may be limited to individuals with tumors that show clear evidence of non-progression, excluding individuals with evidence of increasing tumor volume on serial imaging evaluations.

[0143] In certain embodiments, individuals (e.g., mammals, e.g., human patients) with larger tumor(s) (e.g., tumors that are more likely to compress the brainstem) are excluded from treatment with the compositions described herein because, in some embodiments, these individuals are at higher risk for potentially life-threatening tumor-related complications that may be avoided with the current standard of care of surgical resection and radiation therapy. In certain embodiments, individuals (e.g., mammals, e.g., human patients) with larger tumors (e.g., tumors that are more likely to compress the brainstem) are expressly candidates for treatment with the compositions described herein because, in some embodiments, these individuals are at higher risk for potentially life-threatening tumor-related complications that may be avoided with the current standard of care of surgical resection and radiation therapy in a manner that is less invasive and / or more or equally effective than the current standard of care of surgical resection and radiation therapy.

[0144] In certain embodiments, individuals (e.g., mammals, e.g., humans) with growing tumors whose size is unlikely to affect the brainstem may benefit most from intervention with the compositions described herein (e.g., rAAV-anti-VEGF therapy), while remaining candidates for future surgical resection and / or radiation, if necessary. In certain embodiments, individuals (e.g., mammals, e.g., humans) with growing tumors whose size is likely to affect the brainstem may benefit most from intervention with the compositions described herein (e.g., rAAV-anti-VEGF therapy), while remaining candidates for future surgical resection and / or radiation, if necessary.

[0145] In certain embodiments, provided herein are methods that include introducing into the inner ear of an individual, e.g., a mammal, e.g., a human, an effective amount, e.g., a therapeutically effective amount, of rAAV particles that include a nucleotide sequence of a construct 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 (e.g., a Fab or scFv) operably linked to a signal peptide.

[0146] In certain embodiments, the compositions described herein (e.g., rAAV-anti-VEGF) can be administered in an operating room under sterile conditions supervised by an otic surgeon.

[0147] In some embodiments, provided herein are methods for increasing the level of an antibody in the inner ear and / or inner ear canal of an individual, e.g., a mammal, e.g., a human, in need thereof, comprising introducing into the inner ear of the mammal an effective amount, e.g., a therapeutically effective amount, of rAAV particles comprising nucleotide sequences 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 (e.g., a Fab or scFv) operably 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 inner ear of the individual, e.g., the mammal, e.g., the human.

[0148] In some embodiments, the disclosure provides a method for treating an inner ear disorder in an individual, e.g., a mammal, e.g., a human, in need thereof, comprising introducing into the inner ear of the mammal an effective amount, e.g., a therapeutically effective amount, of rAAV particles 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 introduction results in treatment of the inner ear disorder in the mammal.

[0149] In some embodiments, provided herein are methods for reducing VEGF activity in the inner ear of an individual, e.g., a mammal, e.g., a human, in need thereof, comprising introducing into the inner ear of the mammal an effective amount, e.g., a therapeutically effective amount, of rAAV particles comprising nucleotide sequences 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 (e.g., a 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, wherein the introduction results in reduced VEGF activity in the inner ear of the individual, e.g., a mammal or human.

[0150] In some embodiments, provided herein are methods for treating an otic disease associated with neovascularization, e.g., VS, or neurofibromatosis type II, in the inner ear of an individual (e.g., a mammal, e.g., a human), comprising introducing into the inner ear of the individual an effective amount (e.g., a therapeutically effective amount) of rAAV particles comprising nucleotide sequences 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 (e.g., a 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, wherein the introduction results in treatment of the otic disease associated with neovascularization, e.g., VS, in the inner ear of the individual.

[0151] In some embodiments, provided herein are methods comprising introducing into the inner ear of an individual (e.g., a mammal, e.g., a human) an effective amount (e.g., a therapeutically effective amount) of rAAV particles comprising a nucleotide sequence encoding a soluble VEGF receptor operably linked to a signal peptide.

[0152] In some embodiments, the present disclosure provides a method for increasing the level of soluble VEGF receptor in the inner ear of an individual (e.g., a mammal, e.g., a human) in need thereof, comprising introducing into the inner ear of the individual an effective amount (e.g., a therapeutically effective amount) of an rAAV particle 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 soluble VEGF receptor in the inner ear of the individual.

[0153] In some embodiments, provided herein are methods for treating an inner ear disorder in an individual (e.g., a mammal, e.g., a human) in need thereof, comprising introducing into the inner ear of the individual an effective amount (e.g., a therapeutically effective amount) of an rAAV particle comprising a nucleotide sequence encoding at least a portion of a soluble VEGF receptor operably linked to a signal peptide, wherein the introduction results in treatment of the inner ear disorder in the individual.

[0154] In some embodiments, provided herein are methods for reducing VEGF activity in the inner ear of an individual (e.g., a mammal, e.g., a human) in need thereof, comprising introducing into the inner ear of the individual an effective amount (e.g., a therapeutically effective amount) of an rAAV particle comprising a nucleotide sequence encoding at least a portion of a soluble VEGF receptor operably linked to a signal peptide, wherein the introduction results in a reduction of VEGF activity in the inner ear of the individual.

[0155] In some embodiments, provided herein are methods for treating an otic disease associated with neovascularization, VS, or neurofibromatosis type II in the inner ear (e.g., including the internal auditory canal) of an individual (e.g., a mammal, e.g., a human), comprising introducing into the inner ear of the individual an effective amount (e.g., a therapeutically effective amount) of an rAAV particle comprising a nucleotide sequence encoding at least a portion of a VEGF receptor operably linked to a signal peptide, wherein the introduction results in treatment of an otic disease associated with neovascularization, VS, or neurofibromatosis type II in the inner ear of the individual.

[0156] In other embodiments, the present disclosure also provides kits comprising any of the rAAV particles described herein.

[0157] Additional non-limiting aspects of compositions, kits, and methods are described herein, which can be used in any combination without limitation.

[0158] The present disclosure provides gene therapy methods, e.g., methods using the compositions disclosed herein, for treating individuals with neovascularization-related otic disorders, e.g., VS, by locally expressing secreted anti-VEGF protein in cells of the cochlea and vestibular system that are adjacent to and diffusely connected to the VS tumor environment in the internal auditory canal. In some embodiments, the methods involve gene transfer into the cochlea using rAAV particles containing a construct containing complementary DNA (cDNA) encoding the anti-VEGF protein (rAAV-anti-VEGF). Without wishing to be bound by theory, in some embodiments, it is believed that cochlear and vestibular cells of the inner ear transduced with rAAV-anti-VEGF (e.g., rAAVAnc80-anti-VEGF) can secrete anti-VEGF protein into the perilymph and the interstitium and perineural space of the vestibulocochlear nerve (composed of the superior vestibular nerve, the inferior vestibular nerve, and the cochlear nerve). The majority of VS tumors arise in the outer third of the internal auditory canal, which houses the vestibulocochlear nerve, the portion closest to the cochlea (Figures 1-3). Due to the lack of a barrier to diffusion along this internal auditory canal, the cochlear nerve is bathed in a fluid continuum, with perilymph at its lateral end and CSF at its medial end. Thus, diffusion from the perilymph to the neural interstitium provides a potential pathway for therapeutic anti-VEGF proteins expressed in the perilymph to reach their intended VS targets. While the exact mechanisms by which VEGF inhibitors result in tumor control and regression are not fully understood, in some embodiments, mechanisms include reduced vascular permeability and / or aberrant angiogenesis due to inhibition of endothelial cell proliferation, as well as normalization of tumor vasculature (Brastianos 2009, incorporated herein by reference in its entirety).

[0159] To date, there are no known gene transfer clinical trials involving delivery to the inner ear using rAAV particles in humans; however, a clinical trial evaluating adenovirus particles containing a construct encoding the complementary DNA (cDNA) of human atonal transcription factor (Hath1) for the treatment of severe-to-profound hearing loss was initiated in 2014 and completed in 2019 (Clinicaltrials.gov 2020a [NCT02132130], incorporated herein by reference in its entirety).

[0160] In some embodiments, the delivery approaches disclosed herein include synthetic AAV capsids (e.g., AAVAnc80) for transduction of inner ear cells and / or devices for targeted delivery directly to the cochlea.

[0161] The current standard of care for patients with VS includes several approaches and several therapeutic goals (Doherty 2006; Kaul 2018, each of which is incorporated herein by reference in its entirety). Treatments include imaging / surveillance, surgery, and radiation therapy. While therapeutic goals may include hearing preservation, patients often present with complete or partial hearing loss, and tumor size and growth may necessitate more aggressive interventions that accept hearing loss as an inevitable consequence of treatment. Both surgery and radiation have side effects, but importantly, neither is associated with improved quality-of-life measures compared to observation alone (Carlson 2015, incorporated herein by reference in its entirety). Therefore, there is a clear need for less invasive treatments that can mitigate the impact of tumor growth.

[0162] The clinical efficacy of systemically administered bevacizumab to control tumor growth and associated hearing loss in patients with NF2 is not without significant risks, and the continuous intravenous infusions required to maintain therapeutic efficacy also increase the risk of serious complications. A meta-analysis of studies using bevacizumab for NF2 found a pooled incidence of serious toxicity (grade 3 or 4) of 17% based on meta-analysis of five clinical trial populations including 125 patients (Lu 2019, incorporated herein by reference in its entirety). Hypertension, proteinuria, elevated liver enzymes, arterial thromboembolic events (ATEs), venous thromboembolic events, bleeding, and surgical and wound healing complications have all been associated with high-dose bevacizumab therapy (Chen 2009; Hanna 2019, each of which is incorporated herein by reference in its entirety). Specifically disclosed herein are alternative approaches to treating VS that, for example, do not require high levels of circulating anti-VEGF proteins (e.g., bevacizumab) and, in some embodiments, can lower the patient's risk for events associated with systemic exposure to therapeutic molecules.

[0163] In some embodiments, the use of an intracochlear route of administration to deliver rAAV-anti-VEGF to the inner ear, transduction of inner ear cells, and subsequent expression and / or secretion of anti-VEGF proteins (e.g., bevacizumab, ranibizumab, and / or aflibercept) can generate a sustained depot of therapeutic agent in close proximity to the tumor. In some embodiments, cochlear and vestibular cells of the inner ear transduced with rAAV-anti-VEGF can secrete anti-VEGF proteins into nearby chambers (e.g., perilymph and cells of the modiolus, e.g., spiral ganglion neurons and satellite glial cells) and / or directly into the interstitial fluid of the cochlear nerve. In some embodiments, the lack of a barrier to diffusion along the internal auditory canal leaves the cochlear nerve bathed in a fluid continuum with perilymph at its lateral end (where the majority of VS tumors arise closest to the cochlea [Figures 1-3]) and CSF at its medial end. In some embodiments, diffusion from the perilymph to the neural interstitium provides a potential route for therapeutic anti-VEGF proteins expressed in the perilymph to reach their intended VS targets.

[0164] In some embodiments, the use of an intracochlear route of administration to deliver rAAV-anti-VEGF to the inner ear, transduction of inner ear cells, and subsequent expression and / or secretion of anti-VEGF protein (e.g., bevacizumab, ranibizumab, and / or aflibercept) results in therapeutically irrelevant or undetectable levels of anti-VEGF protein in non-cochlear tissues or fluid compartments. In some embodiments, the anti-VEGF protein is present at therapeutically irrelevant or undetectable levels in serum, CSF, liver, spleen, brainstem, auditory cortex, mandibular lymph nodes, or a combination thereof.

[0165] In some embodiments, local exposure to anti-VEGF proteins at the tumor surface can control tumor growth despite different diffusion pathways for accessing and neutralizing VEGF compared to extravasation of anti-VEGF proteins from the bloodstream. For example, Lichtenbeld et al., 1999 (incorporated herein by reference in its entirety) applied anti-VEGF proteins locally to tumors in mice and observed a significant reduction in vascular permeability, notably at a 20-fold lower dose than systemic administration, which achieved a similar reduction in vascular permeability in mice (Yuan 1996, incorporated herein by reference in its entirety).

[0166] Without wishing to be bound by theory, it is believed that in some embodiments, low-level but sustained exposure to anti-VEGF proteins in the fluid surrounding the VS may stabilize and / or reduce tumor growth through various mechanisms, such as, for example, reducing tumor vascular permeability and normalizing tumor vasculature. In some embodiments, by minimizing circulating levels of anti-VEGF proteins, local delivery of rAAV-anti-VEGF to the ear and the resulting exposure of anti-VEGF proteins in the tumor microenvironment may provide durable therapeutic benefits while minimizing the risk of adverse events associated with systemic administration of anti-VEGF proteins.

[0167] Vestibular schwannoma (VS) Vestibular neuromas (VSs) are benign, usually slow-growing tumors resulting from neoplasia of Schwann cells covering the vestibular-cochlear nerve (also known as the eighth cranial nerve). VSs often arise in the superior or inferior vestibular branches of the vestibular-cochlear nerve, a nerve responsible for transmitting sound and balance information from the inner ear to the brain (see Figures 1-3, for example). These tumors often arise within the internal auditory canal (e.g., immediately adjacent to the inner ear) and may extend into the cerebellopontine angle. Tumors can occur as sporadic unilateral tumors or, more rarely, as bilateral tumors (commonly occurring in the condition neurofibromatosis type 2 (NF2)). VSs are most commonly found along the vestibulocochlear nerve (see Figures 1-3, for example). Small intracanalicular tumors (less than 5 mm wide) can occur, for example, within the outer third of the internal auditory canal, closest to the cochlea (Koen 2020, incorporated herein by reference in its entirety).

[0168] Common symptoms associated with VS include hearing loss, tinnitus, and dizziness. As the tumor continues to grow and extends outside the internal auditory canal and into the cranial cavity, it can compress the brainstem, posing a significant concern for more severe morbidity and survival. The current standard of care is observation only for small or non-growing tumors, while surgical resection and / or radiation therapy are indicated for large and / or aggressive tumors.

[0169] In certain embodiments, the compositions and methods described herein can reduce and / or ameliorate symptoms associated with VS and / or current standard therapies for treating VS, which may include, but are not limited to, hearing loss, hair cell degeneration, altered biochemical milieu of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear opening obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, and / or death.

[0170] It is noteworthy that an increasing number of VS tumors are identified in asymptomatic patients undergoing imaging for other indications (Reznitsky 2019, incorporated herein by reference in its entirety). In some cases, VS symptoms may result from compression of the cochlear nerve and invasion of the vestibular branch of the vestibulocochlear nerve (cranial nerve VIII). The facial nerve is often stretched and dilated by the tumor, but facial paralysis is generally uncommon. In some cases, compression of the nearby trigeminal nerve, which transmits facial sensory information to the brain, can result in facial paresthesia. Despite histological benignity, in some cases, large tumors can compress the brainstem, leading to hydrocephalus, cerebellar herniation, and, rarely, death.

[0171] VS-induced hearing loss is thought to result from, for example, cochlear nerve compression and / or cochlear dysfunction, as supported by the presence of cochlear pathology in most cases. Mechanisms of VS-induced hearing loss are hypothesized to include, for example, hair cell degeneration, alterations in the biochemical environment of the inner ear fluid (e.g., toxic cytokines from tumors), elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear orifice obstruction, intralabyrinthic hemorrhage, and / or disruption of the cochlear vascular supply (Roosli 2012; Dilwali 2015; Remenschneider 2017, each of which is incorporated herein by reference in its entirety).

[0172] Current treatments may reduce the risks associated with tumor growth but are not associated with stabilization of hearing loss or tinnitus and often result in side effects such as unilateral residual hearing loss, intractable headaches, and facial nerve damage (Pedrosa 1994; Sampath 1997; Sardhara 2020, each of which is incorporated herein by reference in its entirety). Although rare, radiation therapy also carries the additional risk of secondary malignancies (Kapurch 2016, incorporated herein by reference in its entirety). Estimates of hearing preservation with current treatment regimens for VS vary, but in a large retrospective comparison of surgical resections, Ansari reported that mean postoperative hearing loss ranged from 40.6 to 82.7%, depending on the surgical approach used and tumor size and location. These include surgical approaches such as the middle cranial fossa and retrosigmoid sinus approaches, which, depending on tumor size, may be preferred over translabyrinthine approaches, particularly to promote greater hearing preservation (Ansari 2012, incorporated herein by reference in its entirety). Guidelines from the American Society of Neurosurgeons recommend that patients considering radiosurgery, microsurgery, or observation be counseled to expect a 50–75% chance of hearing loss within 10 years, stating that each of these treatment options has only a moderately low (defined as >25–50%) chance of hearing preservation after 10 years (Carlson 2018, incorporated herein by reference in its entirety). Currently available standard treatments for VS do not offer the potential for hearing improvement.

[0173] Decision-making regarding the treatment of sporadic VS is complex. The presence of a tumor is usually confirmed by magnetic resonance imaging (MRI), and initial standard of care is based on symptom severity and tumor size. At the time of diagnosis, approximately 20-30% of cases are less than 1 cm, approximately 30% are 1-2 cm (inclusive), and the remaining cases (approximately 40-50%) are greater than 2 cm (Peris-Celda 2019, incorporated herein by reference in its entirety). With the increasing availability and utilization of MRI scanners, these tumors are generally detected earlier (smaller) and more often at an asymptomatic stage (Reznitsky 2019, incorporated herein by reference in its entirety). In certain embodiments, the present disclosure provides compositions and methods that may be particularly suitable for halting or slowing the growth and / or shrinking tumors less than 0.5 cm, less than 1 cm, less than 2 cm, less than 3 cm, less than 4 cm, or less than 5 cm.

[0174] In some cases, VSs exhibit variable and often unpredictable growth rates. The inherent behavior of such tumors is further complicated by variations in imaging modalities, tumor size estimates, and definitions of growth (Kondziolka 2012, incorporated herein by reference in its entirety). Overall tumor growth averages approximately 1 mm / year, but 30–60% of all tumors exhibit slow or no appreciable growth, and of those that do grow, the annual linear rate is 2–3 mm / year (Paldor 2016; Lees 2018, each of which is incorporated herein by reference in its entirety). In some patients with small tumors, treatment consists solely of MRI scans, with additional treatment considered only if the tumor shows measurable growth or if symptoms worsen (MacKeith 2013; Kirchmann 2017, each of which is incorporated herein by reference in its entirety). For some patients whose tumors continue to grow, thus significantly increasing the risk of sequelae such as brainstem compression, current treatment options include surgical resection, radiation (Golfinos 2016, incorporated herein by reference in its entirety), or some combination of these approaches. While these treatments may reduce the risks associated with tumor growth, they are not associated with long-term stabilization of hearing loss (Carlson 2018, incorporated herein by reference in its entirety) or improvement in tinnitus (Sardhara 2020, incorporated herein by reference in its entirety), and often result in side effects such as unilateral residual hearing loss, intractable headaches (Pedrosa 1994, incorporated herein by reference in its entirety), and facial nerve damage (Sampath 1997, incorporated herein by reference in its entirety). Currently, approximately half of all VS cases ultimately require surgical resection and / or radiation. In some embodiments, the present disclosure provides compositions and methods for reducing the risks associated with tumor growth, such as hearing loss, reduced speech understanding, tinnitus, reduced quality of life, brainstem compression, and / or death.In some embodiments, the present disclosure provides compositions and methods that reduce the associated risks of tumor growth, hearing loss, hair cell degeneration, altered biochemical milieu of the inner ear fluid, elevated intralabyrinthic proteins, endolymphatic hydrops, cochlear orifice obstruction, intralabyrinthic hemorrhage, disruption of the cochlear vascular supply, tinnitus, dizziness, intractable headache, facial nerve damage, trigeminal nerve damage, facial paralysis, facial paresthesia, hydrocephalus, cerebellar herniation, and / or death.

[0175] Despite modern improvements in both surgical and radiation therapy, in some embodiments, a conservative approach of observation is advocated; unless the tumor is growing, this remains the best current strategy for preserving residual hearing function and minimizing potential adverse effects from interventional treatments (MacKeith 2013; Kirchmann 2017; Torres Maldonado 2019, each of which is incorporated herein by reference in its entirety). In some embodiments, improved treatments may promote stasis and / or regression of VS, thus avoiding the need for more invasive approaches, including surgical resection or radiation therapy.

[0176] In the United States, the overall incidence of VS is estimated at 1.09–1.98 new cases per 100,000 population, with 3,300–6,300 patients diagnosed with VS annually in the United States (Kshettry 2015; Ostrom 2018, each of which is incorporated herein by reference in its entirety). The recent increase in reported incidence is attributed to a combination of an aging population and continuing improvements in imaging techniques. The current standard of care for patients with VS includes several approaches and several therapeutic objectives (Doherty 2006; Kaul 2018, each of which is incorporated herein by reference in its entirety). Treatments include imaging / surveillance, surgery, and radiation therapy. While therapeutic objectives may include hearing preservation, patients often present with complete or partial hearing loss, and tumor size and growth may necessitate more aggressive interventions, with hearing loss becoming an inevitable consequence of treatment. Both surgery and radiation have side effects, but importantly, neither is associated with improved quality of life measures compared to observation alone (Carlson 2015, incorporated herein by reference in its entirety), so there is a clear need for less invasive treatments that can mitigate the impact of tumor growth.

[0177] Many patients with growing tumors, or tumors that are compressing or may eventually compress the brainstem, cannot tolerate observation alone and may require surgical resection or radiation to prevent neurological function from being affected. In some cases, it is not possible to remove the entire tumor without causing some damage to the facial nerve. In these cases, a subtotal resection is often performed to preserve facial nerve function, but this treatment leaves the tumor with the potential to continue growing. In some embodiments, the compositions and methods described herein can be used as a combination therapy in conjunction with surgical resection and / or radiation therapy. In some embodiments, such a combination therapy approach reduces the risk of facial nerve damage or residual tumor cell growth.

[0178] In some embodiments, the therapeutic approaches described herein utilizing the compositions described herein, e.g., rAAV-anti-VEGF, pharmacologically attenuate tumor growth while avoiding and / or minimizing side effects associated with current standard treatments, such as surgical resection and / or radiation.

[0179] The current standard of care for VS has evolved over the past few decades as advances in imaging techniques have improved our ability to identify and track tumor growth. With the growing recognition that many tumors exhibit slow or slow growth and will not affect hearing as long as low growth rates are maintained, there has been a trend toward a conservative, observational "wait and rescan" approach (MacKeith 2013; Reznitsky 2019, each of which is incorporated herein by reference in its entirety).

[0180] In certain embodiments, the interventions, methods, and / or compositions described herein offer advantages, including the potential to enhance conservative treatment approaches by, for example, halting tumor growth, stabilizing hearing, and / or avoiding the need for more invasive treatment approaches, such as surgical resection and / or radiation therapy. While halting VS growth is believed to provide substantial clinical benefit, in some embodiments, methods and treatments utilizing the compositions described herein (e.g., rAAV-anti-VEGF) may promote not only tumor stasis but also tumor shrinkage, restoration of speech comprehension, and reduction in perceived difficulty with speech comprehension, as shown in studies of VS tumors in NF2 patients treated with bevacizumab (Huang 2018; Plotkin 2019, each of which is incorporated herein by reference in its entirety). Furthermore, studies using systemically administered VEGF inhibitors have shown improvements in NF2-related quality of life, including symptoms associated with VS, in NF2 patients (Plotkin 2019, incorporated herein by reference in its entirety). However, these systemic administrations are often associated with negative side effects. These early findings suggest that anti-VEGF protein therapy may alleviate and potentially combat symptoms associated with VS. In certain embodiments, the compositions described herein (e.g., rAAV-anti-VEGF) may enable less invasive treatments and sustained, localized expression of anti-VEGF protein in diffuse contiguous contact with the tumor, potentially providing patients with otologic diseases associated with neovascularization (e.g., VS) with more focused and improved benefits of anti-VEGF therapy without the side effects of systemic therapy.

[0181] Vascular endothelial growth factor (VEGF) and VEGF inhibitors in VS The development and vascularization of new blood vessels has been found to be important factors in many tumor growth models and may be important for VS growth. VEGF is one of the key regulators of angiogenesis. In certain cases, VEGF protein and its receptors are expressed in sporadic VS tumors (Caye-Thomasen 2003; Plotkin 2009, each of which is incorporated herein by reference in its entirety); for example, in a study of 182 resected sporadic VS tumors, all tumors examined expressed VEGF receptors (Koutsimpelas 2012, incorporated herein by reference in its entirety). In some cases, the expression levels of VEGF protein and / or receptors in this type of schwannoma have been shown to correlate with tumor growth rate and / or growth index measured by serial MRI (Caye-Thomasen 2005; Koutsimpelas 2007, each of which is incorporated herein by reference in its entirety) and microvascular density (Koutsimpelas 2007, incorporated herein by reference in its entirety), suggesting that VEGF expression is involved in VS growth. Furthermore, systemic treatment with a VEGF inhibitor (bevacizumab) has been effective in controlling growth and improving hearing in NF2 patients with VS. Without being bound by theory, it is believed that the use of VEGF inhibitors can control tumor growth, e.g., VS growth, by controlling blood vessel growth, e.g., tumor vascularization.

[0182] The VEGF gene encodes vascular endothelial growth factor (VEGF), formerly 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. Non-limiting examples of proteins and nucleotide sequences encoding wild-type VEGF proteins are provided herein.

[0183] In some embodiments, local exposure to anti-VEGF proteins at the tumor surface has the potential to control tumor growth, despite different diffusion pathways for accessing and neutralizing VEGF compared with extravasation of anti-VEGF proteins from the bloodstream. For example, Lichtenbeld et al., 1999 (incorporated herein by reference in its entirety) locally applied anti-VEGF proteins to tumors in mice and observed a significant reduction in vascular permeability, although, notably, a 20-fold lower dose was used compared with systemic administration, which achieved a similar reduction in vascular permeability in mice (Yuan 1996, incorporated herein by reference in its entirety). Thus, in some embodiments, compositions described herein (e.g., comprising rAAV-anti-VEGF) provide low-level but sustained exposure to anti-VEGF proteins in the fluid surrounding the VS, thus potentially stabilizing and / or reducing tumor growth. In certain embodiments, compositions described herein (e.g., comprising rAAV-anti-VEGF) stabilize and / or reduce tumor growth by reducing tumor vascular permeability and / or normalizing tumor vasculature.

[0184] In certain embodiments, anti-VEGF protein therapies for VS, such as those described herein, attenuate tumor growth without the need for invasive alternatives such as surgical resection and / or radiation therapy, thereby avoiding the complications of, for example, surgical resection and / or radiation therapy. It has been suggested that systemically administered VEGF inhibitors (e.g., bevacizumab) may be effective in stabilizing or reducing VS growth and hearing loss sequelae in patients with neurofibromatosis type 2 (NF2) (tumors resulting from germline NF2 mutations also highly express VEGF and its receptors) (Plotkin 2009; Plotkin 2012; Lu 2019, each of which is incorporated herein by reference in its entirety). However, systemic administration of VEGF inhibitors to control VS growth in NF2 patients can also be associated with adverse effects; in a meta-analysis of five clinical trial participant populations including 125 patients, the pooled incidence of severe toxicity (grade 3 or 4) was 17% (Lu 2019, incorporated herein by reference in its entirety).

[0185] In certain embodiments, compositions described herein (e.g., rAAV-anti-VEGF) can be used in methods of treating individuals (e.g., mammals, e.g., humans) with VS, e.g., by gene transfer to the inner ear to promote expression and secretion of anti-VEGF protein. In certain embodiments, compositions described herein, e.g., rAAV-anti-VEGF, provide local exposure to anti-VEGF protein (e.g., in the inner ear). In certain embodiments, local exposure to anti-VEGF protein at the site of VS (e.g., in the inner ear) limits systemic exposure and / or reduces, e.g., minimizes, the potential for side effects. In certain embodiments, compositions described herein (e.g., rAAV-anti-VEGF) comprise ranibizumab (48 kilodaltons [kDa]), a humanized monoclonal antibody fragment (Fab) derived from a full-length murine anti-human VEGF monoclonal antibody. In certain embodiments, ranibizumab binds to VEGF and inhibits VEGF binding to its receptors VEGFR-1 and / or VEGFR-2, thereby reducing vascular leakage, aberrant angiogenesis, and / or tumor growth (Genentech 2017, incorporated herein by reference in its entirety).

[0186] In certain embodiments, cochlear and vestibular cells of the inner ear are transduced with compositions described herein (e.g., comprising rAAV-anti-VEGF). In certain embodiments, these and / or other cell types may secrete anti-VEGF protein into the perilymph, an inner ear fluid that is diffusely continuous with the interstitium and perineural space of the vestibulocochlear nerve (e.g., composed of the superior and inferior vestibular nerves and cochlear nerve) where the tumor resides. In some embodiments, the majority of VS tumors arise in the outer third of the internal auditory canal (the portion closest to the cochlea), which houses the vestibulocochlear nerve. In some embodiments, the lack of a barrier to diffusion along the internal auditory canal means that the cochlear nerve is bathed in a fluid continuum, with perilymph at its lateral end and CSF at its medial end; thus, diffusion from the perilymph to the neural interstitium provides a potential pathway for therapeutic anti-VEGF proteins expressed in the perilymph to reach their intended VS target (Rask-Andersen 2006, incorporated herein by reference in its entirety). In some embodiments, spiral ganglion neurons and / or their satellite glial cells within the modiolus are transduced and / or transfected with compositions described herein (e.g., comprising rAAV-anti-VEGF), and these cells are positioned to secrete proteins directly into the interstitial fluid of the cochlear nerve.

[0187] In some embodiments, intracochlear administration of a composition described herein (e.g., rAAV-anti-VEGF) has the potential to eliminate the need for future treatment and preserve physiological hearing in individuals (e.g., mammals, e.g., humans) with neovascularization-associated otologic disorders (e.g., VS), regardless of the underlying etiology. In some embodiments, intracochlear administration of a composition described herein (e.g., rAAV-anti-VEGF) has the potential to delay invasive treatment approaches, such as surgical resection and / or radiation therapy, and the associated physiological hearing loss. In some embodiments, intracochlear administration of a composition described herein (e.g., rAAV-anti-VEGF) is followed by subsequent standard of care treatment. In some embodiments, intracochlear administration of a composition described herein (e.g., rAAV-anti-VEGF) is administered before and / or after radiation therapy. In some embodiments, administration of a composition described herein (e.g., rAAV-anti-VEGF) may improve an individual's (e.g., a mammal, e.g., a human's) response to radiation therapy by sensitizing tumors and allowing for lower radiation doses (Koutsimpelas 2012; Gao 2015, each of which is incorporated herein by reference in its entirety).

[0188] As mentioned above, the development and vascularization of new blood vessels have been found to be important factors in the growth of VS (Koutsimpelas 2007; Wong 2010, each of which is incorporated herein by reference in its entirety), and VEGF is one of the major regulators of angiogenesis. In addition to its angiogenic effects, VEGF also provides cellular protection and resistance to radiation-induced apoptosis (Koutsimpelas 2012, incorporated herein by reference in its entirety). Over the past decade, clinical data have emerged demonstrating that VEGF inhibitors can halt or reverse VS growth. Bevacizumab (Avastin®) is currently the only pharmacological agent in the setting of NF2 that has demonstrated preliminary evidence of efficacy in VS patients.

[0189] The first clinical evidence was published over 10 years ago in a seminal paper by Plotkin et al. (Plotkin 2009, incorporated herein by reference in its entirety). In their study, patients with baseline tumor size ranging from 2.2 to 38.7 cm 3 In a study, 10 NF2 patients (6 men, 4 women, median age 25 years) with annual growth rates ranging from 9% to 121% of baseline, most of whom presented with hearing loss, received systemic bevacizumab at 5 mg / kg every 2 weeks for a mean of 12 months (range 3-19 months). After repeated bevacizumab administration, tumors shrank in 9 of the 10 patients, and 6 patients demonstrated a response with at least a 20% reduction in tumor volume on MRI. Responses were maintained in 4 of the 6 patients over a follow-up period of 11-16 months. The median best response for the 9 of the 10 patients with tumor shrinkage was a 26% volume reduction. Of the 7 patients available for audiometric testing, 4 showed improvement, defined as a significant increase in word recognition scores, 2 showed stable hearing loss, and 1 showed progressive hearing loss compared to baseline (Plotkin 2009, incorporated herein by reference in its entirety). Such results may suggest that this approach may reverse the progressive hearing loss seen in patients with persistent tumor growth.

[0190] Subsequent to this initial publication, a larger retrospective review of 31 patients with NF2 who received systemic intravenous bevacizumab (5 mg / kg every 2 weeks for 6–41 months; median duration 14 months) was reported (Plotkin 2012, incorporated herein by reference in its entirety). In this study of a similar patient population, 57% of patients evaluated demonstrated hearing improvement, defined as an increase in word recognition score from baseline, and 55% demonstrated radiographic response, defined as at least a 20% reduction in tumor volume compared to baseline. When evaluating long-term treatment response, hearing stabilized or improved in 90% of patients after 1 year, and this trend persisted in 61% of these patients after 3 years. Furthermore, 88% of patients experienced stable or decreased tumor size after 1 year of treatment, and 54% of these patients remained stable after 3 years (Plotkin 2012, incorporated herein by reference in its entirety).

[0191] Recently, Plotkin et al. published the results of a multicenter, prospective, phase 2 efficacy trial of systemic intravenous bevacizumab in NF2 patients with progressive VS (Plotkin 2019, incorporated herein by reference in its entirety). In this trial, patients (median age 23 years) received systemic bevacizumab at 10 mg / kg every 2 weeks for 6 months, followed by 5 mg / kg every 3 weeks for 18 months. Consistent with previous findings, interim results showed that 41% of participants had improved hearing and 32% had radiographic responses after 6 months of treatment.

[0192] The preliminary efficacy and safety of systemically administered bevacizumab for NF2 patients with S was also examined in a meta-analysis of treatment outcomes from eight clinical trials conducted across the United States and Europe (Killeen 2019; Karajannis 2019; Lu 2019, each of which is incorporated herein by reference in its entirety). Across these eight studies, 161 NF2 patients with 196 schwannomas received bevacizumab at doses ranging from 5 to 10 mg / kg every two weeks (average range across studies: 11 to 22 months) and had their treatment outcomes evaluated. Across these studies, combined data showed radiographic responses (at least 20% volumetric reduction) in approximately 41% of schwannomas, tumor stability in approximately 47% of schwannomas, and tumor progression (at least 20% volumetric increase) in approximately 7% of schwannomas. In patients with available audiometric data, bevacizumab administration was associated with hearing improvement in approximately 20% of individuals, hearing preservation (stabilization of hearing loss) in approximately 69% of individuals, and worsening of hearing loss in approximately 6% of individuals. During the reported follow-up period, 11% of patients required subsequent surgical intervention. Additionally, bevacizumab side effects, such as severe toxicity (including hypertension, proteinuria, and amenorrhea), were also evaluated. Based on a meta-analysis of five cohorts of clinical trial participants involving 125 patients, the pooled incidence of severe toxicity (grade 3 or 4) was 17% (Lu 2019, incorporated herein by reference in its entirety). In certain embodiments, the present disclosure provides methods and compositions suitable for fulfilling the long-standing need to effectively treat otologic disorders associated with neovascularization while potentially avoiding the negative consequences associated with systemic delivery of therapeutic anti-VEGF proteins.

[0193] The above-mentioned clinical data (showing response rates of approximately 30% to over 60% in tumor shrinkage) support the use of systemic VEGF inhibitors to suppress VS growth and improve the associated symptoms of hearing loss, despite the side effects common to systemic use of VEGF inhibitors (Plotkin 2009; Plotkin 2012; Lu 2019; Plotkin 2019, each of which is incorporated herein by reference in its entirety). Without treatment, the average growth rate of VS varies between 0.4 and 2.9 mm / year (Yoshimoto 2005, incorporated herein by reference in its entirety). Studies of up to 212 patients have reported spontaneous tumor regression of 0-11% of tumors (Tschudi 2000; Slattery 2004; Peyre 2013; Schnurman 2019, each of which is incorporated herein by reference in its entirety), or a larger study of 1,261 patients reported spontaneous tumor regression of 3.8% of tumors (Huang 2013, incorporated herein by reference in its entirety). A limitation of systemic anti-VEGF protein therapy has been its unavoidable systemic adverse effects. In certain embodiments, the present disclosure provides methods for administering anti-VEGF proteins. In certain embodiments, methods and compositions utilizing an adeno-associated virus (AAV) delivery mechanism are provided. In certain embodiments, such AAV comprises a recombinant construct and is referred to as recombinant AAV (rAAV). Embodiments of such constructs are described in further detail below. In certain embodiments, compositions are provided for delivering localized anti-VEGF proteins, e.g., rAAV-anti-VEGF, into the cochlea, e.g., adjacent to the location of a tumor, e.g., VS. In some embodiments, intracochlear administration of a VEGF inhibitor, e.g., rAAV-anti-VEGF, reduces side effects associated with systemic delivery of the VEGF inhibitor.

[0194] Although insufficient data exist to assess the biologically active dose range of gene therapy particles delivered via the intracochlear route for the treatment of VS, several mouse models have been used to generate data supporting the biological plausibility that VEGF inhibitors reduce tumor vascular permeability. Using a cranial window model, Yuan et al. implanted various human tumor cell lines and administered a bolus of a neutralizing antibody against VEGF / VPF (vascular permeability factor) or a phosphate-buffered saline control intravenously (Yuan 1996, incorporated herein by reference in its entirety). The authors demonstrated that tumor vascular permeability to albumin in the antibody-treated group was lower than that in the corresponding control group, and that tumor vessels decreased in diameter and eventually disappeared after successive treatments. These data demonstrate that neutralization of endogenous VEGF / VPF can reduce tumor vascular permeability (Yuan 1996, incorporated herein by reference in its entirety). More recently, using both the sciatic nerve model and the intracranial window model, Gao et al. characterized the mechanisms behind anti-VEGF protein treatment for bilateral VS (Gao 2015, incorporated herein by reference in its entirety). This group injected human HEI193 schwannoma cells into either the mouse sciatic nerve sheath or into the space between the pia mater and arachnoid mater of the right hemisphere of cranial window-implanted mice. After tumor size reached 4 mm in diameter, the VEGF inhibitor bevacizumab was administered intraperitoneally (IP) at 5 mg / kg / week. The data showed that bevacizumab alleviated tumor edema, improved neurological function, and transiently normalized tumor vasculature in mice.

[0195] These studies provide proof-of-concept data supporting the scientific rationale for the use of VEGF inhibitors to slow schwannoma progression in mice, consistent with preliminary clinical data in NF2 patients treated systemically with Avastin® (Plotkin 2009; Plotkin 2012; Lu 2019; Plotkin 2019, each of which is incorporated by reference in its entirety). One limitation of these prior studies is that they do not reflect persistent exposure levels at limited target sites.

[0196] In certain embodiments, the methods and compositions disclosed herein (e.g., comprising rAAV-anti-VEGF) provide sustained levels of VEGF inhibitors in a localized target site, e.g., the inner ear. Without wishing to be bound by theory, it is believed that the compositions disclosed herein, e.g., VEGF inhibitors, may enter VS tumor cells through direct uptake from surrounding cisterns, which communicate with the perilymphatic compartment of the inner ear through the neural interstitium, from which the inner ear supply of anti-VEGF protein can diffuse. In tumor-bearing mice, applying anti-VEGF protein locally to tumor tissue, rather than through the bloodstream, resulted in beneficial effects such as reduced vascular permeability (Lichtenbeld 1999, incorporated herein by reference in its entirety).

[0197] In certain embodiments, the present disclosure provides methods and compositions suitable for localized delivery of VEGF inhibitors, such as rAAV-anti-VEGF, to tumor sites. In some embodiments, these methods and compositions have the potential to minimize the risk of serious toxicity documented with systemic administration of VEGF inhibitors while maintaining the benefits of tumor growth control with anti-VEGF proteins. In certain embodiments, the present disclosure provides methods and compositions suitable for transduction of inner ear cells. In some embodiments, transduction of inner ear cells may enable long-term expression of anti-VEGF proteins at and / or near the site of the tumor with minimal systemic exposure.

[0198] VEGF polynucleotide sequence Among other things, the present disclosure provides polynucleotides, such as polynucleotides comprising a VEGF gene or characteristic portions thereof, as well as compositions comprising such polynucleotides, and methods utilizing such polynucleotides and / or compositions.

[0199] In some embodiments, the polynucleotide comprising a VEGF gene or a characteristic portion thereof can be DNA or RNA. In some embodiments, the DNA can be genomic DNA or cDNA. In some embodiments, the RNA can be mRNA. In some embodiments, the polynucleotide comprises exons and / or introns of the VEGF gene.

[0200] In some embodiments, the gene product is expressed from a polynucleotide comprising a VEGF gene or a characteristic portion thereof. In some embodiments, the expression of such a polynucleotide can utilize one or more control elements (e.g., a promoter, an enhancer, a splice site, a polyadenylation site, a translation initiation site, etc.). Thus, in some embodiments, the polynucleotides provided herein can comprise one or more control elements.

[0201] In some embodiments, the VEGF gene is a mammalian VEGF gene. In some embodiments, the VEGF gene is a mouse VEGF gene. In some embodiments, the VEGF gene is a primate VEGF gene. In some embodiments, the VEGF gene is a human VEGF gene. In some embodiments, the VEGF gene is a genomic DNA sequence. In some embodiments, the VEGF gene is an RNA sequence that encodes a protein product. In some embodiments, the VEGF gene is a complementary DNA sequence that encodes a complementary RNA sequence that encodes a protein product.

[0202] In some embodiments, an exemplary human VEGF gene is found at human chromosome location 6p21.1; location NC_000006.12 (43770209..43786487) in the current 2020 assembly, and is also known as VEGF-A at NCBI Reference Sequence No. NG_008732.1. In some embodiments, an exemplary human VEGF gene is a cDNA sequence contained within the VEGF-A gene (e.g., VEGF-A transcript variant 1, transcript variant 2, transcript variant 3, etc.). In some embodiments, an exemplary human VEGF gene is one of at least nine known transcript isoforms of VEGF-A; one of skill in the art will understand that these transcript isoforms may undergo alternative splicing to generate alternative translational isoforms. In some embodiments, an exemplary human VEGF-A gene is a cDNA sequence represented by transcript variant 1, which encodes VEGF isoform A (NCBI Reference Sequence NM_001025366.3). In some embodiments, an exemplary human VEGF-A gene is a cDNA sequence represented by transcript variant 2, which encodes VEGF isoform B (NCBI Reference Sequence: NM_003376.6). In some embodiments, an exemplary human VEGF-A gene is a cDNA sequence represented by transcript variant 3, which encodes VEGF isoform C (NCBI Reference Sequence: NM_001025367.3). In some embodiments, an exemplary human VEGF-A gene is a cDNA sequence represented by transcript variant 4, which encodes VEGF isoform D (NCBI Reference Sequence: NM_001025368.3). In some embodiments, an exemplary human VEGF-A gene is a cDNA sequence represented by transcript variant 5, which encodes VEGF isoform E (NCBI Reference Sequence: NM_001025369.3). In some embodiments, an exemplary human VEGF-A gene is the cDNA sequence represented by transcript variant 6, which encodes VEGF isoform F (NCBI Reference Sequence: NM_001025370.3).In some embodiments, an exemplary human VEGF-A gene is the cDNA sequence represented by transcript variant 7, which encodes VEGF isoform G (NCBI Reference Sequence: NM_001033756.3). In some embodiments, an exemplary human VEGF-A gene is the cDNA sequence represented by transcript variant 8, which encodes VEGF isoform H (NCBI Reference Sequence: NM_001171622.2). In some embodiments, an exemplary human VEGF-A gene is the cDNA sequence represented by transcript variant encoding VEGF isoform R (NCBI Reference Sequence: NM_001204385.2).

[0203] In some embodiments, an exemplary human VEGF gene is found at human chromosome location 11q13.1, location NC_000011.10 (64234584..64239264), and is known as VEGF-B (NCBI Reference Sequence: NG_029823.1). In some embodiments, an exemplary human VEGF gene is a cDNA sequence contained within the VEGF-B gene, e.g., VEGF-B transcript variant 167 (NCBI Reference Sequence: NM_001243733.2) and / or transcript variant 186 (NCBI Reference Sequence: NM_003377.5).

[0204] In some embodiments, an exemplary human VEGF gene is found at human chromosome location 4q34.1-q34.3, location NC_000004.12 (176683538..176792922, complement), and is known as VEGF-C (NCBI Reference Sequence: NG_034216.1). In some embodiments, an exemplary human VEGF gene is a cDNA sequence contained within the VEGF-C gene, e.g., VEGF-C transcript variant 1 (NCBI Reference Sequence: NM_005429.5).

[0205] In some embodiments, an exemplary human VEGF gene is found at human chromosome location Xp22.2, location NC_000023.11 (15345596..15384413, complement) and is known as VEGF-D (NCBI Reference Sequence: NG_012509.1). In some embodiments, an exemplary human VEGF gene is a genomic or cDNA sequence contained within the VEGF-D gene, e.g., VEGF-D transcript variant 1 (NCBI Reference Sequence: NM_004469.5).

[0206] VEGF protein sequence In certain embodiments, the protein of interest is an isoform of the VEGF-A gene. This gene is a member of the PDGF (platelet-derived growth factor) / VEGF (vascular endothelial growth factor) family (PDGF / VEGF). It encodes a heparin-binding protein and typically exists as a disulfide-linked homodimer. This growth factor induces the proliferation and migration of vascular endothelial cells and is essential for both physiological and pathological angiogenesis. Alternatively spliced ​​transcript variants encoding different isoforms have been described. There is substantial evidence that alternative translation initiation from upstream non-AUG (CUG) codons results in additional isoforms. In some embodiments, the protein of interest is an inhibitor of endogenous VEGF-A function.

[0207] Exemplary Human VEGF-A Isoform L-VEGF 206 Precursor (also known as isoform A) amino acid sequence (SEQ ID NO: 1) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQA APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCE CRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0208] Exemplary Human VEGF-A Isoform L-VEGF 189 Precursor (also known as isoform B) amino acid sequence (SEQ ID NO: 2) MTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPGRASETMNFLLSWVHWSLALLLY LHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQH IGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0209] Exemplary Human VEGF-A Isoforms111 Precursor amino acid sequence (SEQ ID NO: 3) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRCDKPRR

[0210] Exemplary Human VEGF-A Isoforms 121 Precursor amino acid sequence (SEQ ID NO: 4) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKCDKPRR

[0211] Exemplary Human VEGF-A Isoforms 145 Precursor amino acid sequence (SEQ ID NO: 5) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVCDKPRR

[0212] Exemplary Human VEGF-A Isoforms 165A Precursor amino acid sequence (SEQ ID NO: 6) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCCRCDKPRR

[0213] Exemplary Human VEGF-A Isoforms 189 Precursor amino acid sequence (SEQ ID NO: 7) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIM RIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0214] Exemplary Human VEGF-A Isoforms 206 Precursor amino acid sequence (SEQ ID NO: 8) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQH IGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR

[0215] In certain embodiments, the protein of interest is an isoform of the VEGF-B gene. This gene encodes a member of the PDGF / VEGF family. VEGF family members regulate blood vessel formation and are involved in the physiological function of endothelial cells. This member is a ligand for VEGFR-1 (vascular endothelial growth factor receptor 1) and NRP-1 (neuropilin-1). In some embodiments, the protein of interest is an inhibitor of the function of endogenous VEGF-B.

[0216] Exemplary Human VEGF-B Isoform VEGFB-167 Precursor Amino Acid Sequence (SEQ ID NO: 9) MSPLLRRLLLAALLQLAPAQAPVSQPDAPGHQRKVVSWIDVYTRATCQPREVVVPLTVELMGTVAKQLVPSCVTVQRCGGCCPDDGLECVPTGQHQVRMQILMIRYPSSQLGEMSLEEHSQCECRPKKKDSAVKPDSPRPLCPRCTQHHQRPDPRTCRCRCRRRSFLRCQGRGLELNPDTCRCRKLRR

[0217] Exemplary human VEGF-B isoform VEGFB-186 precursor amino acid sequence (SEQ ID NO: 10) MSPLLRRLLLAALLQLAPAQAPVSQPDAPGHQRKVVSWIDVYTRATCQPREVVVPLTVELMGTVAKQLVPSCVTVQRCGGCCPDDGLECVPTGQHQVRMQILMIRYPSSQLGEMSLEEHSQCECRPKKKDSAVKPDRAATPHHRPQPRSVPGWDSAPGAPSPADITHPTPAPGPSAHAAPSTTSALTPGPAAAAADAAASSVAKGGA

[0218] In certain embodiments, the protein of interest is an isoform of the VEGF-C gene. The protein encoded by this gene is a member of the PDGF / VEGF family. The encoded protein promotes angiogenesis and endothelial cell proliferation and can also affect vascular permeability. The precursor protein is further cleaved into fully processed forms that can bind to and activate VEGFR-2 and VEGFR-3 receptors. In some embodiments, the protein of interest is an inhibitor of endogenous VEGF-C function.

[0219] Exemplary human VEGF-C precursor amino acid sequence (SEQ ID NO: 11) MHLLGFFSVACSLLAAAALLPGPREAPAAAAAFESGLDLSDAEPDAGEATAYASKDLEEQLRSVSSVDELMTVLYPEYWKMYKCQLRKGGWQHNREQANLNSRTEETIKFAAAHYNTEILKSIDNEWRKTQCMPREVCIDVGKEFGVATNTFFKPPCVSVYRCGGCCNSEGLQCMNTSTSYLSKTLFEITVPLSQGPKPVTISFANHTSC RCMSKLDVYRQVHSIIRRSLPATLPQCQAANKTCPTNYMWNNHICRCLAQEDFMFSSDAGDDDSTDGFHDICGPNKELDEETCQCVCRAGLRPASCGPHKELDRNS CQCVCKNKLFPSQCGANREFDENTCQCVCKRTCPRNQPLNPGKCACECTESPQKCLLKGKKFHHQTCSCYRRPCTNRQKACEPGFSYSEEVCRCVPSYWKRPQMS

[0220] In certain embodiments, the protein of interest is an isoform of the VEGF-D gene. The protein encoded by this gene is a member of the PDGF / VEGF family and is active in angiogenesis, lymphangiogenesis, and endothelial cell proliferation. This secreted protein undergoes complex proteolytic maturation to generate multiple processed forms that bind to and activate VEGFR-2 and VEGFR-3 receptors. This protein is structurally and functionally similar to VEGF-C. In some embodiments, the protein of interest is an inhibitor of endogenous VEGF-D function.

[0221] Exemplary human VEGF-D precursor amino acid sequence (SEQ ID NO: 12) MYREWVVVNVFMMLYVQLVQGSSNEHGPVKRSSQSTLERSEQQIRAASSLEELLRITHSEDWKLWRCRLRLKSFTSMDSRSASHRSTRFAATFYDIETLKVIDEEWQRTQCSPRETCVEVASELGKSTNTFFKPPCVNVFRCGGCCNEESLICMNTSTSYISKQLFEISVPLTSVPE LVPVKVANHTGCKCLPTAPRHPYSIIRRSIQIPEEDRCSHSKKLCPIDMLWDSNKCKCVLQEENPLAGTEDHSHLQEPALCGPHMMFDEDRCECVCKTPCPKDLIQHPKNCSCFECKESLETCCQKHKLFHPDTCSCEDRCPFHTRPCASGKTACAKHCRFPKEKRAAQGPHSRKNP

[0222] VEGF, VEGF-R, and VEGF-binding proteins In some embodiments, the protein of interest is a protein capable of binding to VEGF. In some embodiments, the VEGF binding protein may be or comprise an antibody and / or a fragment thereof. In some embodiments, the VEGF binding protein may be or comprise a vascular endothelial growth factor receptor (VEGFR) protein and / or a fragment thereof.

[0223] As described above, data have been generated that may support the biological plausibility that VEGF inhibitors reduce tumor vascular permeability. In some embodiments, such data were generated using mouse models that have been utilized to generate data supporting the biological plausibility that VEGF inhibitors reduce tumor vascular permeability. In some embodiments, such mouse models are not ideal for assessing the biologically active dose range of gene therapy particles delivered via the intracochlear route, for example, for the treatment of VS. Using a cranial window model, Yuan et al. implanted various human tumor cell lines and administered a neutralizing antibody against VEGF / VPF (vascular permeability factor) or a phosphate-buffered saline control intravenously (Yuan 1996, incorporated herein by reference in its entirety). The authors showed that tumor vascular permeability to albumin in the antibody-treated group was lower than that in the corresponding control group, and that tumor vessels decreased in diameter and eventually disappeared after successive treatments. These data demonstrate that neutralization of endogenous VEGF / VPF can reduce tumor vascular permeability (Yuan 1996, incorporated herein by reference in its entirety). More recently, using both the sciatic nerve model and the intracranial window model, Gao et al. attempted to characterize the mechanism behind anti-VEGF protein treatment of bilateral VS (Gao 2015, incorporated herein by reference in its entirety). This group injected human HEI193 schwannoma cells into either the mouse sciatic nerve sheath or the space between the pia mater and arachnoid mater of the right hemisphere of cranial window-implanted mice. After tumor size reached 4 mm in diameter, the VEGF inhibitor bevacizumab was administered intraperitoneally (IP) at 5 mg / kg / week. The resulting data indicate that bevacizumab alleviates tumor edema, improves neurological function, and transiently normalizes tumor vasculature in mice.

[0224] Collectively, these studies provide proof-of-concept data supporting the scientific rationale for the use of VEGF inhibitors to slow schwannoma progression in mice, consistent with preliminary clinical data in NF2 patients treated systemically with Avastin® (Plotkin 2009; Plotkin 2012; Lu 2019; Plotkin 2019, each of which is incorporated by reference in its entirety). One limitation of these prior studies is that they do not reflect persistent exposure levels at limited target sites.

[0225] In some embodiments, the methods and compositions disclosed herein provide sustained levels of a VEGF inhibitor, e.g., rAAV-anti-VEGF, at a localized target site, e.g., the inner ear of an individual, e.g., a mammal, e.g., a human. Without wishing to be bound by theory, in some embodiments, it is believed that the VEGF inhibitor may enter VS tumor cells via direct uptake from surrounding cisterns, which communicate with the perilymphatic compartment of the inner ear through the neural interstitium, from which the inner ear supply of anti-VEGF protein may diffuse. In tumor-bearing mice, applying anti-VEGF protein locally to tumor tissue, rather than through the bloodstream, resulted in beneficial effects such as reduced vascular permeability (Lichtenbeld 1999, incorporated herein by reference in its entirety).

[0226] Anti-VEGF antibody In some embodiments of any of the antibodies described herein, the antibody is capable of binding (e.g., with any of the binding affinities described herein) to a VEGF antigen (e.g., any of the exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).

[0227] In some embodiments described herein, the antibody is capable of reducing the activity of VEGF (e.g., one or more of any exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D). In some embodiments, the antibody is capable of inhibiting the binding of VEGF (e.g., one or more of any exemplary VEGF proteins described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D) 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 33:1046-1052 (2012); and Ferrara et al. (2005) Biochem Biophys Res Commun 333(2):328-335 (each of which is incorporated by reference in its entirety). In some embodiments, the antibody can reduce downstream signaling (e.g., downstream signaling of a VEGF receptor, e.g., one or more of any exemplary VEGF receptors described herein, e.g., one or more of human VEGFR-1, human VEGFR-2, and human VEGFR-3). In some embodiments, reduced VEGF activity can be detected indirectly, such as by: For example, a change in VS tumor size and / or a VS-related symptom described herein, such as an increase in hearing (e.g., a 1% to about 400% increase in hearing (or any subrange of this range described herein)) or a decrease in VS tumor size (e.g., 1% to 99%, 1% to 95%, 1% to 90%, 1% to 85%, 1% to 80%, 1% to 75%, ... %~70%, 1%~65%, 1%~60%, 1%~55%, 1%~50%, 1%~45%, 1%~40%, 1%~35%, 1%~30%, 1%~25%, 1%~20%, 1%~15%, 1%~10%, 1%~5%, 5%~99%, 5%~95%, 5%~90%, 5%~85%, 5%~80%, 5%~75%, 5%~70%, 5%~65%,5%~60%、5%~55%、5%~50%、5%~45%、5%~40%、5%~35%、5%~30%、5%~25%、5%~20%、5%~15%、5%~10%、10%~99%、10%~95%、10%~90%、10%~85%、10%~80%、10%~75%、10%~70%、10%~65%、10%~60%、10%~55%、10%~50%、10%~45%、10%~40%、10%~35%、10%~30%、10%~25%、10%~20%、10%~15%、15%~99%、15%~95%、15%~90%、15%~85%、15%~80%、15%~75%、15%~70%、15%~65%、15%~60%、15%~55%、15%~50%、15%~45%、15%~40%、15%~35%、15%~30%、15%~25%、15%~20%、20%~99%、20%~95%、20%~90%、20%~85%、20%~80%、20%~75%、20%~70%、20%~65%、20%~60%、20%~55%、20%~50%、20%~45%、20%~40%、20%~35%、20%~30%、20%~25%、25%~99%、25%~95%、25%~90%、25%~85%、25%~80%、25%~75%、25%~70%、25%~65%、25%~60%、25%~55%、25%~50%、25%~45%、25%~40%、25%~35%、25%~30%、30%~99%、30%~95%、30%~90%、30%~85%、30%~80%、30%~75%、30%~70%、30%~65%、30%~60%、30%~55%、30%~50%、30%~45%、30%~40%、30%~35%、35%~99%、35%~95%、35%~90%、35%~85%、35%~80%、35%~75%、35%~70%、35%~65%、35%~60%、35%~55%、35%~50%、35%~45%、35%~40%、40%~99%、40%~95%、40%~90%、40%~85%、40%~80%、40%~75%、40%~70%、40%~65%、40%~60%、40%~55%、40%~50%、40%~45%、45%~99%、45%~95%、45%~90%、45%~85%、45%~80%、45%~75%、45%~70%、45%~65%、45%~60%、45%~55%、45%~50%, 50%~99%, 50%~95%, 50%~90%, 50%~85%, 50%~80%, 50%~75%, 50%~70%, 50%~65%, 50%~60%, 50%~55%, 55%~99%, 55%~95%, 55%~90%, 55%~85%, 55%~80%, 55%~75%, 55%~70%, 55%~65%, 55%~ 60%, 60%~99%, 60%~95%, 60%~90%, 60%~85%, 60%~80%, 60%~75%, 60%~70%, 60%~65%, 65%~99%, 65%~95%, 65%~90%, 65%~85%, 65%~80%, 65%~75%, 65%~70%, 70%~99%, 70%~95%, 70%~90%, 70%~85%, 7 and / or the severity of one or more symptoms of VS or neurofibromatosis type II in a mammal compared to the severity of one or more symptoms of VS or neurofibromatosis type II (e.g., hearing loss, tinnitus, vertigo, reduced quality of life, etc.), and / or the size of, respectively, VS or neurofibromatosis type II in a mammal prior to administration of any of the rAAV particles described herein. In some embodiments, the reduction in VEGF activity can be detected in an in vitro assay.

[0228] In some embodiments, the antibody may be a humanized antibody, a chimeric antibody, or a multivalent antibody. In some embodiments, the antibody may be an scFv-Fc, a VHH domain, a VNAR domain, an (scFv)2, a minibody, or a BiTE. In some embodiments, the antibody may be a DVD-Ig, a dual affinity retargeting antibody (DART), a triomab, a kih IgG with a common LC, a crossmab, or an ortho-Fab. IgG, 2-in-1-IgG, IgG-ScFv, scFv2-Fc, bi-nanobody, tandem antibody, DART-Fc, scFv-HAS-scFv, DNL-Fab3, DAF (2-in-1 or 4-in-1), DutaMab, DT-IgG, knob-in-hole common LC, knob-in-hole assembly, charge-paired 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, KIH The antibody may be an IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, nanobody, nanobody-HSA, diabody, TandAb, sc diabody, sc diabody-CH3, diabody-CH3, triple body, miniantibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFV2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock-and-lock bispecific antibody, ImmTAC, HSA body, sc diabody-HAS, tandem scFv, IgG-IgG, Cov-X-body, and scFv1-PEG-scFv2.

[0229] Further examples of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments. Further examples of antibodies include antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG (e.g., human or humanized IgG1, IgG2, IgG3, or IgG4)), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA (e.g., human or humanized IgA1 or IgA2)), antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.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).

[0230] In some embodiments, any antibody described herein can specifically bind to VEGF. In some embodiments, any antibody described herein can specifically bind to PDGF / VEGF.

[0231] VHH domains are single monomeric variable antibody domains that can be found in Camelidae. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. Non-limiting embodiments of VHH domains and VNAR domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014;Muyldermans,J.Biotechnol.74:277-302,2001;Muyldermans et al.,Trends Biochem.Sci.26:230-235,2001;Muyldermans,Ann.Rev.Biochem.82:775-797,2013;Rahbarizadeh et al.,Immunol.Invest.40:299-338,2011;Van Audenhove et al.,EBioMedicine 8:40-48,2016;Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009;Vincke et al.,Methods Mol. Biol. 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009, each of which is incorporated herein by reference in its entirety.

[0232] In some embodiments, an "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0233] In some embodiments, a "Fab" fragment contains the heavy and light chain variable domains of an Fv fragment, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.

[0234] In some embodiments, an "F(ab')2" fragment comprises two Fab fragments linked near the hinge region by a disulfide bond.

[0235] In some embodiments, "dual variable domain immunoglobulin" or "DVD-Ig" refers to a multivalent and multispecific binding protein as described, for example, in 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 (each of which is incorporated by reference in its entirety).

[0236] In some embodiments, a Drug Affinity Responsive Target Stability (DARTS) assay is described and performed, for example, as shown in Garber, Nature Reviews Drug Discovery 13:799-801, 2014 (incorporated herein by reference in its entirety).

[0237] In some embodiments, the dissociation constant (KD) of any antibody described herein is less than 1×10 M (e.g., less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, less than 1×10 M, less than 0.5×10 M, or less than 1×10 M), e.g., as measured using surface plasmon resonance (SPR) in phosphate buffered saline against a VEGF protein (e.g., any VEGF protein described herein, e.g., one or more of mature human VEGF-A, mature human VEGF-B, mature human VEGF-C, and mature human VEGF-D).

[0238] In some embodiments, the KD of any antibody described herein is about 1×10 M to about 1×10 M, about 0.5×10 M, ... , about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, or about 0.5 × 10 M (inclusive); about 0.5 × 10 M to about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, or about 1 × 10 M (inclusive). ;Approx. 1×10-11M~Approx. 1×10-5M, Approx. 0.5×10-5M, Approx. 1×10-6M, Approx. 0.5×10-6M, Approx. 1×10-7M, Approx. 0.5 x 10-7M, Approx. 1 x 10-8M, Approx. 0.5 x 10-8M, Approx. 1 x 10-9M, Approx. 0.5 x 10-9M, Approx. 1 x 10-10M , or about 0.5×10-10M (including both extreme values); about 0.5×10-10M to about 1×10-5M, about 0.5×10-5M, about 1×1 0-6M, approx. 0.5 x 10-6M, approx. 1 x 10-7M, approx. 0.5 x 10-7M, approx. 1 x 10-8M, approx. 0.5 x 10-8M, approx. 1 x 1 0-9M, about 0.5 x 10-9M, or about 1 x 10-10M (inclusive); about 1 x 10-10M to about 1 x 10-5M, about 0.5 x 10-5M, about 1 x 10-6M, about 0.5 x 10-6M, about 1 x 10-7M, about 0.5 x 10-7M, about 1 x 10-8M, about 0.5 x 10-8M, about 1 x 10-9M, or about 0.5 x 10-9M (inclusive); about 0.5 x 10-9M to about 1 x 10-5M, about 0.5 x 10-5M, about 1 x 10-6M, about 0.5 x 10-6M, about 1 x 10-7M, about 0.5 x 10-7M, about 1 x 10-8M, about 0.5 × 10 M, or about 1 × 10 M (inclusive); about 1 × 10 M to about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, or about 0.5 × 10 M (inclusive); about 0.5 × 10 M to about 1 x 10 M, about 0.5 x 10 M, about 1 x 10 M, about 0.5 x 10 M, about 1 x 10 M, about 0.5 x 10 M, or about 1 x 10 M (inclusive); about 1 x 10 M to about 1 x 10 M, about 0.5 x 10 M, about 1 x 10 M, about 0.5 x 10 M, about 1 x 10 M, or about 0.5 × 10 M (inclusive); about 0.5 × 10 M to about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, about 0.5 × 10 M, or about 1 × 10 M (inclusive); about 1 × 10 M to about 1 × 10 M, about 0.5 × 10 M, about 1 × 10 M, or about 0.5 × 10 M (inclusive); about 0.5 × 10 M to about 1 × 10 M, about 0.5 × 10 M, or about 1 × 10 M (inclusive); about 1 × 10 M to about 1 × 10 M, or about 0.5 × 10 M (inclusive); or about 0.5 × 10 M to about 1 × 10 M (inclusive).

[0239] A variety of different methods known in the art can be used to determine the KD value of any antibody described herein (e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and bimolecular binding kinetics assays, etc.).

[0240] In some embodiments of any antibody described herein, the half-life of the antibody in a subject (e.g., a human) is 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). See, e.g., Leabman et al., MAbs. 5(6):896-903, 2013 (incorporated herein by reference in its entirety). In some embodiments, an antibody described herein has one or more amino acid substitutions in the Fc region that reduce mammalian half-life, and a control antibody lacks at least one (e.g., lacks all) of such one or more amino acid substitutions in the Fc region.

[0241] In some embodiments, the antibody that specifically binds to VEGF is bevacizumab (Avastatin®). Bevacizumab (full-size antibody, approximately 150 kDa) inhibits all isoforms of VEGF-A. Bevacizumab was approved by the U.S. Food and Drug Administration (FDA) in 2004 for colon cancer, with an intravenous (IV) dose of 4.0-7.5 mg / kg for 2-3 weeks (plasma half-life 21 days) and an intravitreal (IVT) dose of 1.25 mg in 0.05 mL (half-life 5.6 days). The KD of bevacizumab for VEGF165 (VEGF-A) is 58 pM. See, e.g., WO2017 / 050825, incorporated herein by reference in its entirety.

[0242] In some embodiments, the antibody that specifically binds to VEGF is ranibizumab (Lucentis®). Ranibizumab (approximately 50 kDa) inhibits all isoforms of VEGF-A. Ranibizumab was approved by the FDA for ophthalmic use in 2006, with an intravenous (IV) dose of 4.0-7.5 mg / kg over 2-3 weeks (plasma half-life 0.5 days) and an intravitreal (IVT) dose of 0.5 mg in 0.05 mL (half-life 3.2 days). Ranibizumab inhibits VEGF-A 165 The KD for VEGF-A isoform 165 (represented by SEQ ID NO: 6) is 46 pM. See, e.g., WO2014 / 178078 (incorporated herein by reference in its entirety).

[0243] In some embodiments, the antibody that specifically binds to VEGF is sevacizumab (APX003 / SIM-BD0801) or a characteristic portion thereof.

[0244] In certain embodiments, the anti-VEGF protein coding sequence contained within the compositions described herein (e.g., rAAV-anti-VEGF) is selected from VEGF inhibitors approved for the treatment of pathological vascularization in the retina, glioblastoma, and other cancers, including, for example, bevacizumab (Avastin®); aflibercept (Eylea®); dib-aflibercept (Zaltrap®); brolucizumab (Beovu®); and / or ranibizumab (Lucentis®). Additionally, in some embodiments, biosimilars of many of these products, as well as Razumab®, a biosimilar of ranibizumab, may be utilized.

[0245] Ranibizumab, aflibercept, and brolucizumab are each approved for repeated intravitreal administration for wet age-related macular degeneration (AMD), and ranibizumab and aflibercept are also approved for repeated intravitreal administration for retinal vein occlusion (RVO), diabetic macular edema (DME), and diabetic retinopathy (DR). Targeted local delivery and the associated reduced systemic exposure have been shown to offer improved safety profiles (including lower rates of thromboembolic events) compared with intravenous VEGF inhibitor treatment regimens. Gene therapy versions of these therapeutic agents are currently in clinical development for AMD and DME (Clinicaltrials.gov 2020b, 2020c, and 2020d [NCT03066258, NCT03748784, and NCT04418427], each of which is incorporated herein by reference in its entirety). Aflibercept is a recombinant fusion protein (97 kDa) consisting of portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1, and has been shown to be effective when delivered locally to the eye by repeated intravitreal injections in clinical trials for wet AMD and DME. Like bevacizumab, dib-aflibercept is approved for intravenous infusion and carries similar risks and warnings regarding bleeding and wound healing (Sanofi-Aventis US 2020, incorporated herein by reference in its entirety).

[0246] In a study of patients with wet age-related macular degeneration (AMD), local delivery of clinical doses of anti-VEGF proteins, such as bevacizumab and ranibizumab, administered via intravitreal injection was associated with significant improvements in visual acuity at 4 months; however, at the same time point, VEGF plasma levels were significantly reduced only in the bevacizumab-injected cohort (Carneiro 2012, incorporated herein by reference in its entirety). In certain embodiments, the methods and compositions described herein provide a solution to the reduced plasma VEGF levels that may be associated with systemic delivery of bevacizumab and / or acute local delivery. Certain studies have demonstrated results consistent with findings from pharmacokinetic studies, for example, in rabbits, where bevacizumab, but not ranibizumab, was detected in serum after intravitreal injection (Bakri 2007, incorporated herein by reference in its entirety). A review of clinical safety data from pivotal trials in AMD suggests that repeated intravitreal administration of ranibizumab for up to two years is not associated with significant safety risks (Schmidt-Erfurth 2010, incorporated herein by reference in its entirety).

[0247] Ranibizumab Ranibizumab is a humanized monoclonal antibody fragment (Fab) that, when delivered locally into the eye via intravitreal administration, has similar clinical efficacy and a comparable incidence of thromboembolic events to aflibercept (Genentech 2017, incorporated herein by reference in its entirety). Ranibizumab is an IgG1 Fab that binds to and neutralizes VEGF. Ranibizumab is believed to bind to all known isoforms of the human VEGF-A protein and inhibit its biological activity by preventing its interaction with its cognate receptors (VEGFR-1 and VEGFR-2). Ranibizumab is sold under the brand name Lucentis®. It has been approved by the FDA for indications such as macular edema after retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema. Compared to bevacizumab, ranibizumab exhibits over eight-fold higher binding capacity and over 66-fold higher binding affinity (Klettner 2008; Yang 2014, each of which is incorporated herein by reference in its entirety). These differences result in ranibizumab's IC50 for VEGF-induced endothelial proliferation being six-fold lower than that of bevacizumab, and ranibizumab exerts clinically relevant effects on VEGF activity down to 0.37 nanomolar (nM) (approximately 17 nanograms per milliliter [ng / ml] of ranibizumab; Yang 2014, incorporated herein by reference in its entirety). Importantly, ranibizumab also lacks an Fc region, allowing the molecule to avoid Fc recycling and making it significantly smaller (48 kDa) than full-size antibodies (149 kDa) (Meyer 2011, incorporated herein by reference in its entirety). In some embodiments, this smaller molecular size may be advantageous for improving diffusion to target sites, the ability to extravasate into the tumor interstitium, and / or the efficiency of diffusion to target sites within the tumor (Xenaki 2017, incorporated herein by reference in its entirety). In some embodiments, the compositions described herein comprise a coding sequence for ranibizumab for development into an rAAV gene therapy product.In some embodiments, compositions described herein (e.g., rAAV-anti-VEGF) can be delivered to the cochlea, and intracochlear administration can result in lower systemic exposure, thereby providing an improved safety profile compared to intravenous administration.

[0248] In certain embodiments, the present disclosure provides a composition comprising ranibizumab, an anti-VEGF protein lacking an Fc region. The antibody Fc region found in bevacizumab is believed to allow bevacizumab to cross biological barriers through Fc receptor-mediated transport and activate immune responses (Kim 2009; Meyer 2011, each of which is incorporated herein by reference in its entirety). Certain studies suggest that compared to bevacizumab, ranibizumab may exhibit 17-fold higher binding capacity and 6-fold higher binding affinity at high dilutions (Ferrara 2006; Klettner 2008, each of which is incorporated herein by reference in its entirety), suggesting higher specific activity at low concentrations. In some embodiments, ranibizumab's smaller molecular size (48 kDa compared to 149 kDa for bevacizumab) may also be advantageous in improving diffusion to target sites, ability to extravasate into the tumor interstitium, and / or efficiency of diffusion to target sites within the tumor (Xenaki 2017, incorporated herein by reference in its entirety).

[0249] In some embodiments, the anti-VEGF proteins described herein that specifically bind to VEGF and / or its antigen-presenting fragments are antibodies. In some embodiments, such antibodies comprise an immunoglobulin light chain variable domain that is or comprises a sequence 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 immunoglobulin light chain variable domain of ranibizumab, and / or comprise an immunoglobulin heavy chain variable domain that is or comprises a sequence 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 immunoglobulin heavy chain variable domain of ranibizumab.

[0250] In some embodiments, the anti-VEGF proteins described herein that specifically bind to VEGF and / or antigen-presenting fragments thereof are antibodies. In some embodiments, such antibodies specifically comprise an immunoglobulin light chain variable domain that is or includes the immunoglobulin light chain variable domain of ranibizumab and / or an immunoglobulin heavy chain variable domain that is or includes the immunoglobulin heavy chain variable domain of ranibizumab. In some embodiments, the antibody comprises an immunoglobulin light chain variable domain that is or includes the sequence of the immunoglobulin light chain variable domain of ranibizumab, except that it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions (represented by SEQ ID NO: 20), and / or an immunoglobulin heavy chain variable domain that is or includes the sequence of the immunoglobulin light chain variable domain of ranibizumab, except that it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions (represented by SEQ ID NO: 16, 17, or 18). In some embodiments, the antigen-binding domain comprises the three CDRs of the immunoglobulin light chain variable domain of ranibizumab and / or the three CDRs of the immunoglobulin heavy chain variable domain of ranibizumab.

[0251] Exemplary Ranibizumab Heavy Chain Nucleotide Sequence (SEQ ID NO: 13) GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTACGACTTCACCCACTACGGCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCACTGGTACTTTGACGTGTGGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCGGCAAG

[0252] Exemplary ranibizumab heavy chain nucleotide sequence (SEQ ID NO: 14) GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTACGACTTCACCCACTACGGCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCACTGGTACTTTGACGTGTGGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGGCGCTCTGACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACAAG

[0253] Exemplary ranibizumab heavy chain nucleotide sequence (SEQ ID NO: 15) GAGGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTGCCGCTTCTGGCTACGACTTCACCCACTACGGCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGC GAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACGGCACCAGCCACTGGTACTTTGACGTGT GGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGTCTACCAGCGGAGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATTCTGGCGCTCT GACAAGCGGCGTGCACACCTTTCCAGCTGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGC

[0254] Exemplary Ranibizumab Heavy Chain Amino Acid Sequence (SEQ ID NO: 16) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGK

[0255] Exemplary ranibizumab heavy chain amino acid sequence (SEQ ID NO: 17) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL

[0256] Exemplary Ranibizumab Heavy Chain Amino Acid Sequence (SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDV WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS

[0257] Exemplary Ranibizumab Light Chain Nucleotide Sequence (SEQ ID NO: 19) GACATCCAGCTGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATCACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAAAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTCACAAGCAGCC TGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGTACAGCACCGTGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAAG CGGACTGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAGCGGCAACAGCCAAG AGAGCGTGACAGAGCAGGACTCCAAGGATAGCACCTATAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAATGT

[0258] Exemplary Ranibizumab Light Chain Amino Acid Sequence (SEQ ID NO: 20) DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0259] Exemplary cleavable polypeptide comprising heavy and light chain ranibizumab nucleotide sequences (SEQ ID NO: 103)

[0260] Exemplary cleavable polypeptide comprising heavy and light chain ranibizumab (SEQ ID NO: 21) MYRMQLLSCIALSLALVTNSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHW YFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGKRKR RGSGEGRGSLLTCGDVEENPGPMYRMQLLSCIALSLALVTNSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0261] Bevacizumab Bevacizumab, a humanized monoclonal full-length antibody against VEGF, is approved for intravenous infusion for the treatment of glioblastoma, colorectal cancer, lung cancer, renal cancer, cervical cancer, and ovarian cancer. However, the major drawbacks of bevacizumab treatment are the need for continuous regular intravenous infusions and the side effects associated with high doses administered in the systemic circulation, including hypertension, proteinuria, elevated liver enzymes, arterial thromboembolic events (ATEs), venous thromboembolic events, bleeding, and surgical and wound healing complications. Currently, bevacizumab is the only pharmacological agent with demonstrated preliminary clinical evidence of efficacy in patients with VS.

[0262] In some embodiments, the anti-VEGF proteins described herein that specifically bind to VEGF and / or its antigen-presenting fragments are antibodies. In some embodiments, such antibodies comprise an immunoglobulin light chain variable domain that is or includes a sequence 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 immunoglobulin light chain variable domain of bevacizumab, and / or comprise an immunoglobulin heavy chain variable domain that is or includes a sequence 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 immunoglobulin heavy chain variable domain of bevacizumab.

[0263] In some embodiments, the anti-VEGF proteins described herein that specifically bind to VEGF and / or its antigen-presenting fragments are antibodies. In some embodiments, such antibodies comprise an immunoglobulin light chain variable domain that is or includes the immunoglobulin light chain variable domain of bevacizumab and / or an immunoglobulin heavy chain variable domain that is or includes the immunoglobulin heavy chain variable domain of bevacizumab. In some embodiments, the antibody comprises an immunoglobulin light chain variable domain that is or includes the sequence of the immunoglobulin light chain variable domain of bevacizumab, except that it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, and / or an immunoglobulin heavy chain variable domain that is or includes the sequence of the immunoglobulin heavy chain variable domain of bevacizumab, except that it contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions.

[0264] In some embodiments, the anti-VEGF proteins described herein that specifically bind to VEGF and / or its antigen-presenting fragments are antibodies. In some embodiments, such antibodies comprise an immunoglobulin light chain (e.g., comprising an immunoglobulin light chain variable domain and an immunoglobulin light chain constant domain) that is or includes the immunoglobulin light chain constant domain of bevacizumab, and / or an immunoglobulin heavy chain (e.g., comprising an immunoglobulin heavy chain variable domain and an immunoglobulin heavy chain constant domain) that is or includes the immunoglobulin heavy chain constant domain of bevacizumab. In some embodiments, the antibody comprises an immunoglobulin light chain constant domain that has or includes the sequence of the immunoglobulin light chain constant domain of bevacizumab, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, and / or comprises an immunoglobulin heavy chain constant domain that has or includes the sequence of the immunoglobulin heavy chain constant domain of bevacizumab, except that it includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions.

[0265] In some embodiments, the first antigen-binding domain comprises three CDRs from an immunoglobulin light chain variable domain of bevacizumab and / or three CDRs from an immunoglobulin heavy chain variable domain of bevacizumab, hi some embodiments, the second antigen-binding domain comprises three CDRs from an immunoglobulin light chain variable domain of bevacizumab and / or three CDRs from an immunoglobulin heavy chain variable domain of bevacizumab.

[0266] Exemplary Bevacizumab Nucleotide Sequence (SEQ ID NO: 22)

[0267] Exemplary cleavable polypeptide comprising heavy and light chain bevacizumab amino acid sequences (SEQ ID NO: 23) MYRMQLLSCIALSLALVTNSEVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKRKRRGSGEGRGSLLTCGDVEENPGPMYRMQLLSCIALSLALVTNSDIQMTQSPSSLSASVGDRVTITCSASQDISN YLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0268] Exemplary Bevacizumab Heavy Chain Nucleotide Sequence (SEQ ID NO: 108)

[0269] Exemplary Bevacizumab Heavy Chain Amino Acid Sequence (SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0270] Exemplary Bevacizumab Light Chain Nucleotide Sequence (SEQ ID NO: 109) GACATCCAGATGACACAGAGCCCCAGCAGCCTGTCTGCCTCTGTGGGAGACAGAGTGACCATCACCTGTAGCGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAAAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTCACAAGCAGCCTGCACTCCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAAGCGGACTGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAATGCCCTGCAGAGCGGCAACAGCCAAGAGAGCGTGACAGAGCAGGACTCCAAGGATAGCACCTATAGCCTGAGCAGCACCCTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAATGT

[0271] Exemplary bevacizumab light chain amino acid sequence (SEQ ID NO: 25) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0272] VEGF TRAP A soluble VEGF receptor (also referred to herein as a VEGF TRAP) is a polypeptide comprising a portion of the extracellular region of one or more (e.g., two or three) mammalian VEGF receptor(s) (e.g., one or more of VEGFR-1, VEGFR-2, and VEGFR-3) operably linked to a signal peptide (e.g., any of the exemplary signal peptides described herein), wherein the soluble VEGF receptor is capable of specifically binding to one or more mammalian VEGF protein(s) (e.g., one or more (e.g., two, three, or four) of VEGF-A, VEGF-B, VEGF-C, and VEGF-D, e.g., one or more (e.g., two, three, or four) of human wild-type VEGF-A, human wild-type VEGF-B, human wild-type VEGF-C, and human wild-type VEGF-D).

[0273] In some examples, the soluble VEGF receptor comprises a portion of the extracellular domain of VEGFR-1 (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 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 250 amino acids, about 10 amino acids to about 200 amino acids, About 10 amino acids to about 150 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 50 amino acids, about 50 amino acids to about 732 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 to about 150 amino acids, about 50 to about 100 amino acids, about 100 to about 732 amino acids, about 100 to about 700 amino acids, about 100 to about 650 amino acids, about 100 to about 600 amino acids, about 100 to about 550 amino acids, about 100 to about 500 amino acids, about 100 to about 450 amino acids, about 100 to about 400 amino acids, about 100 to about 350 amino acids, about 100 to about 300 amino acids, about 100 to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 732 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids,About 200 amino acids to about 732 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 732 amino acids, about 250 amino acids to about 700 amino acids, about 250 amino acids to about 650 Amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 550 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, 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, about 300 amino acids to about 732 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about About 450 amino acids, about 300 to about 400 amino acids, about 300 to about 350 amino acids, about 350 to about 732 amino acids, about 350 to about 700 amino acids, about 350 to about 650 amino acids, about 350 to about 600 amino acids, about 350 to about 550 amino acids, about 350 to about 500 amino acids, about 350 to about 450 amino acids, about 350 to about 400 amino acids, about 400 to about 732 amino acids, about 400 to about 700 amino acids, about 400 to about 650 amino acids, about 400 Amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 732 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 732 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 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) (e.g., a continuous sequence from wild-type human VEGFR-1 (e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) extracellular domains from wild-type human VEGFR-1 (e.g., SEQ ID NO: 27, 29, 31, or 33) A contiguous sequence comprising an immunoglobulin-like domain, or a sequence that is 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 a contiguous sequence from wild-type human VEGFR-1, e.g., a sequence that is 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 contiguous sequence of SEQ ID NO: 27, 29, 31, or 33.

[0274] In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-2 (e.g., from about 20 amino acids to about 745 amino acids, or any subrange within this range described herein) (e.g., a contiguous sequence from wild-type human VEGFR-2 (e.g., a contiguous sequence comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2 (e.g., SEQ ID NO: 35)), or a contiguous sequence from wild-type human VEGFR-2 that is at least 80% (e.g., at least Also includes sequences that are 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, e.g., 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 the contiguous sequence in SEQ ID NO:35.

[0275] In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-1 (e.g., any portion of the extracellular region of VEGFR-1 described herein) and a portion of the extracellular region of VEGFR-2 (e.g., any portion of the extracellular region of VEGFR-2 described herein). For example, the soluble VEGF receptor can comprise one or more (e.g., 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-1 and one or more (e.g., 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-2 (e.g., aflibercept).

[0276] In some examples, the soluble VEGF receptor comprises a portion of the extracellular region of VEGFR-3 (e.g., from about 20 amino acids to about 751 amino acids, or any subrange within this range described herein) (e.g., a contiguous sequence from wild-type human VEGFR-3 (e.g., a contiguous sequence comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) immunoglobulin-like domains in the extracellular region from wild-type human VEGFR-3 (e.g., SEQ ID NO: 37, 39, or 41)), or a contiguous sequence from wild-type human VEGFR-3 that is at least 80% identical (e.g., at least Sequences that are 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, e.g., 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 the contiguous sequence in SEQ ID NO: 37, 39, or 41.

[0277] Non-limiting examples of extracellular domains of different mammalian VEGFR-1, different mammalian VEGFR-2, and different mammalian VEGFR-3 are described herein. Non-limiting examples of protein and nucleotide sequences encoding wild-type VEGF receptor proteins are provided below. As one skilled in the art can appreciate, substitutions at amino acid positions that are conserved between species are likely to result in changes in protein function, while substitutions at amino acid positions that are not conserved between species are unlikely to affect protein function.

[0278] The VEGFR-1 gene, found on human chromosome 13q12.3, encodes 33 exons, including members of the vascular endothelial growth factor receptor (VEGFR) family. VEGFR family members are receptor tyrosine kinases (RTKs) containing an extracellular ligand-binding region with seven immunoglobulin (Ig)-like domains, a transmembrane segment, and a tyrosine kinase (TK) domain within the cytoplasmic domain. This protein binds VEGF-A, VEGF-B, and placental growth factor (PGF), and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblasts, and peripheral blood monocytes. Multiple transcript variants encoding different isoforms have been found in this gene. Isoforms include full-length transmembrane receptor isoforms and truncated soluble isoforms.

[0279] Exemplary Human VEGFR-1 Isoform 1 cDNA Sequence (SEQ ID NO:26)

[0280] Exemplary human VEGFR-1 isoform 1 precursor amino acid sequence (SEQ ID NO: 27)

[0281] This variant (2), also known as sFlt1 or sVEGFR-1, differs in the 3' coding region and 3' UTR compared to variant 1. The encoded soluble protein (isoform 2) has a short, unique C-terminus and lacks the transmembrane and cytoplasmic regions of isoform 1.

[0282] Exemplary Human VEGFR-1 Isoform 2 (also known as sVEGFR-1) cDNA Sequence (SEQ ID NO: 28)

[0283] Exemplary Human VEGFR-1 Isoform 2 (also known as sVEGFR-1) Precursor Amino Acid Sequence (SEQ ID NO:29) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKK FPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQV LETVAGKRSYRLSMKVKAFPSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGK NKMASTLVVADSRISGIYICIASNKVGTVGRNISFYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRGEHCNKKAVFSRISKFKSTRNDCTTQSNVKH

[0284] This variant (3) differs in the 3' coding region and 3' UTR compared to variant 1. The encoded soluble protein (isoform 3) has a short, unique C-terminus and lacks the transmembrane and cytoplasmic regions of isoform 1.

[0285] Exemplary Human VEGFR-1 Isoform 3 cDNA Sequence (SEQ ID NO: 30)

[0286] Exemplary Human VEGFR-1 Isoform 3 cDNA Sequence (SEQ ID NO: 31) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKR IIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFPSPE VVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDAGNYTILLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKMASTLVVADSRISGIYICIASNKVGTVGRNIS FYITDVPNGFHVNLEKMPTEGEDLKLSCTVNKFLYRDVTWILLRTVNNRTMHYSISKQKMAITKEHSITLNLTIMNVSLQDSGTYACRARNVYTGEEILQKKEITIRDQEAPYLLRNLSDHTVAISSSTTLDCHANGVPEPQITWFKNNHKIQQEPELYTSTSTSPSSSSSPLSSSSSSSSSSSS

[0287] This variant (4) differs in the 3' coding region and 3' UTR compared to variant 1. The encoded soluble protein (isoform 4) has a short, unique C-terminus and lacks the transmembrane and cytoplasmic domains of isoform 1.

[0288] Exemplary Human VEGFR-1 Isoform 4 cDNA Sequence (SEQ ID NO: 32)

[0289] Exemplary Human VEGFR-1 Isoform 4 cDNA Sequence (SEQ ID NO: 33) MVSYWDTGVLLCALLSCLLLTGSSSGSKLKDPELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPF VEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPD EKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKAFITVKHRKQQVLETVAGKRSYRLSMKVKAFSPEVVWLKDGLPATEKSARYLTRGYSLIIKDVTEEDAGNYTI LLSIKQSNVFKNLTATLIVNVKPQIYEKAVSSFPDPALYPLGSRQILTCTAYGIPQPTIKWFWHPCNHNHSEARCDFCSNNEESFILDADSNMGNRIESITQRMAIIEGKNKLPPANSSFMLPPTSFSSNYFHFLP

[0290] The VEGFR-2 gene, found on human chromosome 4q12, encodes 30 exons containing members of the vascular endothelial growth factor receptor family (VEGFR) and is one of two genes encoding receptors for VEGF-A. This receptor, known as a kinase insert domain receptor, is a type III receptor tyrosine kinase. It functions as a key mediator of VEGF-A-induced endothelial proliferation, survival, migration, tubular morphogenesis, and sprouting. Signaling and trafficking of this receptor are regulated by multiple factors, including Rab GTPases, P2Y purine nucleotide receptors, integrin αVβ3, and T cell protein tyrosine phosphatases.

[0291] Exemplary Human VEGFR-2 cDNA Sequence (SEQ ID NO: 34) CAGCTCTGGCTCCTTGTCTCCCAGGCTGATCCTTTATTCAGAATACCACAAAGAAAGGACATTCAGCTCAAGGCTCCCTGCCGTGTTGAAGAGTTCTGACTGCACAAACCAGCTTCTGGTTTCTTCTGGAATGAATACCCTCATATCTGTCCTGATGTGATATGTCTGAGACTGAATGCGGG AGGTTCAATGTGAAGCTGTGTGGTGTCAAAGTTCAGGAAGGATTTTACCCTTTTGTTCTTCCCCCTGTCCCCAACCCACTCTCACCCCGCAACCCATCAGTATTTTAGTTATTTGGCCTCTACTCCAGTAAACCTGATTGGGTTTGTTCACTCTCTGAATGATTATTAGCCAGACTTCA AAATTATTTTATAGCCCAAATTATAACATCTATTGTATTATTTAGACTTTTAACATATAGAGCTATTTCTACTGATTTTTGCCCTTGTTCTGTCCTTTTTTTCAAAAAAGAAAATGTGTTTTTTGTTTGGTACCATAGTGTGAAATGCTGGGAACAATGACTATAAGACATGCTATGGCACA TATATTTATAGTCTGTTTATGTAGAAACAAATGTAATATATTAAAGCCTTATATATAATGAACTTTGTACTATTCACATTTTGTATCAGTATTATGTAGCATAACAAAGGTCATAATGCTTTCAGCAATTGATGTCATTTTATTAAAGAACATTGAAAAACTTGAAAAAAAAAAAAAAAAAA

[0292] Exemplary human VEGFR-2 precursor amino acid sequence (SEQ ID NO: 35)

[0293] The VEGFR-3 gene is found at human chromosome location 5q35.3 and encodes 35 exons that contain members of the vascular endothelial growth factor receptor family (VEGFR). This gene encodes the tyrosine kinase receptor for vascular endothelial growth factors C and D. This protein is thought to be involved in lymphangiogenesis and maintenance of lymphatic endothelium.

[0294] Exemplary Human VEGFR-3 Isoform 1 cDNA Sequence (SEQ ID NO: 36) GTGGGATCCACTCAGGAAGGCTCGGGGCTGGCACAAAGGACCACCCAGCATTGCCCTGTGCCACCCAGCACTCAGTGGACATTCTGGGGACCTGCCTTCAGCCTTTTCCTGCCCTGTGCCTGACATCAGCACCCTGGCTGGTCAGAATGCCGCCCTCCCAGAGGAGCAGCCGAGAGATCCCCTGAAGGCTGGAGGCATTCTGCTCAGGACCCCTATCCCAGCTCACAGTGCCCAACCATCTCACCAGGAGAAAGAGCCACATCCCCACGTTAGGACCACGGAGACTGACCACCACCCTGACCCCCCAAACCCACGCACCAGACGCTTGCAGGACAGGCGCCGCGCAGCGGGCAGGGGCTTGCCCGGCCGACCCTCCCCTCCCCACCTCCCCCACTGCGCGTTACTCCAGGATATGCCGAGTGCACGTATAAGGTCATCTTCGTCGTCCCCGTGGACCTCCCCCTTCCTCTGCACGTCGTCCAACGTGGGACTGGCGTGTCAGGCTTCCCTGGGAGGATCTGGAGGTTGTTCTCTGCAGAGAACCAGCCTGGCTCCTGGCGCGCACCTCTGCTCCCTTCTCCTCACTACCCACCCACGCATGTACCGGGAAAAAAACTACTATGCCCTTCTAGACCATGTTCTGAGAAAAGATCGAAAATATTTAACAAGAGATAATAATAAATCTGATGCCGGTCTTTGTGTGTGTTGCGGA

[0295] Exemplary human VEGFR-3 isoform 1 precursor amino acid sequence (SEQ ID NO: 37)

[0296] This variant (2) contains an alternative 3'-terminal exon compared to variant 1. As a result, isoform (2) has a shortened C-terminus compared to isoform 1.

[0297] Exemplary Human VEGFR-3 Isoform 2 cDNA Sequence (SEQ ID NO: 38)

[0298] Exemplary human VEGFR-3 isoform 1 precursor amino acid sequence (SEQ ID NO: 39)

[0299] This variant (3) contains an alternative 3'-terminal exon compared to variant 1. As a result, isoform (3) has a shortened C-terminus compared to isoform 1.

[0300] Exemplary Human VEGFR-3 Isoform 2 cDNA Sequence (SEQ ID NO: 40)

[0301] Exemplary human VEGFR-3 isoform 1 precursor amino acid sequence (SEQ ID NO: 41)

[0302] fusion proteins A fusion protein or chimeric protein is a protein created by joining two or more genes that originally encoded separate proteins. Translation of the fusion gene produces a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion proteins are engineered, and the resulting proteins may have underlying polypeptides with different functions and / or physicochemical patterns. In some embodiments, the anti-VEGF protein compositions described herein comprise fusions of one or more VEGF-R proteins or characteristic portions thereof described herein.

[0303] For example, antibody fusion proteins can combine an antibody targeting a specific antigen with a protein that can modify the immune response or induce direct damage to cancer cells. For example, cytolytic fusion proteins enhance the antibody's efficacy in eliminating cancer cells by conjugating the antibody to a toxin. These so-called "immunotoxins" derive their potency from the toxin and their specificity from the antibody or antibody fragment to which they are attached. In certain embodiments, such fusion proteins can include a polypeptide (e.g., an antibody and / or a characteristic portion thereof) fused with a cytokine to generate an "immunocytokine." Such fusion proteins can combine a tumor-targeting antibody (e.g., an anti-VEGF antibody) with a cytokine (a mediator of the innate immune response). In some such embodiments, this can increase local activation of the immune system in the tumor microenvironment, potentially aiding in the elimination of cancer cells and / or tumors (e.g., VS).

[0304] In some embodiments, any of the anti-VEGF proteins (e.g., antibodies, soluble VEGF receptors, and / or characteristic VEGF-binding portions thereof) described herein can include 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 and / or soluble VEGF receptor in a mammal, e.g., compared to the half-life of an otherwise identical antibody and / or soluble VEGF receptor that does not contain at least one of the one or more amino acid substitutions. Methods for determining the half-life of an antibody and / or soluble VEGF receptor in a mammal are well known in the art. Non-limiting examples of point mutations in Fc mutations that can decrease the half-life of an antibody and / or soluble VEGF receptor are described in Leabman et al., MAbs 5(6):896-903, 2013 (incorporated herein by reference in its entirety).

[0305] Aflibercept In certain embodiments, the anti-VEGF protein described herein is aflibercept. Aflibercept is a recombinant fusion protein consisting of portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept is a dimeric glycoprotein with a protein molecular weight of approximately 97 kilodaltons (kDa) and may contain post-translational glycosylation, which may comprise an additional 15% of the total molecular weight, resulting in a total molecular weight of 115 kDa. Aflibercept is FDA-approved and is indicated for the treatment of neovascular (wet) age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, metastatic colorectal cancer, and diabetic retinopathy.

[0306] In certain embodiments, aflibercept functions as a VEGF-TRAP. In contrast to certain exemplary antibody-based VEGF binding strategies described herein, such as those utilizing ranibizumab and / or bevacizumab, in some embodiments, a VEGF-TRAP incorporates the second binding domain of the VEGFR-1 receptor and the third domain of the VEGFR-2 receptor. By fusing these extracellular protein sequences to the Fc segment of a human IgG framework, a chimeric protein with extremely high VEGF binding affinity (Kd approximately 1 pM) is generated. Similar to ranibizumab and bevacizumab, a VEGF-TRAP such as aflibercept binds to all isomers of the VEGF-A family, as well as VEGF-B and placental growth factor. In certain embodiments, the aflibercept protein is conjugated to an appropriate secretory signal sequence, such as IL2SS (described below). In certain embodiments, the VEGF-TRAP protein product is represented by SEQ ID NO: 43 or 44.

[0307] Exemplary Aflibercept Nucleotide Coding Sequence (SEQ ID NO: 42)

[0308] Exemplary Aflibercept Amino Acid Sequence (SEQ ID NO: 43) MYRMQLLSCIALSLALVTNSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNY LTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0309] Exemplary Aflibercept Extracellular (EC) Domain Amino Acid Sequence (SEQ ID NO: 112) MYRMQLLSCIALSLALVTNSSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNY LTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

[0310] Exemplary Aflibercept Amino Acid Sequence (SEQ ID NO: 44) SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLS PSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0311] Exemplary Fragment, Crystallizable (Fc) Nucleotide Sequence (SEQ ID NO: 110) GACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCAAAGCCTAAGGACACCCTGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGCCACGAGGATCCCGAAGTGAAGTTCAATT GGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCTATCGAG AAAACCATCAGCAAGGCCAAGGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCAGAGAACA ACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCAGAAGTCTCTGAGCCTGTCTCCTGGC

[0312] Exemplary sequence of amino acids(Fc) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0313] construct Among other things, the present disclosure provides that some polynucleotides described herein are polynucleotide constructs. Polynucleotide constructs according to the present disclosure include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viral constructs (e.g., lentiviral, retroviral, adenoviral, and adeno-associated viral constructs) incorporating polynucleotides including anti-VEGF proteins, e.g., anti-VEGF antibodies and / or soluble VEGF receptors (e.g., anti-VEGF-TRAP proteins (e.g., fusion proteins such as aflibercept)). One of skill in the art would be able to select appropriate constructs and cells for producing any polynucleotide described herein. In some embodiments, the construct is a plasmid (e.g., a circular DNA molecule capable of autonomous replication inside a cell). In some embodiments, the construct can be a cosmid (e.g., the pWE or sCos series).

[0314] In some embodiments, the construct is a viral construct. In some embodiments, the viral construct is a lentiviral, retroviral, adenoviral, or adeno-associated viral construct. In some embodiments, the construct is an adeno-associated viral (AAV) construct (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety). A simplified example of a WT AAV genome is depicted in panel (A) of Figure 5, while a simplified example of a recombinant AAV (rAAV) construct is depicted in panel (B) of Figure 5. In some embodiments, the viral construct is an adenoviral construct. In some embodiments, the viral construct may also be based on or derived from an alphavirus. Alphaviruses include Sindbis (and VEEV) virus, Aura virus, Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus, Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Nudum virus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Southern elephant seal virus, and Tonate virus. virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, and Wataroa virus.Generally, the genomes of such viruses encode nonstructural proteins (e.g., replicons) that are translatable in the cytoplasm of host cells, as well as structural proteins (e.g., capsids and envelopes). Ross River virus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEEV) have all been used to generate viral constructs for delivery of coding sequences. By combining the envelope glycoproteins of an alphavirus and the capsid of a retrovirus, pseudotyped viruses can be formed. Examples of alphavirus constructs can be found in U.S. Patent Publication Nos. 20150050243, 20090305344, and 20060177819; the constructs and methods for their production are incorporated herein by reference in their entireties.

[0315] The constructs provided herein may be of different sizes. In some embodiments, the constructs are plasmids and may comprise a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some embodiments, the construct is a plasmid and can have a total length in the range of about 1 kb 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 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 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.

[0316] In some embodiments, the construct is a viral construct and can have a total nucleotide number of up to 10 kb. In some embodiments, the viral construct may be from about 1 kb to about 2 kb, 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 1 kb to about 9 kb, from about 1 kb to about 10 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 2 kb to about 9 kb, from about 2 kb to about 10 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 3 kb to about 9 kb. 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 kb 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 about 9 kb to about 10 kb.

[0317] In some embodiments, the construct is a lentiviral construct and can have a total nucleotide number of up to 8 kb. In some examples, the lentiviral construct 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, or from about 3 kb to about 4 kb. The total number of nucleotides may be 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 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 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb 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.

[0318] In some embodiments, the construct is an adenoviral construct and can have a total nucleotide number of up to 8 kb. In some embodiments, the adenoviral construct 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, or from about 3 kb to about 4 kb. The total number of nucleotides may be in the range of 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 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 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0319] Any of the constructs described herein may further comprise a regulatory sequence selected from the group of regulatory sequences, e.g., transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and / or additional untranslated regions that may house pre- or post-transcriptional regulatory and / or control elements. In some embodiments, the promoter may be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of regulatory sequences are described herein.

[0320] AAV particles Among other things, the present disclosure provides AAV particles comprising an AAV capsid described herein, including a recombinant AAV (rAAV) construct encoding an anti-VEGF protein, such as an anti-VEGF antibody and / or an anti-VEGF-TRAP protein. In some embodiments, AAV particles may be described as having a serotype that describes the construct strain and capsid strain. For example, in some embodiments, an AAV particle may be described as AAV2, in which case the particle has an AAV2 capsid and a construct comprising characteristic AAV2 inverted terminal repeats (ITRs). In some embodiments, AAV particles may be described as pseudotypes in which the capsid and construct are derived from different AAV strains, e.g., AAV2 / 9 refers to an AAV particle comprising a construct utilizing AAV2 ITRs and an AAV9 capsid. This same nomenclature can also be used for rAAV capsids, constructs, and / or particles.

[0321] In certain embodiments, gene therapy using rAAV particles is a promising treatment for inner ear disorders for several reasons: (1) the inner ear, including the auditory and vestibular sensory epithelium, has altered immune surveillance mechanisms similar to those in the central nervous system (Fujioka 2014, incorporated herein by reference in its entirety); (2) the sensory and supporting cells of the organ of Corti in the cochlea are postmitotic, which may allow for long-term expression after a single administration of rAAV; and (3) the combined clinical experience of rAAV delivery via multiple routes of administration in both adults and children suggests a strong safety profile of rAAV as a delivery vehicle, particularly when delivered locally and / or at low- to moderate-dose levels.

[0322] Clinical trials utilizing rAAV particles began over 20 years ago, and rAAV particles have been administered to hundreds of participants in dozens of clinical trials at systemic doses up to approximately 1E15 vg or greater (Flotte 1996; Flotte 2013; Parente 2018; Wang 2019, each of which is incorporated herein by reference in its entirety). The number of studies using rAAV particles for in vivo gene transfer has steadily increased. Their safety profile, combined with their high transduction efficiency across a wide range of target tissues, has established rAAV particles as the platform of choice for in vivo gene therapy (Wang 2019, incorporated herein by reference in its entirety). rAAV technology has been successfully applied clinically to a variety of conditions, including coagulation disorders, hereditary blindness, and neurodegenerative diseases (Colella 2018; Wang 2019, each of which is incorporated herein by reference in its entirety).

[0323] In certain embodiments, compositions comprising rAAV particles containing a construct encoding an anti-VEGF protein are referred to as rAAV-anti-VEGF. In certain compositions in which the Anc80 capsid is utilized, such compositions may be referred to as rAAVAnc80-anti-VEGF. In some embodiments, these compositions may also be referred to as AK-anti-VEGF. In some embodiments, rAAVAnc80-anti-VEGF particles comprise specific rAAV constructs, for example, may include the construct represented by panel (A) of Figure 6. Such constructs are referred to herein as VH / VL constructs and / or rAAVAnc80-VH / VL constructs. H / V L Illustratively referred to as rAAVAnc80-V H / V LThe construct encodes ranibizumab. In some embodiments, the rAAVAnc80-anti-VEGF particles comprise a construct represented by panel (B) of Figure 6, which may be referred to as an antibody GFP construct (ABGFP) and / or rAAVAnc80-ABGFP; an exemplary rAAVAnc80-ABGFP construct encodes ranibizumab or bevacizumab transcriptionally linked to GFP. In some embodiments, the rAAVAnc80-anti-VEGF construct comprises a construct represented by panel (C) of Figure 6, which may be referred to as an antibody (AB) construct and / or rAAVAnc80-AB; an exemplary rAAVAnc80-AB construct encodes bevacizumab. In some embodiments, the rAAVAnc80-anti-VEGF construct comprises the construct represented by panel (D) of Figure 6, which may be referred to as anti-VEGF-TRAP (TRAP) and / or rAAVAnc80-TRAP; an exemplary rAAVAnc80-TRAP construct encodes aflibercept. In some embodiments, the compositions disclosed herein, e.g., rAAV-anti-VEGF, are gene therapy compositions intended for the treatment of VS patients. In some embodiments, the compositions disclosed herein comprise AAVAnc80, the predicted common ancestor of adeno-associated virus (AAV) particle serotypes 1, 2, 8, and 9 (Zinn 2015, incorporated herein by reference in its entirety), as a delivery vehicle for a transgene encoding the sequence of ranibizumab, an antibody fragment (Fab) previously optimized for VEGF inhibition following intraocular administration (Genentech 2017, incorporated herein by reference in its entirety). In some embodiments, the compositions disclosed herein are non-enveloped, icosahedral virions approximately 26 nanometers (nm) in diameter. In some embodiments, the adeno-associated viruses on which the compositions disclosed herein, e.g., the synthetic AAVAnc80 capsid sequence, are based are non-pathogenic, single-stranded DNA genome-containing, helper virus-dependent members of the Parvoviridae family.

[0324] Recombinant AAV (rAAV) particles retain the same overall capsid structure as wild-type adeno-associated viruses, but encapsulate a genome lacking viral protein-coding sequences, replacing them with a therapeutic gene expression cassette. The only viral-derived sequences are the inverted terminal repeat (ITR) sequences, which are required to direct genome replication and packaging during particle production. Removal of the viral coding sequences maximizes the packaging capacity of rAAV particles and contributes to reduced immunogenicity and cytotoxicity when delivered in vivo (Wang 2019, incorporated herein by reference in its entirety).

[0325] AAVAnc80 is a rationally designed AAV capsid whose sequence was inferred by ancestral sequence reconstruction (Zinn 2015, incorporated herein by reference in its entirety). The first reported use of AAVAnc80 in the mammalian inner ear revealed high transduction efficiency in cochlear and vestibular hair cells and evidence of restoration of hearing, cochlear, and vestibular function in knock-in mouse models (Landegger 2017; Pan 2017, each of which is incorporated herein by reference in its entirety). Subsequent studies have confirmed that AAVAnc80 transduce cochlear and vestibular cells with significantly higher efficiency than other AAV serotypes in mice (Landegger 2017; Tao 2018; Yoshimura 2018; Omichi 2020, each of which is incorporated by reference in its entirety) and in non-human primates (Andres-Mateos 2019, incorporated by reference in its entirety).

[0326] In some embodiments, the compositions disclosed herein, e.g., rAAV-anti-VEGF, comprise two components: an AAVAnc80 (also referred to as Anc80L65; Zinn 2015, incorporated herein by reference in its entirety) capsid and a single-stranded rAAV DNA genome encoding an anti-VEGF protein, e.g., rAAV-VH / VL (e.g., ranibizumab), rAAV-AB (e.g., bevacizumab), rAAV-ABGFP (e.g., a GFP-tagged antibody, e.g., GFP-tagged ranibizumab or bevacizumab), and / or rAAV-TRAP (e.g., aflibercept) encapsidated by the AAVAnc80 capsid. In some embodiments, a composition disclosed herein, e.g., rAAV-anti-VEGF, comprises two components: an AAVAnc80 (also referred to as Anc80L65; Zinn 2015, incorporated herein by reference in its entirety) capsid and a single-stranded DNA genome of 3000-5000 nucleotides, excluding ITR sequences, encapsidated by the AAVAnc80 capsid (e.g., as shown by panels (A)-(D) of Figure 6).

[0327] In some embodiments, the compositions disclosed herein, e.g., recombinant construct DNA genome, contain a eukaryotic expression cassette flanked by AAV2 ITR sequences and encoding the following promoter and regulatory sequences: a cytomegalovirus (CMV) early enhancer element; a chicken β-actin (CβA) gene sequence located between the 5' flanking region and the proximal region of the second exon; and a 3' splice acceptor sequence from the rabbit β-globin (RβG) gene (commonly referred to as the CAG promoter) (Miyazaki 1989; Niwa 1991; Orban 2009, each of which is incorporated herein by reference in its entirety). In some embodiments, this hybrid regulatory element is followed by a bicistronic cassette encoding the ranibizumab heavy and light chains, separated by furin and protease 2A (T2A) cleavage sites from the S. asigna virus. In some embodiments, a 20 amino acid human interleukin-2 (IL-2) leader sequence is located upstream of each Fab chain to facilitate secretion of the protein. In some embodiments of the compositions disclosed herein, a bovine growth hormone polyadenylation site is present downstream of the anti-VEGF protein coding sequence.

[0328] The current standard of care for VS has evolved over the past few decades as advances in diagnostic imaging have improved our ability to identify and track tumor growth. With the growing recognition that many tumors exhibit slow or low growth rates and will not affect hearing as long as low growth rates are maintained, there has been a trend toward a conservative, observational "wait and rescan" approach (MacKeith 2013; Reznitsky 2019, each of which is incorporated herein by reference in its entirety). In some embodiments, an important potential benefit of the compositions disclosed herein, e.g., rAAV-anti-VEGF, is their potential to enhance this conservative treatment approach, e.g., by halting tumor growth, stabilizing hearing, and / or avoiding the need for more invasive treatment approaches, such as surgical resection and / or radiation therapy. Without being bound by theory, in some embodiments, it is believed that arresting VS growth provides substantial clinical benefit, and as shown in studies of bevacizumab-treated VS tumors in NF2 patients (Huang 2018; Plotkin 2019, each of which is incorporated herein by reference in its entirety), compositions disclosed herein, such as rAAV-anti-VEGF treatment, may promote not only tumor stasis but also tumor shrinkage, restoration of speech comprehension, and / or reduction in perceived difficulty with speech comprehension. Furthermore, studies using systemically administered VEGF inhibitors have shown improvements in NF2-related quality of life, including symptoms associated with VS in NF2 patients (Plotkin 2019, incorporated herein by reference in its entirety). In some embodiments, these clinical data suggest that rAAV-anti-VEGF administration and sustained, localized expression of anti-VEGF protein in diffuse continuity with the tumor may provide these additional benefits to patients with VS.

[0329] In some embodiments, intracochlear administration of a composition disclosed herein, such as rAAV-anti-VEGF, has the potential to eliminate the need for future treatment and preserve physiological hearing in patients with VS, regardless of the underlying etiology. In some embodiments, the compositions disclosed herein may delay invasive treatment approaches, such as surgical resection and / or radiation therapy, and the associated decline in physiological hearing. In some embodiments of any method disclosed herein, the method further comprises administration of standard of care. In some embodiments, anti-VEGF protein treatment may also potentially benefit subjects who subsequently undergo radiation therapy, as it may improve a subject's response to radiation therapy by sensitizing tumors and allowing for lower radiation doses (Koutsimpelas 2012; Gao 2015, each of which is incorporated herein by reference in its entirety).

[0330] AAV constructs The present disclosure provides polynucleotide constructs comprising anti-VEGF antibodies and / or soluble VEGF receptors (e.g., anti-VEGF TRAP proteins, such as the fusion protein aflibercept). In some embodiments described herein, polynucleotides comprising anti-VEGF antibodies and / or soluble VEGF receptors are referred to as constructs and may be packaged within AAV capsids to produce rAAV particles. In certain embodiments, rAAV constructs may also be referred to as vectors and may be quantified as vector genomes (vg), e.g., vg per milliliter (mL). A simplified schematic of a WT AAV genome is depicted in panel (A) of Figure 5, and a simplified schematic of a rAAV construct is depicted in panel (B) of Figure 5.

[0331] In some embodiments, polynucleotide construct comprises one or more components derived from or modified from naturally occurring AAV genome construct.In some embodiments, the sequence derived from AAV construct is AAV1 construct, AAV2 construct, AAV3 construct, AAV4 construct, AAV5 construct, AAV6 construct, AAV7 construct, AAV8 construct, AAV9 construct, AAV2.7m8 construct, AAV8BP2 construct, AAV293 construct or AAVAnc80 construct.Other exemplary AAV constructs that can be used herein are known in the art. See, e.g., Kanaan et al., Mol. Ther. Nucleic Acids 8:184-197, 2017; Li et al., Mol. Ther. 16(7):1252-1260, 2008; Adachi et al., Nat. Commun. 5:3075, 2014; Isgrig et al., Nat. Commun. 10(1):427, 2019; and Gao et al., J. Virol. 78(12):6381-6388, 2004 (each of which is incorporated by reference in its entirety).

[0332] In some embodiments, the provided constructs include a coding sequence, e.g., an anti-VEGF gene or a characteristic portion thereof, one or more regulatory and / or control sequences, and optionally 5' and 3' AAV-derived inverted terminal repeats (ITRs). In some embodiments in which 5' and 3' AAV-derived ITRs are utilized, the polynucleotide construct may be referred to as a recombinant AAV (rAAV) construct. In some embodiments, the provided rAAV construct is packaged into an AAV capsid to form an rAAV particle.

[0333] In some embodiments, the AAV-derived sequences (included in the polynucleotide construct) 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, 1990, incorporated herein by reference in its entirety). A typical AAV2-derived ITR sequence is approximately 145 nucleotides in length. In some embodiments, at least 80% (e.g., at least 85%, at least 90%, or at least 95%) of a typical ITR sequence is incorporated into the constructs provided herein. The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., Cold Spring Harbor Laboratory, New York, 1989; and K. Fisher et al., J. Virol. 70:520 532, 1996, each of which is incorporated herein by reference in its entirety). In some embodiments, any coding sequence and / or construct described herein is flanked by 5' and 3' AAV ITR sequences. AAV ITR sequences can be obtained from any known AAV, including currently identified AAV types.

[0334] In some embodiments, polynucleotide constructs of the pattern described in accordance with the present disclosure and known in the art (see, e.g., Asokan et al., Mol. Ther. 20:699-7080, 2012, incorporated herein by reference in its entirety) typically consist of a coding sequence or portion thereof, at least one and / or regulatory sequence, and optionally 5' and 3' AAV inverted terminal repeats (ITRs). In some embodiments, the provided constructs can be packaged into capsids to create rAAV particles. The rAAV particles can be delivered to selected target cells. In some embodiments, the provided constructs include an additional, optional coding sequence that is a nucleic acid sequence heterologous to the construct sequence (e.g., an inhibitory nucleic acid sequence), which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor), or other gene product of interest. In some embodiments, the nucleic acid coding sequence is operably linked to components that enable and / or control the transcription, translation, and / or expression of the coding sequence in cells of the target tissue.

[0335] As shown in panel (A) of Figure 5, the unmodified AAV endogenous genome contains two open reading frames, "cap" and "rep," flanked by ITRs. As shown in panel (B) of Figure 5, an exemplary rAAV construct similarly contains a coding region, e.g., ITRs flanking a coding sequence (e.g., an anti-VEGF gene). In some embodiments, the rAAV construct also contains conventional regulatory elements operably linked to the coding sequence in a manner that allows for its transcription, translation, and / or expression in cells transfected with the plasmid construct or infected with a virus produced by the present disclosure. In some embodiments, the rAAV construct optionally contains a promoter (shown in panel (B) of Figure 5), an enhancer, an untranslated region (e.g., 5'UTR, 3'UTR), a Kozak sequence, an internal ribosome entry site (IRES), a splicing site (e.g., acceptor site, donor site), a polyadenylation site (shown in panel (B) of Figure 5), or any combination thereof. Such additional elements are further described herein.

[0336] In some embodiments, the construct is a rAAV construct. In some embodiments, the rAAV construct can comprise at least 500 bp, at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb, at least 3.5 kb, at least 4 kb, or at least 4.5 kb. In some embodiments, the rAAV construct can comprise at most 7.5 kb, at most 7 kb, at most 6.5 kb, at most 6 kb, at most 5.5 kb, at most 5 kb, at most 4.5 kb, at most 4 kb, at most 3.5 kb, at most 3 kb, or at most 2.5 kb. In some embodiments, the rAAV construct may comprise about 1 kb 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 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

[0337] Any construct described herein can further comprise regulatory and / or control sequences, such as control sequences selected from the group of transcription initiation sequences, transcription termination sequences, promoter sequences, enhancer sequences, RNA splicing sequences, polyadenylation (poly(A)) sequences, Kozak consensus sequences, and / or any combination thereof. In some embodiments, the promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein.

[0338] Exemplary Construction Components Inverted terminal repeats (ITRs) The AAV-derived sequences of the construct typically include cis-acting 5' and 3' ITRs (see, e.g., BJ Carter, "Handbook of Parvoviruses," ed., P. Tijsser, CRC Press, pp. 155-168 (1990) (incorporated herein by reference in its entirety)). Generally, ITRs are capable of forming hairpins. The ability to form hairpins may contribute to the self-priming ability of ITRs, allowing for primase-independent synthesis of the second DNA strand. ITRs can also assist in efficient encapsidation of the rAAV construct in rAAV particles.

[0339] In some embodiments, rAAV particles (e.g., AAV2 / Anc80 particles) of the present disclosure can include rAAV constructs that include coding sequences (e.g., anti-VEGF genes) and associated elements flanked by 5' and 3' AAV ITR sequences. In some embodiments, the ITRs are or include approximately 145 nucleic acids. In some embodiments, all or substantially all of the ITR-encoding sequences are used. AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the ITRs are AAV2 ITRs.

[0340] An example of a construct molecule used in this disclosure is a "cis-acting" construct containing a transgene, in which a selected transgene sequence and associated regulatory elements are flanked by 5' or "left" and 3' or "right" AAV ITR sequences. The designations 5' and left refer to the position of the ITR sequence relative to the entire construct, read from left to right in the sense direction. For example, in some embodiments, the 5" or left ITR is the ITR that is closest to the promoter (as opposed to the polyadenylation sequence) of a given construct when the construct is linearly drawn in the sense direction. Similarly, the designations 3' and right refer to the position of the ITR sequence relative to the entire construct, read from left to right in the sense direction. For example, in some embodiments, the 3' or right ITR is the ITR that is closest to the polyadenylation sequence (as opposed to the promoter sequence) of a given construct when the construct is linearly drawn in the sense direction. The ITRs provided herein are depicted in 5' to 3' order according to the sense strand. Therefore, those skilled in the art will understand that when converting from sense to antisense, the 5' or "left" ITR can also be described as a 3' or "right" ITR. Furthermore, it is well within the capabilities of those skilled in the art to convert a given sense ITR sequence (e.g., a 5' / left AAV ITR) to an antisense sequence (e.g., a 3' / right ITR sequence). Those skilled in the art will understand how to modify a given ITR sequence for use as either a 5' / left or 3' / right ITR, or an antisense version thereof.

[0341] For example, an ITR (e.g., a 5' ITR) can have the sequence set forth in SEQ ID NO: 45. In some embodiments, an ITR (e.g., a 3' ITR) can have the sequence set forth in SEQ ID NO: 46. In some embodiments, an ITR contains one or more modifications, such as truncations, deletions, substitutions, or insertions, as known in the art. In some embodiments, an ITR contains fewer than 145 nucleotides, e.g., 127, 130, 134, or 141 nucleotides. For example, in some embodiments, an ITR comprises 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 nucleotides. In some embodiments, an ITR (e.g., a 5' ITR) can have the sequence set forth in SEQ ID NO: 47. In some embodiments, an ITR (e.g., a 3' ITR) can have the sequence set forth in SEQ ID NO: 48.

[0342] A non-limiting example of a 5' AAV ITR sequence is SEQ ID NO: 45. A non-limiting example of a 3' AAV ITR sequence is SEQ ID NO: 46. A non-limiting example of a 5' AAV ITR sequence is SEQ ID NO: 47. A non-limiting example of a 3' AAV ITR sequence is SEQ ID NO: 48. In some embodiments, an rAAV construct of the disclosure comprises a 5' AAV ITR and / or a 3' AAV ITR. In some embodiments, the 5' AAV ITR sequence is SEQ ID NO: 45. In some embodiments, the 3' AAV ITR sequence is SEQ ID NO: 46. In some embodiments, the 5' and 3' AAV ITRs (e.g., SEQ ID NOs: 45 and 46, or 47 and 48) flank a portion of a coding sequence, e.g., all or a portion of an anti-VEGF gene (e.g., SEQ ID NOs: 13, 14, 15, 19, 22, 42, and / or 103). The ability to modify these ITR sequences is within the skill of one in the art (see, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual," 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J. Virol. 70:520 532 (1996), each of which is incorporated herein by reference in its entirety). In some embodiments, the 5' ITR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 5' ITR sequence represented by SEQ ID NO:45 or 47. In some embodiments, the 3' ITR sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the 3' ITR sequence represented by SEQ ID NO:46 or 48.

[0343] Exemplary 5' AAV ITR (SEQ ID NO: 45) TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0344] Exemplary 3' AAV ITR (SEQ ID NO: 46) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGGGAGTGGCCAA

[0345] Exemplary 5' AAV ITR (SEQ ID NO: 47) CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0346] Exemplary 3' AAV ITR (SEQ ID NO: 48) AGGAACCCCTAGTGATGGAGTTGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0347] promoter In some embodiments, a construct (e.g., an rAAV construct) comprises a promoter. The term "promoter" refers to a DNA sequence that is recognized by an enzyme / protein that can promote and / or initiate transcription of an operably linked gene (e.g., an anti-VEGF gene). For example, a promoter typically refers to a nucleotide sequence to which, for example, RNA polymerase and / or any associated factors can bind and initiate transcription therefrom. Thus, in some embodiments, a construct (e.g., an rAAV construct) comprises a promoter operably linked to one of the non-limiting example promoters described herein.

[0348] In some embodiments, the promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some embodiments, the promoter is an RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some embodiments, the promoter is an RNA polymerase III promoter, including, but not limited to, the HI promoter, the human U6 promoter, the mouse U6 promoter, or the porcine U6 promoter. Generally, the promoter is one that can promote transcription in inner ear cells. In some embodiments, the promoter is a cochlear-specific promoter or a cochlear-directed promoter. In some embodiments, the promoter is a hair cell-specific promoter or a supporting cell-specific promoter.

[0349] Various promoters that can be used herein are known in the art.Non-limiting examples of promoters that can be used herein include: human EF1α, human cytomegalovirus (CMV) (US Pat. No. 5,168,062, which is incorporated herein by reference in its entirety), 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, retrovirus LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin 2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, mouse MX gene, glucose-regulated protein (GRP78 and GRP94), alpha-2-macroglobulin, vimentin, MHC class I gene H-2κb, HSP70, proliferin, tumor necrosis factor, thyroid-stimulating hormone a gene, immunoglobulin light chain, T cell receptor, HLA DQa and DQ, interleukin 2 receptor, MHC class II, MHC class II HLA-DRa, 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), troponin I (TN I), Duchenne muscular dystrophy, human immunodeficiency virus, and gibbon ape leukemia virus (GALV) promoter. Further examples of promoters are known in the art. See, e.g., Lodish, Molecular Cell Biology, Freeman and Company, New York 2007, each of which is incorporated by reference in its entirety. In some embodiments, the promoter is a CMV immediate early promoter.In some embodiments, the promoter is a CAG promoter or a CAG / CBA promoter. In some embodiments, the promoter comprises or consists of SEQ ID NO: 49. In some embodiments, the promoter comprises or consists of SEQ ID NO: 50. In certain embodiments, the promoter comprises a CMV / CBA enhancer / promoter construct exemplified in SEQ ID NO: 51. In certain embodiments, the promoter comprises a CMV / CBA enhancer / promoter construct exemplified in SEQ ID NO: 52. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified in SEQ ID NO: 53. In certain embodiments, the promoter comprises a CAG promoter or a CMV / CBA / SV-40 enhancer / promoter construct exemplified in SEQ ID NO: 54. In some embodiments, the promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the promoter sequence represented by SEQ ID NO: 49 or 50. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence represented by SEQ ID NO: 51, 52, 53, or 54.

[0350] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a nucleic acid encoding a protein (e.g., an anti-VEGF protein), causes transcription of RNA from the nucleic acid in a cell under most or all physiological conditions.

[0351] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41:521-530, 1985, incorporated herein by reference in its entirety), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFl-α promoter (Invitrogen).

[0352] Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of specific physiological states, such as acute phase, specific differentiation states of cells, or only in replicating cells. Inducible promoters and induction systems are available from a variety of commercial sources, including but not limited to, Invitrogen, Clontech, and Ariad. Further examples of inducible promoters are known in the art.

[0353] Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088, incorporated herein by reference in its entirety); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA 93:3346-3351, 1996, incorporated herein by reference in its entirety), the tetracycline repression system (Gossen et al, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992, incorporated herein by reference in its entirety), the tetracycline inducible system (Gossen et al, Science 268:1766-1769, 1995; also, Harvey et al. al, Curr. Opin. Chem. Biol., 2:512-518, 1998 (each of which is incorporated herein by reference in its entirety), the RU486 inducible system (Wang et al, Nat. Biotech. 15:239-243, 1997 and Wang et al., Gene Ther. 4:432-441, 1997, each of which is incorporated herein by reference in its entirety), and the rapamycin inducible system (Magari et al. J Clin. Invest. 100:2865-2872, 1997, each of which is incorporated herein by reference in its entirety).

[0354] The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and / or tissues (e.g., transcription of a specific gene occurs only in cells that express a transcriptional regulatory and / or control protein that binds to the tissue-specific promoter). In some embodiments, regulatory and / or control sequences confer tissue-specific gene expression. In some cases, tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner.

[0355] In some embodiments, the tissue-specific promoter is a cochlear-specific promoter. In some embodiments, the tissue-specific promoter is a cochlear hair cell-specific promoter. Non-limiting examples of cochlear hair cell-specific promoters include, but are not limited to, the ATOH1 promoter, the POU4F3 promoter, the LHX3 promoter, the MYO7A promoter, the MYO6 promoter, the α9ACHR promoter, and the α10ACHR promoter. In some embodiments, the promoter is a cochlear hair cell-specific promoter, such as the PRESTIN promoter or the ONCOMOD promoter. See, e.g., Zheng et al., Nature 405:149-155, 2000; Tian et al., Dev. Dyn. 23 1:199-203, 2004; and Ryan et al., Adv. Otorhinolaryngol. 66:99-115, 2009 (each of which is incorporated herein by reference in its entirety).

[0356] In some embodiments, the tissue-specific promoter is an ear cell-specific promoter. In some embodiments, the tissue-specific promoter is an inner ear cell-specific promoter. Non-limiting examples of inner ear non-sensory cell-specific promoters include, but are not limited to, GJB2, GJB6, SLC26A4, TECTA, DFNA5, COCH, NDP, SYN1, GFAP, PLP, TAK1, or SOX21. In some embodiments, the cochlear non-sensory cell-specific promoter can be an inner ear supporting cell-specific promoter. Non-limiting examples of inner ear supporting cell-specific promoters include, but are not limited to, SOX2, FGFR3, PROX1, GLAST1, LGR5, HES1, HES5, NOTCH1, JAG1, CDKN1A, CDKN1B, SOX10, P75, CD44, HEY2, LFNG, or S100b.

[0357] In some embodiments, the provided rAAV construct comprises a promoter sequence selected from a CAG, CBA, CMV, or CB7 promoter. In some embodiments of any of the therapeutic compositions described herein, the first and / or sole rAAV construct further comprises at least one promoter sequence selected from a cochlear and / or inner ear-specific promoter.

[0358] Exemplary CBA Promoter (SEQ ID NO: 49) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGG CGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0359] Exemplary CBA Promoter (SEQ ID NO: 50) GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGCGCGCCAGGCGGGGC GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0360] Exemplary CMV / CBA Enhancer / Promoter (SEQ ID NO: 51) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0361] Exemplary CMV / CBA enhancer / promoter (SEQ ID NO: 52) GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

[0362] Exemplary CAG enhancer / promoter (SEQ ID NO: 53)

[0363] Exemplary CAG enhancer / promoter (SEQ ID NO: 54)

[0364] In certain embodiments, the promoter is the endogenous human ATOH1 enhancer-promoter set forth in SEQ ID NO: 55. In some embodiments, the enhancer-promoter sequence is at least 85%, 90%, 95%, 98%, or 99% identical to the enhancer-promoter sequence represented by SEQ ID NO: 55.

[0365] Exemplary Human ATOH1 Enhancer-Promoter (SEQ ID NO: 55)

[0366] In certain embodiments, the promoter is the endogenous human SLC26A4 immediate promoter set forth in SEQ ID NO: 56 or 57. In certain embodiments, the promoter is the endogenous human SLC26A4 enhancer-promoter set forth in SEQ ID NO: 58, 59, or 60. In some embodiments, the enhancer-promoter sequence is at least 85...

Claims

1. A construct comprising a coding sequence operably linked to a promoter, said coding sequence encoding a vascular endothelial growth factor (VEGF) binding agent or a portion thereof.

2. The construct of claim 1 , wherein the promoter is a CAG promoter, a CBA promoter, a CMV promoter, or a CB7 promoter.

3. The construct of claim 1 or 2, wherein the VEGF binding agent is an antibody or a fragment thereof or comprises an antibody or a fragment thereof.

4. The construct of claim 3 , wherein the antibody fragment is a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment, an Fd fragment, an Fd′ fragment, a complementarity determining region (CDR), a single chain Fv, or an Fc domain.

5. The construct of any one of claims 1 to 4, wherein the VEGF binding agent is or comprises an immunoglobulin heavy chain, an immunoglobulin light chain, or a combination thereof.

6. the VEGF-binding agent being (i) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; (ii) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20; or (iii) combinations thereof; The construct according to any one of claims 1 to 5, comprising:

7. The construct of claim 6 , wherein the VEGF binding agent is or comprises ranibizumab.

8. The coding sequence is (i) a nucleic acid sequence comprising SEQ ID NO: 13; (ii) a nucleic acid sequence comprising SEQ ID NO: 19, or (iii) combinations thereof; The construct according to any one of claims 1 to 3 or 5 to 6, comprising:

9. The construct of any one of claims 1 to 8, wherein the coding sequence comprises one or more nucleic acid sequences each encoding a signal peptide.

10. The construct of claim 9 , wherein at least one nucleic acid sequence encodes an interleukin 2 (IL2) signal peptide.

11. The construct of any one of claims 1 to 10, wherein the coding sequence comprises one or more sequences encoding a self-cleaving peptide.

12. The construct of claim 11 , wherein the self-cleaving peptide is the thosea asigna virus 2A (T2A) peptide.

13. A construct according to any one of claims 1 to 3 or 5 to 12, wherein the coding sequence is or comprises the nucleic acid sequence set forth in SEQ ID NO:

21.

14. The construct of any one of claims 1 to 13, further comprising two AAV inverted terminal repeats (ITRs) flanking the coding sequence and promoter.

15. The construct of claim 14 , wherein the two AAV ITRs are or are derived from AAV2 ITRs.

16. 16. The construct of claim 14 or 15, wherein the two AAV ITRs comprise a 5'ITR comprising the nucleic acid sequence set forth in SEQ ID NO: 45 or 47 and a 3'ITR comprising the nucleic acid sequence set forth in SEQ ID NO: 46 or 48.

17. The construct of claim 1 , wherein the construct comprises a nucleic acid sequence set forth in any of SEQ ID NOs: 90, 91, 92, or 106.

18. An AAV particle comprising a construct according to any one of claims 1 to 17.

19. 20. The AAV particle of claim 18, further comprising an AAV capsid that is, or is derived from, an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-rh8, AAV-rhlO, AAV-rh39, AAV-rh43, or AAV Anc80 capsid.

20. 20. The AAV particle of claim 19, wherein the AAV capsid is an AAV Anc80 capsid, optionally an AAV Anc80L65 capsid.

21. 21. The AAV particle of claim 19 or 20, wherein the AAV capsid comprises a polypeptide set forth in SEQ ID NO: 113 or SEQ ID NO:

114.

22. A composition comprising a construct according to any one of claims 1 to 17 or an AAV particle according to any one of claims 18 to 21.

23. 23. The composition of claim 22, wherein the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

24. Approximately 1×10 １１ vg / mL ~ approx. 1 x 10 １５ 24. The composition of claim 22 or 23, formulated in a dosage of vg / mL.

25. Approximately 1×10 １０ ~Approx. 1×10 １３ The composition of any one of claims 22 to 24, formulated in a dose of vg / cochlea.

26. The composition of any one of claims 22 to 25, administered in a volume of about 0.01 mL to 0.1 mL.

27. A method comprising contacting an inner ear cell with a construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26.

28. Cells, (i) a construct according to any one of claims 1 to 17, and (ii) one or more constructs comprising an AAV Rep gene, an AAV Cap gene, an AAV VA gene, an AAV E2a gene, and an AAV E4 gene. The method of claim 1, comprising contacting said

29. A method comprising introducing a construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26 into the inner ear of a subject.

30. 30. The method of claim 29, further comprising measuring the hearing level of the subject.

31. 31. The method of claim 30, further comprising comparing the hearing level of the subject to a reference hearing level.

32. 32. The method of claim 31, wherein the hearing level of the subject is measured after introduction of a construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26, and the reference hearing level is the hearing level of the subject measured before introduction of a construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26.

33. The method of any one of claims 29 to 32, further comprising measuring a level of a vascular endothelial growth factor (VEGF) binding agent or a portion thereof in the subject.

34. 34. The method of claim 33, further comprising comparing the level of the vascular endothelial growth factor (VEGF) binding agent or portion thereof in the subject to a reference level of the vascular endothelial growth factor (VEGF) binding agent or portion thereof.

35. The method of any one of claims 29 to 34, further comprising measuring the volume of a tumor in the subject.

36. 36. The method of claim 35, further comprising comparing the subject's tumor volume to a reference tumor volume.

37. A method for treating hearing loss comprising administering to a subject in need thereof a construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26.

38. A method of treating an inner ear disorder in a mammal, comprising administering to a subject in need thereof a construct described in any one of claims 1 to 17, an AAV particle described in any one of claims 18 to 21, or a composition described in any one of claims 22 to 26.

39. 39. The method of claim 38, wherein the inner ear disorder is vestibular schwannoma or neurofibromatosis type II.

40. A method of treating vestibular schwannoma in a mammal, comprising administering to a subject in need thereof a construct described in any one of claims 1 to 17, an AAV particle described in any one of claims 18 to 21, or a composition described in any one of claims 22 to 26.

41. A construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26 for use in the treatment of an otic disease characterized by neovascularization and / or one or more symptoms associated with said otic disease.

42. A construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26, for use in the treatment of an inner ear disorder.

43. 43. The construct for use according to claim 42, wherein the inner ear disorder is vestibular schwannoma or neurofibromatosis type II.

44. A construct according to any one of claims 1 to 17, an AAV particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26, for use in the treatment of schwannoma.

45. Use of a construct according to any one of claims 1 to 17, a particle according to any one of claims 18 to 21 or a composition according to any one of claims 22 to 26 for the manufacture of a medicament for the treatment of an otic disease characterized by neovascularization and / or one or more symptoms associated with said otic disease.

46. Use of a construct according to any one of claims 1 to 17, a particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26 for the manufacture of a medicament for treating an inner ear disorder.

47. 47. The use of claim 46, wherein the inner ear disorder is vestibular schwannoma or neurofibromatosis type II.

48. Use of a construct according to any one of claims 1 to 17, a particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26 for the manufacture of a medicament for treating vestibular schwannoma.

49. 49. The method of any one of claims 37 to 40, the AAV construct for use of any one of claims 41 to 44, or the use of any one of claims 45 to 48, wherein the mammal is a human.

50. A kit comprising a construct according to any one of claims 1 to 17, a particle according to any one of claims 18 to 21, or a composition according to any one of claims 22 to 26.

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